Articles

Molecular Structure and Function of the Glycine Receptor Chloride Channel

Published Online:https://doi.org/10.1152/physrev.00042.2003

Abstract

The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four α-subunits (α1 to α4) and one β-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from α2 to α1β is completed by around postnatal day 20 in the rat. The β-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning GlyR molecular structure and function. Most of the information has been obtained from homomeric α1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.

I. INTRODUCTION

A. Scope of This Review

Ligand-gated ion channels permit cells to respond rapidly to changes in their external environment. They are particularly well known for mediating fast neurotransmission in the nervous system. The glycine receptor (GlyR) is a membrane-embedded protein that contains an integral Cl-selective pore. When glycine binds to its site on the external receptor surface, the pore opens allowing Cl to passively diffuse across the membrane. The GlyR is a member of the pentameric ligand-gated ion channel (LGIC) family, of which the nicotinic acetylcholine receptor cation channel (nAChR) is the prototypical member. Other members of this family include the cation-permeable serotonin type 3 receptor (5-HT3R), anion-permeable GABA type A and C receptors (GABAAR and GABACR), recently identified cation-permeable zinc and GABA receptors (34, 86), as well as invertebrate anion-permeable glutamate and histidine receptors (130). Note that glycine also directly activates a cation-selective ion channel of the excitatory glutamate receptor family (62). The structural and functional properties of this receptor class have recently been reviewed (94) and are not considered here.

Glycine was first proposed as an inhibitory neurotransmitter on the basis of a detailed analysis of its distribution in the spinal cord (16). Subsequent electrophysiological studies demonstrated a strychnine-sensitive hyperpolarizing action of glycine on spinal neurons (80, 395). This hyperpolarization was soon discovered to be mediated by an increase in Cl conductance (81, 82, 396). The receptors responsible for these actions were subsequently purified by strychnine affinity chromatography (291, 292), and the first GlyR subunit was cloned in 1987 (138).

Current research into the GlyR can be divided into two major strands. The first involves the investigation of the molecular mechanisms of GlyR trafficking and clustering at synapses. This area is currently the subject of intense investigation, and recent progress has been covered in several authoritative reviews (189, 190, 219). The second research strand is concerned with understanding the molecular structure and function of the GlyR. Research has intensified in this area over the past few years, and the purpose of this review is to present a coherent view of recent findings. Much of our understanding of GlyR structure-function has been gained by comparison with the structurally homologous nAChR. Hence, this review makes frequent references to research on the nAChR, particularly in areas where knowledge of the GlyR is deficient.

B. Glycine as an Inhibitory (and Excitatory) Neurotransmitter

When the GlyR is activated, the resulting Cl flux moves the membrane potential rapidly toward the Cl equilibrium potential. Depending on the value of the equilibrium potential relative to the cell resting potential, the Cl flux may cause either a depolarization or a hyperpolarization. The GlyR is generally known as an inhibitory receptor because the Cl equilibrium potential is usually close to or more negative than the cell resting potential. Subthreshold depolarizations can inhibit neuronal firing if they are accompanied by an increase in membrane conductance that shorts out excitatory responses, a phenomenon termed “shunting inhibition.” However, in embryonic neurons, the intracellular Cl concentration is raised substantially, with the effect that GlyR activation causes a strong, suprathreshold depolarization. These large glycine-induced depolarizations gate a calcium influx that is necessary for the development of numerous specializations, including glycinergic synapses (190). The switch to the mature neuron phenotype is mediated by the expression of a K+-Cl cotransporter, KCC2, which lowers the internal Cl concentration, thereby shifting the Cl equilibrium potential to more negative values and converting the actions of the GlyR from excitatory to inhibitory (355).

II. DIVERSITY, DISTRIBUTION, AND FUNCTION OF GLYCINE RECEPTOR SUBUNITS

A. Molecular Diversity

Betz and colleagues (291, 292) originally purified the rat spinal cord GlyR by affinity chromatography on aminostrychnine-agarose columns. Oligonucleotides designed from peptide sequences of purified receptors were then used to probe a rat spinal cord cDNA library, resulting in isolation of cDNA clones corresponding to the 48 kDa (α1) and 58 kDa (β) subunits (137, 138). Subsequently, cDNAs of the rat α2- and α3-subunits were cloned by homology screening (9, 199, 201). The α4-subunit, which does not appear to exist in the rat or human, was first identified in the mouse (254) and has subsequently been found in the chick (157) and zebrafish (91). While the α-subunit genes are highly homologous, with primary structures displaying 80–90% amino acid sequence identity, the β-subunit has a sequence similarity of ∼47% with the α1-subunit (137).

The rat α1-subunit has a splice variant, termed α1ins, which contains an eight-amino acid insert in the large intracellular loop (244) that contains a possible phosphorylation site (see sect. viA). Alternative splicing of the rat α2-subunit generates two splice variants, α2A and α2B (199, 201). The α2B-variant differs from α2A by the V58I and T59A amino acid substitutions. Another version of the α2-subunit, termed α2*, incorporates a single amino acid substitution (G167E) that confers strychnine insensitivity (202). Two transcripts of the human α3-gene, termed α3L and α3K, have been characterized (280). The α3K-variant lacks a 15-amino acid segment in the large intracellular loop that exists in both the rat α3- and the human α3L-subunits. A splice variant of the zebrafish α4-subunit contains a 15-amino acid insert in the ligand-binding domain (91). Although a human β-subunit gene polymorphism has been described (264), it does not appear to result in a coding mutation.

All GlyR genes cloned to date share a similar exon-intron organization with the coding region spread over nine exons (254, 264, 346). This common organization suggests a phylogenetic gene duplication (345).

B. Distribution and Function in the Rat Nervous System

1. Distribution of functional GlyRs

A) DISTRIBUTION OF STRYCHNINE AND GLYCINE BINDING SITES.

Autoradiographic localization of [3H]strychnine binding sites was first studied by Zarbin et al. (431) in the rat. Strychnine binding sites were shown to exist at high levels in the spinal cord and medulla and at lower levels in the pons, thalamus, and hypothalamus, while being virtually absent in higher brain regions. In the spinal cord, their distribution is relatively diffuse throughout the gray matter. In contrast, GlyRs in the brain stem are highly localized to discrete nuclei, notably the trigeminal nuclei, the cuneate nucleus, the gracile nucleus, the hypoglossal motor nucleus, the reticular nuclei, and cochlear nuclei (301). The retina, which also has a high concentration of strychnine sites (297), is considered as a separate case, below. Together, these areas comprise a subset of the distribution as determined by glycine autoradiography (270) or glycine immunoreactivity (310), a mismatch that is understandable given that glycine is also associated with glutamatergic synapses (270). An advantage of strychnine autoradiography is that it reveals the presence of surface-expressed receptors, but a disadvantage is that its limited resolution does not permit the ultrastructural localization of strychnine binding sites. Thus strychnine autoradiography does not necessarily define the distribution of glycinergic synapses.

B) DISTRIBUTION OF GLYR IMMUNOREACTIVITY.

Many studies have examined the immunocytochemical localization of GlyRs at the light and electron microscopic levels using generic GlyR α-subunit monoclonal antibodies. At the light microscopic level, there is a strong correlation between the distribution patterns revealed by GlyR immunoreactivity and strychnine autoradiography (17, 370). However, some significant differences have been observed. First, immunolabeling reveals the existence of GlyRs in the cerebellum (17, 361) and olfactory bulb (383), whereas none was seen using strychnine binding (431). Second, the substantia gelatinosa in the spinal cord is strongly labeled by strychnine (431), but not by GlyR antibodies (23). The reasons for the differential labeling are yet to be clarified.

Electron microscopic immunoreactivity reveals that GlyRs at central synapses are concentrated into regions closely apposed to presynaptic terminals (13, 23, 370, 371, 383), strongly suggesting a functional role. It is of interest to note that this approach has demonstrated the colocalization of GlyRs and GABAARs at individual postsynaptic densities in the spinal cord (43, 127, 369) and cerebellum (100).

C) DISTRIBUTION OF FUNCTIONAL GLYCINERGIC SYNAPSES.

Most central nervous system neurons are inhibited by glycine (279). Of course, the mere presence of functional GlyRs, especially on dissociated or cultured neurons, does not imply a physiological role. However, it has recently been proposed that the activation of nonsynaptic GlyRs in embryonic cortical neurons may be important for development, and that taurine released from local glial cells may be the endogenous ligand (114). Similarly, nonsynaptic GlyRs on hippocampal CA3 neurons are proposed to be held in a tonically active state by locally released taurine or β-alanine (273). The idea that glycinergic ligands may act on nonsynaptic GlyRs to mediate processes of physiological importance certainly warrants further attention. Traditionally, however, a functional role for GlyRs in neurons has required the demonstration of strychnine-sensitive synaptic currents.

There is abundant evidence for the existence of functional glycinergic synapses in the retina (see below) in spinal cord motor reflex pathways (219) and in spinal cord pain sensory pathways (4). Glycinergic neurotransmission has also been demonstrated in various brain stem nuclei. For example, it has been well characterized in several brain stem nuclei of the central auditory pathways. In the medullary cochlear nucleus, which receive inputs directly from the auditory nerve, glycinergic synapses occur onto stellate cells (108) and bushy cells (230). In the trapezoid body, a subsequent major relay station in the auditory pathway, GlyRs are located presynaptically at calyceal synapses onto principal cells (373). The prominent output from this nuclei extends to the superior olivary complex of the pons, where glycinergic synapses are also found (194, 351). Functional glycinergic synapses also exist on neurons in the medullary trigeminal (421), abducens (325), and hypoglossal motor nuclei (248, 347, 375). In the cerebellum, glycinergic synapses mediate inhibitory neurotransmission between Lugaro cells and Golgi cells in the cerebellar cortex (93), and between interneurons and principal cells in the deep cerebellar nuclei (182). This list may expand as other brain stem nuclei are characterized in detail.

It is relevant to note that glycine may not be the sole inhibitory neurotransmitter at many of these synapses. Mixed GABA-glycine synapses may mediate neurotransmission at individual synapses in the spinal cord (174), brain stem (194, 283, 325), and cerebellum (100). Interestingly, GlyR activation appears to be able to inhibit GABAARs via a phosphorylation-dependent mechanism (229). This process may be important for regulating inhibitory synaptic current magnitude at mixed GABA-glycine synapses. There is evidence that the GABAergic component of inhibitory neurotransmission at mixed synapses may be upregulated in individuals suffering from heritable disorders of glycinergic neurotransmission (see sect. vii).

Finally, presynaptic GlyRs have been functionally characterized at calyceal synapses (373) and on terminals synapsing onto rat spinal sensory neurons (172). Surprisingly, in both cases GlyR activation is excitatory, leading to increased neurotransmitter release.

2. Distribution of GlyR subunits

In situ hybridization was the first approach employed to localize the distribution of individual GlyR subunits in the rat. An advantage of this approach is its subtype specificity, but a disadvantage is that transcript expression does not necessarily imply the surface expression of functional receptors. Expression of α1-subunit mRNA in adult rats was highest in the brain stem nuclei and spinal cord, but it was also found in the superior and inferior colliculi and in regions of the thalamus and hypothalamus (245, 331). It was notably absent from cortical regions. Thus, with few exceptions, its distribution is similar to that of functional GlyRs as described above. In the rat, expression is detectable at embryonic day 15 and increases to a maximum at around postnatal day 15, without substantial changes in its distribution (245). Northern blot analysis reveals that α1ins shares a similar distribution (244).

Prenatally, transcripts of the α2-subunit gene are found throughout most of the central nervous system. However, postnatally they decline sharply with little label remaining by postnatal day 20 (9, 245, 392). Detectable amounts of α2-transcripts do persist into adulthood, however, notably in the retina (see below), auditory brain stem nuclei (293), and some higher brain regions (245). The α2A-isoform is expressed more abundantly than α2B during development, although the α2B-isoform is present at higher levels in the adult (199).

The distribution and developmental changes in α3-transcripts generally resemble those of α1-transcripts, with the exception that α3-expression is much less intense at all developmental stages (245). As with the α1-subunit, its expression intensity increases postnatally to reach a maximum at around 3 wk (245). The α3L- and α3K-variants share similar distribution patterns (280). GlyR α4-subunit transcripts are expressed at very low levels (if at all) in the adult rat (293), although they are strongly expressed in the spinal cord, dorsal root ganglia, sympathetic ganglia, and the male genital ridge of the chick (157).

GlyR β-subunit transcripts are distributed widely throughout the embryonic and adult central nervous system (125, 245). Although present at low levels prenatally, expression increases dramatically after birth and persists into adulthood (245). The reason for this broad expression profile is somewhat puzzling given that these subunits do not form functional receptors in the absence of α-subunits.

3. Developmental switch from α2 to α1β

Becker et al. (27) showed using protein expression that fetal GlyRs are predominantly α2-homomers, whereas adult receptors are predominantly α1β-heteromers. Such a switch is also supported by the mRNA expression patterns described above. In the neonatal rat, the α1-, α2-, and β-subunits exist in abundance, implying a mixture of receptor isoforms, but the switch towards the adult isoform is complete by around postnatal day 20 (27, 121, 392). The sparse expression of the α3- and α4-subunits suggests they may also be included in a minority of adult GlyRs. Recent evidence suggests this switch may not be as complete as originally thought and that α2-subunit expression may remain at significant levels throughout adulthood in the retina (see below) and auditory brain stem (293). Although the mechanism responsible for triggering the developmental switch is not known, it does not seem to require the activation of the GlyRs themselves (247).

Given that α2-subunits alone are expressed in embryonic neurons, is it possible that homomeric α2-GlyRs may mediate synaptic transmission? Takahashi et al. (361) showed that the single-channel conductance and kinetic properties of recombinant homomeric α2- and α1-GlyRs were similar to those of native GlyRs in rat spinal neurons at embryonic day 20 and postnatal day 22, respectively. They also demonstrated an increased decay rate of the glycinergic inhibitory postsynaptic currents (IPSCs) over the same period that was consistent with the change in channel kinetic properties (361). Subsequent studies have supported these findings (12, 347). However, it is unlikely that homomeric α2-GlyRs mediate inhibitory neurotransmission for the following reasons. First, because β-subunits are required for GlyR postsynaptic clustering (188, 259), it is not certain how the α2-homomers would undergo the prerequisite aggregation at postsynaptic densities. Second, a recent study has found that α2-homomeric GlyRs activate too slowly to effectively mediate synaptic transmission (246). Given their wide distribution throughout the nervous system during development, and the fact that Cl fluxes are excitatory in developing neurons (114, 355), it seems more likely that homomeric α2-GlyRs mediate nonsynaptic cell-to-cell communication that could be important for neuronal differentiation and synaptogenesis (190). Glycinergic synapses in immature neurons are probably comprised of α2β-heteromeric GlyRs (219). The single-channel conductance of synaptic GlyRs from embryonic neurons is consistent with such a conclusion (12, 347).

4. A special case: the retina

This is considered separately because the profile of GlyR subunit distribution is atypical and has been mapped in detail and because a specific role for glycinergic synapses has been proposed. GABA and glycine both function as inhibitory neurotransmitters in the retina (143, 176, 297, 390). In situ hybridization and immunohistochemistry both show that GlyR α1-, α2-, α3-, and β-subunits have different patterns of distribution in the adult rat (136, 146, 330). The α1-subunit is distributed predominantly on bipolar cells and on some ganglion cells in the inner plexiform layer (136, 144, 145, 231). The α2-subunit is distributed on amacrine cells and on almost all ganglion cells, whereas the α3- and β-subunits are distributed widely throughout the inner plexiform layer (136). A detailed study in the mouse concluded that α1-subunits are associated with synapses in the rod pathway between AII amacrine cells and off-cone bipolar cells, whereas α3-subunits are restricted to cone pathways (159). Together these results indicate a spatial distribution of GlyR subunit composition throughout the adult retina. Consistent with this picture, Enz and Bormann (105) detected mRNA for all four GlyR subunits in RNA from whole retina, but mRNA for only α1- and β-subunits in RNA isolated from individual rod bipolar cells.

Electron microscopy has confirmed that the punctate immunoreactivity seen with the light microscope is due to clusters of GlyRs at the postsynaptic densities (146, 330). Consistent with these anatomical studies, electrophysiological investigations in the rat have revealed the presence of glycinergic inhibitory postsynaptic potentials (IPSPs) in identified amacrine cells (119), ganglion cells (153, 302, 368), and rod bipolar cells (77, 99, 303).

The synaptic distribution of GlyR subunits is spatially distinct from that of GABAAR subunits, although individual ganglion cells may possess both types of synapse (195, 329). Recent studies have begun to address the possibility that glycinergic and GABAergic transmission may have distinct physiological roles. Although functional differences between GABAergic and glycinergic IPSPs in retinal neurons have been demonstrated (119, 302), the physiological significance remains unknown. However, structural studies have provided evidence that the GABA and glycine synaptic pathways participate in different functional circuits. In particular, glycinergic synapses are thought to play a specific role in the transmission of dark-adjustment signals through the off-channel of the rod pathway from amacrine cells to off-bipolar cells and hence to off-ganglion cells (146, 330, 391). This circuit contributes to the switch from day to night vision.

C. Distribution and Function in Other Tissues

1. Spermatozoa

The front of the mammalian sperm head contains a large secretory vesicle termed the acrosome. The process of fertilization is initiated when the sperm head contacts the outer coat, or zona pellucida, of the egg. A zona pellucida-specific glycoprotein, ZP3, forms the sperm receptor. Its interaction with sperm initiates a complex intracellular signaling mechanism inside the sperm that culminates in a calcium elevation that is thought to be mediated at least partly by an influx through voltage-gated calcium channels (115). This event, termed the acrosome reaction (AR), results in the release of acrosome hydrolytic enzymes by exocytosis. These enzymes induce various protein modifications to ensure that the sperm remains tightly bound to the zona pellucida while fusion takes place between the sperm and egg plasma membranes.

The activation of GlyRs and GABAARs in the sperm plasma membrane appears to be essential for the AR (257). There is considerable evidence that GlyRs exist in sperm plasma membranes. For example, immunochemical studies have demonstrated the existence of GlyR α- and β-subunit protein in porcine, mouse, and human sperm (49, 258, 332). An immunofluorescence study localized the α-subunits to cell membranes in the periacrosomal regions of live mouse sperm (332). In addition, strychnine binding studies have revealed the presence of GlyRs in hamster sperm (232).

Functional evidence for GlyR involvement has also been demonstrated. For example, glycine initiated the AR in a manner that was inhibited by strychnine or a GlyR α-subunit antibody (49, 332). Furthermore, studies using fura 2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the ZP3-mediated calcium influx (49). Finally, sperm from homozygous spasmodic and spastic mice (which possess defective GlyR α1- and β-subunits, respectively) are deficient in their ability to undergo the AR (333).

Thus the GlyR is likely to have a central role in the AR. However, two questions remain about this process. What is the concentration of Cl inside sperm? Presumably, it must be high enough to force an outward (i.e., depolarizing) Cl flux upon GlyR activation. Second, what is the glycine concentration in the oviduct where fertilization takes place? Do the GlyRs remain tonically active holding the sperm in a depolarized state preceding the AR?

Harvey et al. (157) found that the α4-subunit gene is expressed on the developing male genital ridge of the chick and proposed that GlyRs containing this subunit may contribute to the development of immature spermatogonia.

2. Endocrine pancreas

A pancreatic cell line, GK-P3, expresses functional GlyRs. When activated, these receptors cause a depolarization that increases the intracellular calcium concentration (393). A glycine receptor antibody displayed immunoreactivity with GK-P3 cells and with isolated rat pancreatic islet cells (393) prompting the authors to surmise that GlyRs may also be expressed in islet cells in vivo. However, there is as yet no electrophysiological evidence for GlyRs in pancreatic islet cells.

3. Adrenomedullary chromaffin cells

High-affinity [3H]strychnine binding sites have been shown to exist in catecholamine-secreting chromaffin cells of the adrenal medulla (415, 416). The same group subsequently demonstrated that glycine can stimulate significant catecholamine secretion from chromaffin cells in both in vitro and in vivo assays (414, 417). The presence of GlyR α3-subunit mRNA (but not α1 or α2) was also demonstrated by RT-PCR from RNA extracted from rat adrenal glands. However, direct electrophysiological evidence for glycine-activated currents in chromaffin cells is conspicuously absent to date.

4. Kupffer cells and other macrophages

A variety of pharmacological evidence, summarized in Reference 184, suggests that GlyRs may at least partially mediate the anti-inflammatory effects of glycine in macrophages and leukocytes. Research on GlyR involvement in these processes has focused on Kupffer cells, which are specialized macrophages found in the liver. Glycine has been shown to reduce the magnitude of lipopolysaccharide-induced calcium transients in these cells in a strychnine-dependent manner (122, 167). Similar observations have also been made in neutrophils (398) and hepatic parenchymal cells (304). Recent evidence from Western blots, RT-PCR, and RNAse protection assays indeed suggest the presence of GlyR α1-, α2-, α4-, and β-subunits in Kupffer cells (122).

5. Neural stem progenitor cells

Strychnine-sensitive glycine-gated currents are present in postnatal, nestin-positive neural stem progenitor cells (278). Consistent with this observation, RT-PCR and immunocytochemical methods revealed the presence of α1-, α2-, and β-subunit RNA transcripts and α-subunit protein, respectively.

III. STRUCTURE AND ASSEMBLY

A. General Structural Features

The nAChR is the most intensively investigated member of the LGIC family. Consequently, most of the structural features of the GlyR have been deduced from its homology with this receptor. LGIC receptors contain five subunits arranged pseudo-symmetrically around a central ion-conducting pore. The membrane topologies of all LGIC subunits are similar. This topology includes a large NH2-terminal extracellular domain that contains the agonist binding sites. A defining feature of LGIC subunits is the conserved cysteine loop in this domain. All GlyR subunits also harbor a second cysteine loop (309) that incorporates a principal glycine-binding domain. As discussed in detail below, the crystal structure of acetylcholine-binding protein (AChBP) provides an excellent model for understanding the structure of this domain (52).

Hydropathy analysis originally predicted an arrangement of four α-helical transmembrane domains (TM1–TM4) per subunit. Although evidence exists for the inclusion of β-sheet in the TM regions (134), the recent elucidation of the crystal structure of the Torpedo nAChR TM domains provides an overwhelming argument in favor of the original four α-helical model (267). This structure, determined by cryoelectron microscopy to a resolution of 4 Å by Miyazawa et al. (267), is a major advance in the field. Finally, TM3 and TM4 are linked by a large, poorly conserved, intracellular domain that contains phosphorylation sites and other sites for mediating interactions with cytoplasmic factors. The structure and function of each of these regions is now considered in detail.

B. Transmembrane Domains

1. Spatial organization

The principal role of the TM domains is to provide a sealed barrier to separate the ion permeation pathway from the apolar region of the lipid bilayer. In most ion channels of known structure, this is achieved by a close packing of amphipathic α-helices at angles close, but not quite perpendicular, to the plane of the membrane (353). This arrangement also applies to LGIC receptors, with each subunit contributing an α-helical TM2 domain to the lining of a single central water-filled pore. The TM1, TM3, and TM4 domains surround TM2 and provide the interface with the lipid bilayer, thereby isolating TM2 from direct contact with the surrounding hydrophobic environment. Viewed from the synapse, TM1–TM4 are arranged consecutively in a clockwise manner, with TM1 and TM3 located closest to TM2 (267). In the nAChR, the TM domains splay outwards towards the extracellular membrane surface and extend about two helical rotations (∼10 Å) beyond the hydrophobic membrane core. As noted by Miyazawa et al. (267), the extracellular spaces between the splayed helices appear to afford a lateral pathway (in addition to the large central outer vestibule pathway) for ions to access the pore.

The remainder of this section attempts to relate the Miyazawa TM domain structure with an abundance of earlier information that also bears upon TM domain structure and function. However, before doing so, it is worth briefly considering three functionally based techniques that have been of particular value in defining the secondary structure of ion channel pore-lining domains.

2. Methods for probing TM domain secondary structure

A) SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD.

The substituted cysteine accessibility method (SCAM) was initially applied as a means of identifying the secondary structure of ion channel pore-lining domains (5, 8). The method entails introducing cysteine residues one at a time into the protein domain of interest. Cysteine reactivity is then assayed by exposure to highly soluble, sulfhydryl-specific reagents, generally methanethiosulfonate (MTS) derivatives (180). If a functional property of the channel is irreversibly modified upon exposure to such a reagent, the cysteine is assumed to be exposed at the water-accessible protein surface. If every second residue is reactive, then the secondary structure is interpreted as β-sheet (8), whereas if every third or fourth residue is exposed, the structure is interpreted as α-helical (7). This approach is now applied more widely to probe structural changes in extramembranous domains (e.g., Ref. 234). However, a drawback of applying this approach outside the pore is that a lack of functional modification does not necessarily mean that the residue has not reacted. In other words, negative results cannot be interpreted. However, this limitation is less likely to apply in the spatially restricted environment of an ion channel pore, where attachment of a large side chain is more likely to affect current flow, thus providing a generally more reliable measure of cysteine reactivity. Various extensions to this technique have also proven useful. For example, by determining changes in cysteine reactivity in various functional states (e.g., closed, open, and desensitized), it may be possible to draw conclusions about state-dependent structural changes. Similarly, by comparing state-dependent reaction rates of positively and negatively charged reagents, it may be possible to estimate the local electrostatic potential (289, 405). The originators of SCAM have provided an excellent review of its capabilities and limitations (180).

B) TRYPTOPHAN SCANNING MUTAGENESIS.

This approach involves introducing tryptophan residues one at a time into the domain of interest. Because tryptophan side chains are bulky, it is assumed that if they protrude into the relatively fluid lipid bilayer they should be less likely to disrupt receptor structure and function than if they protrude towards the protein interior (71). Experimentally, one or more basic functional properties (e.g., agonist EC50) of each mutant receptor is measured, and then a correlation is drawn between the position of the introduced tryptophan and the severity of the functional consequence. As with SCAM, any resulting periodicity is interpreted as β-sheet or α-helix.

C) HYDROPHOBIC REAGENT REACTIVITY.

n this approach, employed extensively by Blanton and colleagues in the nAChR (22, 40, 41), labeled hydrophobic reagents are incubated with the receptor. The identity of the residues that are covalently modified by these compounds is then determined using biochemical assays. Residues thus identified are assumed to be exposed to the lipid bilayer. Again, any resulting periodicity is interpreted in terms of secondary structure.

3. TM1

By connecting directly with the NH2-terminal domain, TM1 is ideally located to act as a linkage between the ligand-binding site and the channel activation gate. Hence, an unequivocal understanding of its structure and relationship with TM2 is essential. The Torpedo nAChR crystal structure identifies TM1 as an α-helix that is initiated at the residue corresponding to Y222 of the α1-GlyR and enters the membrane at around M227. As stated above, it is likely that water-filled space surrounds the extracellular portion of this helix. In support of this, an aqueous tyrosine-specific reagent labeled two tyrosines (Y223, Y228) in TM1 of the α1-GlyR (222). Furthermore, SCAM analysis on the nAChR revealed several extramembranous TM1 residues that are accessible to modification by hydrophilic reagents (432). Several lines of evidence implicate the extramembranous TM1 residues in LGIC gating (e.g., Refs. 6, 38, 102, 363, 432). Throughout the membrane-embedded portion of the nAChR TM1 there appears to be a distinct absence of van der Waals contacts with TM2, implying that a water-filled pocket separates the respective domains (267). However, by homology with nAChR, there may be a hydrophobic bond linking I234 (or L237) and M12′ in TM2 of the α1-GlyR. At its intracellular end, the TM1 α-helix is probably terminated by the aspartic acid at position 247.

4. TM2

Affinity labeling experiments employing pore-blocking substances first suggested an α-helical open state structure of the nAChR TM2 (reviewed in Ref. 18). The Torpedo nAChR structure of Miyazawa et al. (267) confirms the long-held view that this domain forms an α-helix throughout its entire length (267). As summarized in Figure 1A, an α-helical structure is also strongly supported by SCAM analysis. Indeed, the luminal exposure patterns as determined by SCAM and the Miyazawa structure are entirely in agreement. SCAM also reveals a highly conserved pattern of residue exposure in the nAChR, GABAAR, and 5HT3R (Fig. 1A), suggesting that a similar pattern applies to all LGIC members.

FIG. 1.

FIG. 1.Pore-lining residues in the α1 glycine receptor (GlyR). A: sequence alignments of the TM2 domains of indicated LGIC subunits. Positively charged residues are shaded in blue, and negatively charged residues are shaded in yellow. Note that only cationic LGICs have a negatively charged residue at −1′. Circles denote pore-lining residues as identified by SCAM analysis (7, 234, 341, 409) or by cryoelectron microscopic analysis in the nicotinic acetylcholine receptor (nAChR) (267). In the case of the serotonin (5-HT3) receptor, dark circles denote those residues identified as pore-lining by both Refs. 288 and 316, whereas light circles denote residues identified as pore-lining by Ref. 316 only. Additional GlyR α1-subunit residues that are assumed by structural homology with other LGICs to line the pore are identified by squares. B: helical net representation of GlyR α1-residues with the putative pore-lining residues denoted by a white background. C: hypothetical cross-sectional view through the α1 GlyR pore. Pore-lining residues are indicated by the white backgrounds. The exposure pattern of residues deeper than G2′ cannot currently be modeled.


To facilitate comparison between different LGIC members, a common TM2 numbering system is used (265). This system assigns position 1′ to the putative cytoplasmic end of TM2 and 19′ to the outermost residue (Fig. 1A). (Note that these assignments are confirmed by the Miyazawa TM domain structure.) A complete SCAM analysis of the GlyR TM2 is yet to be published. However, experiments conducted to date indicate the following GlyR α1-subunit residues line the pore: G2′, T6′, R19′, and A20′ (234, 341). By homology with other LGICs (7, 234, 341, 409), the following residues are also likely to line the pore: T7′, L9′, T10′, T13′, S16′ and G17′ (Fig. 1A). When viewed on an α-helical net, these residues form a hydrophilic “strip” along one side of an otherwise hydrophobic α-helix (Fig. 1B). A predicted cross-section through the α1-GlyR pore is shown in Figure 1C. The pore exposure pattern of residues intracellular to G2′ cannot currently be predicted for anionic LGICs because, as discussed in detail in section ivB, they contain an additional proline at position −2′ that is likely to induce structural disruptions around the internal pore boundary. Because the GlyR β-subunit TM2 has an unusually low sequence homology with all other LGIC TM2 domains (Fig. 1A), it will be of interest to determine whether it also shares the consensus residue exposure pattern.

Structural analysis shows TM2 to be kinked radially inwards, attaining a minimum pore diameter at its midpoint (267). Miyazawa et al. (267) propose that this constriction facilitates a tight hydrophobic coupling between TM2 residues of neighboring subunits at two levels in the central region of the pore. The first contact is thought to occur between L9′ of one subunit and the 10′ residue of the adjacent subunit. In all GlyR α-subunits the 10′ residue is a threonine, but in the β-subunit it is a serine. The second intersubunit contact occurs between residues homologous to Q14′ and T13′ in neighboring TM2s of the GlyR α1-subunit (or E14′ and S13′ in the β-subunit).

Although the 19′ position defines the external border of membrane-embedded portion TM2, the α-helical structure extends into extracellular space for another 2.5 turns before terminating at the residue corresponding to V280 in the α1-GlyR (267). SCAM analyses on the GlyR and GABAAR confirm that most extramembranous TM2 residues have extensive contact with water (37, 234). In fact, the SCAM analysis on the GABAAR even predicted an α-helical structure for these residues (37). The TM2-TM3 linker is formed by the α1-subunit residues, V280 to D284.

The roles of TM2 in forming the channel gate, in controlling ionic selectivity, and in forming the binding sites for agents of physiological and pharmacological importance are considered in sections iv and vi.

5. TM3

According to the Miyazawa TM domain structure, the α1-GlyR TM3 α-helical domain starts at I285, with the membrane-embedded portion extending from A288 to H311 (267). There is strong support from functionally based techniques that at least the external (NH2-terminal) half of this domain forms an amphipathic α-helix. For example, evidence from the nAChR using lipophilic probes identified an α-helical like periodicity in the lipid-facing residues (40). A tryptophan scanning analysis identified an identical periodicity in the same set of residues (76). In addition, SCAM analysis of the GABAAR using water-soluble reagents also supported an α-helical periodicity in the outer half of TM3 (400, 401). A satisfying aspect of these studies was that the water-facing residues were generally displaced by one from the lipid-facing residues (see Ref. 223 for review). Consistent with structural predictions (267), the SCAM results strongly suggest that this portion of the domain contributes to the lining of a water-filled pocket distinct from the channel pore. Recent SCAM studies on the GABAAR in the absence and presence of pharmacological agents suggest that this pocket changes conformation as the channel gates (400402). An abundance of evidence from both the GABAAR and GlyR, reviewed in section vi, provide a strong case that residues in this pocket form binding sites for alcohols and volatile anesthetics. The Miyazawa TM domain structure reveals that residues from TM3 are closely apposed to residues from both TM2 and TM4 at several points throughout their lengths (267). However, TM3 appears to contact TM1 only towards the intracellular membrane surface.

6. TM4

In addition to direct structural evidence (267), several lines of functional evidence imply that this domain forms an α-helix throughout its entire length. First, the pattern of lipid-exposed residues is consistent with an α-helical periodicity as determined by both hydrophobic probes in the nAChR (40, 41) and tryptophan scanning mutagenesis in the GABAAR (171). Second, proteolytic studies on GlyR α1-homomers did not identify cleavages in membrane-associated fragments of this domain (221), a result that is also consistent with an α-helical structure. By structural homology with the nAChR, the α1-GlyR TM4 is likely to be initiated at K385 and terminated at V418, with the intramembranous portion extending from K389 to I408 (267). Thus the α-helix extends about 2.5 turns beyond the external membrane boundary. Although TM4 is closely apposed to TM3 throughout its length, its contact TM1 appears confined to its intracellular half (267).

C. NH2-Terminal Ligand-Binding Domain

1. Structural homology with AChBP

The fresh water snail, Lymnaea stagnalis, produces and stores a soluble AChBP in glial cells located near to cholinergic synapses. When released by acetylcholine stimulation, AChBP buffers the acetylcholine in the synaptic cleft (350). This protein forms a stable homopentamer and binds acetylcholine, δ-tubocurarine, and α-bungarotoxin with much the same affinity as does the α7-homomeric nAChR (350). AChBP comprises 210 amino acids and, although it lacks the TM domains, it provides a full-length model of the NH2-terminal ligand-binding domain of LGICs. It also incorporates the signature cysteine loop that is a unique feature of the LGIC family. It shares a 20–24% amino acid sequence homology with nAChR subunits and a 17% homology with the GlyR α1 subunit (Fig. 2). The crystal structure of this protein (52) represents a major breakthrough in our understanding of LGIC structure and function. Due to both its significant sequence homology and to its functional similarity with the α7-homomeric nAChR, its structure is considered an accurate template of the NH2-terminal ligand-binding domain of the nAChR and, by inference, of other LGIC members.

FIG. 2.

FIG. 2.Amino acid sequence alignment of AChBP and the human α1 and β GlyR subunits. Secondary structural elements of AChBP and Torpedo nAChR are shown in gray, with β-strands represented by arrows and helices represented by cylinders (52, 267). Membrane-spanning sections of the TM α-helices are shaded in dark gray. The NH2-terminal α-helix is labeled by α, and two short polyproline helices are identified by 310. Cross-linked cysteines are indicated by the black brackets. The approximate locations of the AChBP binding domains are identified by red lines (and labeled AF, as appropriate) with known glycine and strychnine binding residues boxed in black. The numbered assembly boxes are outlined in green. Residues on the plus and minus sides of the interface are shaded yellow and blue, respectively. Zinc-coordinating histidines are shown in pink. See Ref. 276 for justification of the sequence alignment used in the zinc-coordinating region. Putative glycosylation sites are shaded in green, and putative phosphorylation sites are shaded in red and labeled accordingly. A region of the TM3-TM4 domain involved in determining single-channel conductance in 5-HT3Rs is shaded in gray. The TM3 insertion domain, the gephyrin binding domain, and SH3 homology domains are boxed and labeled in light blue, dark blue, and orange, respectively.


In three dimensions, AChBP forms a hollow cylinder with an external diameter of 80 Å, a height of 62 Å, and an inside diameter of 18 Å. Its size and general shape are in good agreement with that previously determined from electron diffraction images of Torpedo nAChRs (266). A model of the GlyR α1-subunit ligand-binding domain based on the AChBP structure is shown in Figure 3. Each of the five subunits is positioned in a radially symmetrical manner around the central pore. When viewed from above (i.e., from the synapse, looking towards the membrane), the protein is said to resemble “a 5-bladed windmill toy” (52). Individual subunits contain an α-helix near the NH2-terminal extremity and then a series of 10 β-sheets with short 310 helices following the second and third β-sheets. The β-sheets 1–7 form a “twisted β-sandwich” with β-sheets 8–10, resulting in 2 separate hydrophobic cores. Together the β-sheets form a modified immunoglobulin fold. Pockets are present at the subunit interfaces, approximately midway between the top andbottom of the protein, and abundant evidence (reviewed in Refs. 74, 179) identifies these as ligand binding sites. This pocket is lined by three loops from one subunit that form the “principal” (or +) side of the ligand-binding pocket, whereas three β-sheets from the adjacent subunit form the “complementary” (or −) side of the pocket. Viewed from the top of the complex, the complementary side of each AChBP binding site is situated anticlockwise relative to the principal side (52). The AChBP binding site opens to the outside of the complex and, unlike the Torpedo nAChR electron diffraction images (266), there is no entry to the binding site from the central pore side of the protein.

FIG. 3.

FIG. 3.Homology model of the α1 GlyR ligand-binding domain. A: backbone ribbon representation of the α1 GlyR viewed along the 5-fold axis of symmetry from the synaptic cleft. The sequence alignment used to generate this model is shown in Fig. 2. Different colors indicate different subunits. Dotted lines mark the subunit interfaces with +/− signs indicating the subunit faces that contribute to the interface. The putative inhibitory zinc-binding region is shown in blue with H107 and H109 side-chains shown as bonds in pink. [Model from Nevin et al. (276).] B: stereo view of the inhibitory zinc-binding site (as proposed in Ref. 276) viewed from within the vestibule lumen along the direction of the arrow in A. Only two subunits are shown for clarity. Side-chains of H107 and H109 from the + face (right) and H107, H109, E110, and T112 from the − face (left) are shown as bonds, with standard coloring according to atom. The pink sphere indicates the location of bound zinc. (Image courtesy of Dr. Brett Cromer and Prof. Michael Parker.)


The AChBP structure reconciles many years of biochemical and electrophysiological investigations into the structure and function of the nAChR. As discussed later in sections v and vi, it also reconciles an accumulation of structure-function data from the GlyR. In particular, it provides an excellent basis for understanding the glycine and strychnine binding sites and the zinc inhibitory site.

It was recently proposed that sections of the α1-GlyR NH2-terminal domain between residues 158–165 and 181–191 may be associated with the plasma membrane (223). Because the AChBP domain corresponding to 158–165 is located at a subunit interface well away from the membrane, a direct membrane interaction seems unlikely. However, residues 181–191 indeed lie toward the lowest point of the structure, between β-sheets 8 and 9, and thus could conceivably dip into the membrane.

Apart from the direct polypeptide chain linkage between β-sheet 10 and TM1, structural and functional evidence suggest 2 likely points of contact between the ligand-binding domain and the transmembrane domain. These regions are the conserved cysteine loop and the loop linking β-sheets 1 and 2 of AChBP. This later loop is also known as “loop 2.” Both loops have been proposed to interact closely with the TM2-TM3 linker domain (181, 267), and the nature of these proposed interactions is considered in more detail in section vD.

2. Glycosylation

As shown in Figure 2, the GlyR α1-subunit contains a glycosylation consensus site at N38, with other α-subunits containing similar sites at the homologous positions. The GlyR α2-subunit contains additional consensus sites at N45 and N76 (199). On the other hand, the β-subunit contains consensus sites at N33 and N220 (137). The first suggestion that the α1-subunit may be glycosylated was the finding that mutations to N38 prevented surface expression of functional α1-GlyRs (10, 200). Recently, it was found that glycosylation of α1-subunits is a necessary prerequisite for homomeric receptor assembly and that receptor assembly is required for transit from the endoplasmic reticulum to the Golgi apparatus and subsequently to the cell membrane (140). The question of whether β-subunits are glycosylated remains to be addressed.

D. Large Intracellular Domain

As the large TM3-TM4 domain is poorly conserved among LGIC members, both in terms of its length and amino acid sequence, it is likely to exhibit considerable structural variation as well. The only structural information to date suggests that the Torpedo nAChR intracellular domains form a hanging gondola-type structure with transverse holes (or “portals”) connecting the pore with the cytoplasm (266). Because these portals are approximately the same size as a permeating ion plus its first hydration shell (266), they are ideally suited to influence ion permeation. Indeed, it has recently been shown that the deletion of three positively charged residues in the 5-HT3AR TM3-TM4 domain dramatically increases the pore unitary cation conductance (183), implying that these residues may frame the portals. The homologous region of the GlyR α1-subunit is denoted by gray shading in Figure 2. The GlyR β-subunit has an unusually large internal domain, comprising 130 residues, whereas the α1-subunit contains 86 residues (Fig. 2). The intracellular domains of both α- and β-subunits contain a variety of sites that mediate interactions between the GlyR and cytoplasmic factors. These putative interaction sites are now considered.

1. Ubiquitination domain

Under appropriate conditions, intracellular ubiquitin molecules can covalently attach themselves to specific lysine side chains on the cytoplasmic protein surface. In fact, multiple ubiquitin molecules can attach themselves end to end in a piggyback manner, resulting in a condition termed “polyubiquitination.” Ubiquitination or polyubiquitination precipitates the internalization and degradation of many protein types, including surface-expressed α1-GlyRs (55). Following internalization, the ubiquitin molecules induce the 49-kDa GlyR α1-subunit to be proteolytically nicked into a glycosylated (i.e., NH2-terminal) 35-kDa fragment and a 17-kDa COOH-terminal fragment. These fragment sizes are consistent with the ubiquitination domain lying in the large intracellular domain. The TM3-TM4 domain contains a total of 10 lysine residues (Fig. 2), several of which probably need to be individually ubiquitinated before GlyRs can be endocytosed (55). This mechanism is likely to be important in regulating the number of surface-expressed GlyRs per postsynaptic density.

2. SH3-binding motif

Because prolines induce kinks into peptide chains, regular spacing of these residues can form helical structures known as polyproline (PII) helices. Circular dichroism studies reveal the GlyR α1-subunit to contain a significant fraction (9%) of this structure (58). A certain class of protein-protein interaction sites, termed SH3 domains, are formed from PII helices (290). As recently noted (58), GlyR α1- and β-subunits both contain SH3 consensus sequences in their large intracellular domains (Fig. 2). Although the role of these domains has yet to be investigated, they may be involved in GlyR trafficking or cytoskeletal attachment.

3. Phosphorylation sites

The locations of phosphorylation consensus sites in the α1- and β-subunits are shown in Figure 2. The evidence that phosphorylation of these sites is able to modulate GlyR function is considered in section viiA.

4. Gephyrin binding domain

This important molecule has long been known to copurify with the native GlyR as a 93-kDa protein (300). Kirsch and Betz (188) showed that it mediates the clustering of GlyRs at postsynaptic sites. Gephyrin interacts with a large and growing number of binding partners, suggestive of a high degree of complexity in the regulation of GlyR clustering. A description of the interactions of gephyrin with molecules other than the GlyR is beyond the scope of this review. Developments in this area are moving rapidly, and recent progress has been covered in several excellent reviews (189, 190, 219). The GlyR gephyrin contact site was isolated to an 18-amino acid domain in the central region of the β-subunit TM3-TM4 loop (259) (Fig. 2). Insertion of the gephyrin binding domain into the α1-subunit promotes the clustering of α1-homomeric GlyRs (220, 259). A site-directed mutagenesis study isolated gephyrin binding activity to multiple hydrophobic residues in this domain (191). A hydropathy plot suggests that this region forms an irregular amphipathic helix, with the putative gephyrin-binding residues located along the hydrophobic side.

5. Basic cluster required for TM3 integration

A positively charged cluster, RFRRKRR, located close to the intracellular boundary of TM3 in the α1-subunit (Fig. 2), appears to be important for the correct membrane insertion of TM3. It was found that neutralization of positive charges in this cluster prevented the correct translocation of TM3-TM4 into the lumen of the endoplasmic reticulum (328). This effect was rectified by deleting positive charges from TM2-TM3. From these results, it was concluded that the basic cluster is necessary to compensate for the positively charged residues in TM2-TM3 which would otherwise preclude the correct membrane insertion of TM3 (328).

E. Receptor Assembly

1. Subunit stoichiometry and arrangement

The subunit composition of the GlyR was determined by cross-linking its polypeptides with cross-linking reagents of various specificities and lengths (209). Because the size of the largest cross-linked product totaled approximately five times the mean individual subunit size, functional membrane GlyRs were concluded to comprise pentamers. Of course, since a pentameric subunit arrangement was well-established in other LGIC members (and now in AChBP), it is scarcely conceivable that the GlyR quaternary structure would have been different. Oddly, however, evidence for a trimeric α1-homomeric subunit composition has recently been deduced on the basis of both laser scattering and single particle electron microscopic analyses (413). A substantial measure of credibility must be granted to this study as the same group also proposed a pentameric GABAAR structure using the same techniques. These are the only GlyR images published to date, and further investigation into the basis of these findings appears warranted. One possibility is that the structure is distorted by a closely associated protein.

Although GlyRs almost certainly comprise pentameric subunit complexes, the stoichiometry and subunit arrangement of heteromeric receptors are less certain. An invariant 3α:2β stoichiometry was proposed based on the observation that α-subunits predominated over β-subunits in all tissues examined (209). Although no more direct evidence in favor of any particular stoichiometry has ever been presented, the 3α:2β ratio is a long-held dogma in the field. Even if this stoichiometry is correct, there is no compelling rationale for distinguishing a side-by-side β-subunit arrangement from one whereby the β-subunits are separated by an α-subunit. Knowledge of this is particularly important for understanding GlyR molecular pharmacology because binding sites of all types are located at subunit interfaces (74), and the number of α-α, α-β, β-α, and β-β interfaces per receptor cannot currently be determined. A careful reanalysis of subunit stoichiometry and arrangement in αβ-heteromeric GlyRs is overdue.

2. Intersubunit contact points and assembly domains

GlyR α1- and α2-subunits appear to be able to coassemble in a random binomial manner that is dependent only on the relative abundance of each subunit (200). However, when β-subunits are present, the α:β subunit stoichiometry appears to be invariant, as inferred from the monotonic nature of the glycine dose-response (200). The regions of the GlyR β-subunit responsible for this behavior have been investigated in detail (140, 200). The initial characterization involved coexpressing α1-subunits with chimeric subunits made from α1- and β-subunits. Fixed assembly was found to require the extracellular, but not the TM or intracellular regions, of the β-subunit (200). To further delineate the regions responsible for subunit assembly, certain divergent domains (termed “assembly boxes”) in the β-subunit NH2-terminal domain were mutated back towards the α1-subunit sequences to see whether they permitted the individual chimeric subunits to express as homomers. Indeed, the introduction of various combinations of boxes permitted homomeric assembly (200). The important boxes involved in this process are shown in Figure 2. As a minimum requirement, box 1 has to combine with either box 3 or box 2 plus box 8 to result in α1-homomer formation (140, 200). The critical α1-subunit residues that must be set towards the α1-subunit sequence are as follows: box 1 (P35, N38, S40) and box 3 (L90, S92), or box 2 (P79) and box 8 (N125, Y128) (140). Note that N38 is a glycosylation site. The intersubunit contact points between AChBP subunits and their predicted counterparts in the GlyR α1-subunit are shown in Figure 2. It is apparent that the boxes do not directly form the interfaces, although boxes 3 and 8 lie directly adjacent to interface sites. This suggests that the box mutations act allosterically to modulate the interface conformation. The roles of the interface residues themselves in controlling subunit assembly have yet to be investigated.

3. Effects of high receptor density

The injection of increasing amounts of α1-subunit cDNA into Xenopus oocytes results in a progressive increase in both maximum current (Imax) and glycine sensitivity (90, 362). A recent study has provided a possible explanation for this. When α1-GlyRs containing introduced gephyrin binding domains were clustered using gephyrin, they exhibited an additional extremely fast desensitizing current component (220). Although the glycine EC50 of the peak current was similar to that of unclustered receptors, the EC50 of the plateau current was significantly reduced. Fast (submillisecond) solution application to small (HEK293) cells was required to see the fast desensitizing component. Since such rapid solution exchange is difficult to achieve with Xenopus oocytes, it seems possible that the fast desensitizing component was missed in the earlier study. These effects could be the result of direct interactions between adjacent receptors, allosteric actions of gephyrin on the α-subunit, or depletion of a cytoplasmic cofactor necessary for normal receptor function.

4. Coassembly with other LGIC subunits

Functional evidence suggests that the GABAC ρ1-subunit can coassemble with glycine α1- and α2-subunits in vitro (287). This study showed that the recombinant expression of a mutant ρ1-subunit with the α1- or α2-subunits caused a change in the gating of GABA-induced currents. Because homomeric GlyRs were not activated by GABA, it was proposed that the change in pharmacology must have been due to coassembly of ρ1- with α-GlyR subunits. This finding raises the intriguing possibility that such heteromeric receptors might also exist in vivo.

IV. STRUCTURE AND FUNCTION OF THE PORE

A. Functional Properties of the Pore

1. Ionic selectivity

Although GlyRs are strongly selective for anions over cations, they have a small but measurable permeability to K+ and Na+ (45, 186). Native GlyRs expressed in cultured neurons have a permeability sequence of SCN > NO3 > I > Br > Cl > F (45, 107). This sequence is in proportion to the ionic hydration energies, implying that the removal of waters of hydration is the major barrier to ion channel entry. Because electrostatic interactions with pore sites are, by inference, less important, this sequence corresponds to a “weak field strength” binding site. The GlyR pore also exhibits anomalous mole-fraction behavior, suggesting the presence of at least two interacting binding sites (45, 107). The permeability has also been probed with large organic anions. From space-filling models of the molecules tested, the narrowest part of the pore is estimated to have a diameter of at least 5.2 Å in spinal neuron GlyRs (45), 5.5–6.0 Å in hippocampal neuron GlyRs (107) and 5.22–5.45 Å in recombinant GlyRs (324). By comparison, cationic LGICs also possess a weak field strength binding site, but they have not been shown to display anomalous mole fraction effects and have a significantly larger minimum pore diameter of at least 7.5 Å (225).

2. Single-channel conductance

GlyRs display multiple unitary conductance states (45, 46). A useful comparison of GlyR conductance states in native neuronal GlyRs, as well as in various recombinant subunit configurations, is presented in Table 1 of Reference 307. Recombinant α1-GlyRs exhibit five conductance states ranging between 20 and 90 pS, with the 90-pS state occurring with the greatest frequency. The α2- and α3-GlyRs share the same conductance states but also exhibit a frequently visited 110-pS conductance level. This characteristic is conferred to the α1-GlyR by the G2′A (α1 → α2) mutation (46). Coexpression of α1-subunits with the β-subunit eliminates the highest conducting levels, leaving a 45-pS state as the most frequently occurring state (46). Incorporation of the E20′S (β → α1) mutation into the β-subunit confers α1-GlyR-like conductance levels to heteromeric GlyRs. Because neither mutation abolishes the preexisting conductance states, it is difficult to determine whether their actions are mediated allosterically or via direct interactions with permeating ions. The relative probabilities of entering the predominant conductance states do not vary with agonist concentration (25, 374).

As mentioned above, elimination of a series of three positively charged residues in the 5-HT3AR TM3-TM4 domain resulted in a dramatic increase in the single-channel conductance (183). It was proposed these residues might line a narrow portal that links the pore with the cytosol. It is yet to be established whether the homologous residues in the GlyR α- or β-subunits influence the unitary chloride conductance. However, since both positively and negatively charged residues line the corresponding domain in the GlyR α1-subunit (Fig. 2), and the precise alignment is unknown, it is difficult to predict what this influence might be.

B. Molecular Determinants of Ion Selectivity and Conductance

Much of the groundwork for our current understanding of GlyR permeation mechanisms has come from studies on the nAChR. One classic experiment showed that the open-channel blocker QX-222 reached as far as the 6′ position when applied externally in the open state (61, 224), implying that the most constricted section of the pore was located more internally than this point. Because residues in the narrowest part of the pore are more likely to influence both unitary conductance and ion selectivity, this in turn implied that the residues near the intracellular boundary of TM2 controlled both processes. A recent SCAM study has provided the most definitive functional evidence to date for a drastic pore constriction between the −2′ to the +2′ positions (404). It should be noted, however, that the Miyazawa TM domain structure shows the pore widening considerably from the +2′ to −2′ positions (267). The reason for this apparent mismatch is not yet understood.

Charged residues are obvious candidates as ionic selectivity sites. As shown in Figure 1A, the nAChR α1-subunit contains four charged residues in the vicinity of TM2: a negatively charged aspartic acid at −4′ (the “cytoplasmic ring”), negatively charged glutamic acids at −1′ (the “intermediate ring”), and 20′ (the “outer ring”) and a positively charged lysine at 0′. Three lines of evidence indicate that K0′ faces away from the pore: 1) charge-reversal mutations have no effect on single-channel conductance (168), 2) the pore has a strong negative electrostatic potential in this region (289), and 3) the Miyazawa TM domain structure unequivocally shows K′0 facing away from the pore (267). Although charge-reversal mutations to the cytoplasmic and outer rings significantly affect single-channel conductance (168, 169), neither site appears to significantly impede the access of negatively charged molecules to deeper regions of the pore (404). However, mutations to the intermediate ring strongly influence both single-channel conductance (168) and pore effective diameter (387). In addition, mutations to 2′ residues also affect cation selectivity and effective diameter (reviewed in Ref. 225). Thus residues at the −1′ and +2′ positions, which are separated by one α-helical turn in the narrowest part of the pore, form the main selectivity filter. Structural predictions indicate that both side chains have extensive exposure to the pore (267).

By comparing TM2 sequences between the α7-nAChR and the α1-GlyR, Galzi et al. (126) sought to determine the minimum number of GlyR residues required to switch the nAChR pore from cation selective to anion selective. The minimum requirement was found to be the E-1′A and V13′T mutations as well as the insertion of a proline between −2′ and −1′ (i.e., −2′P). The same mutations achieved a similar effect in the 5-HT3R (149). Because the E-1′A and V13′T mutations alone do not convert selectivity, they are considered to be “permissive” rather than “essential” for anion permeability (73). Interestingly, the proline insertion site was not critical: selectivity reversal was also effected by inserting it into the −4′ position (73). Together these results imply that selectivity reversal required a change in either the lumen geometry or in the exposure profile of side chains lining the selectivity filter.

The reverse triple mutation (A-1′E, T13′V and deletion of −2′P) was subsequently found to convert α1-GlyR selectivity from anionic to cationic (185). However, the resultant channels had a low conductance (3 pS at −60 mV) and converted the ratio of permeabilities (PCl/PNa) from 24 to 0.3, implying that Cl permeation may have been reduced without a concomitant increase in Na+ permeation. A stronger case for an increase in cation permeability was made by the same group when they showed that the reverse double mutation (A-1′E and deletion of −2′P) decreased the PCl/PNa further to 0.13, while increasing the unitary cation conductance to 7 pS at −60 mV (186, 272). Furthermore, these mutations increased the effective pore diameter of this GlyR to approximately the value seen in the nAChR. Curiously, however, selectivity reversal was also achieved by incorporating only the A-1′E mutation into both the α1-GlyR and the ρ1-GABAAR (186, 406). From this result, it is tempting to speculate that anion selectivity is associated with a net positive electrostatic charge caused by both the elimination of the negative charge at E-1′ and an enhanced pore exposure of R0′.

However, one problem with the “net positive charge” model of anion permeation is that the anion selectivity of the triple mutant α7-nAChR is not affected by the charge-eliminating K0′Q mutation (73). In addition, the E-1′A mutation alone did not increase anion permeability (73). Indeed, these two observations prompted Corringer et al. (73) to suggest that the anion-selective nAChR pore may be lined by polar groups from the peptide backbone. Further insight into this issue was recently provided by the finding that the ρ1-GABAAR anion-to-cation selectivity switch was conferred by the charge-reversing mutation R0′E, but not by the charge-eliminating mutations R0′C or R0′M (406). It appears feasible to reconcile all these findings by proposing that a net negative charge in the −1′ region is associated with cation selectivity, whereas a net neutral or positive charge is associated with anion selectivity. In the event of a neutral potential in this region, positive dipoles of local polar groups may confer anion permeability. A critical test of this proposal would be to quantitate the GlyR pore electrostatic potential profile using SCAM (289, 405). It would also be informative to investigate the effects of a variety of substitutions at the −1′ and 0′ positions to differentiate the effects of side chain charge, size, hydrophilicity, and hydrophobicity on ion selectivity and pore effective diameter. Unfortunately, however, mutations to these residues have a habit of precluding functional receptor expression.

The influence of β-subunits on ion permeation has yet to be investigated in detail. As shown in Figure 1A, α- and β-subunits have a low sequence homology throughout TM2. Of particular note, the β-subunit includes an alanine-for-proline substitution at −2′ and a proline-for-glycine substitution at position 2′. These substitutions suggest the β-subunit secondary structure may be different in the pore selectivity filter. However, as summarized above, experiments to date suggest the β-subunit does not induce drastic changes in permeation characteristics.

The effect on permeation of the outer ring of charge, formed by R19′, has also been investigated in the α1-GlyR. Elimination of this charge by the human startle disease mutations R19′L and R19′Q caused a decrease in the unitary Cl conductance (208, 305). More recently, it was shown that the R19′A and R19′E mutations increased the unitary cation conductance in cation-selective GlyRs and changed the rectification properties of the pore (272). Together, these results are consistent with an electrostatic contribution of R19′ to ion permeation. However, R19′ does not preclude the entry of positively charged molecules into the GlyR pore (341, 343) and is more likely to affect conductance by concentrating anions in the outer vestibule (186). The β-subunit E20′S mutation, which neutralizes a negative charge at the adjacent position, may also increase the single-channel conductance via an electrostatic mechanism (46).

An impressive array of α1-GlyR pore functional properties was reconciled by a three-dimensional Brownian dynamics simulation study (284). This study used a model of the pore based on the TM2 primary structure and the best available estimates of pore diameter prior to publication of the Miyazawa study (267). The selectivity filter diameter was permitted to vary between 6 and 8.3 Å, but the charge at R0′ was held constant at +0.375e. Under these conditions, the first Cl experiences a deep energy minimum near R0′. A second Cl entering the pore experiences a weaker energy minimum near the pore midpoint. These two ions exist in equilibrium. A third entering Cl abolishes these energy wells, allowing the innermost ion to escape into the intracellular solution. In light of the preceding discussion, it would be interesting to determine the effects on permeation of variations to the charge at R0′.

C. The Channel Activation Gate

This gate refers to the physical barrier that stops ions from traversing the pore in the unliganded state. There is no information available to date as to its location in the GlyR. There are, however, two schools of thought as to its location in other LGIC members. One proposal, supported by the structural analyses of Unwin, Miyazawa, and colleagues (266, 267, 376), is that the TM2 domains are kinked inwards to form a centrally located gate near the highly conserved L9′ residue. However, the results of SCAM experiments on the GABAAR and nAChR are inconsistent with the gate lying more externally than the 2′ position (7, 289, 409). A particularly detailed study on the nAChR, which probed the accessibility of cysteines introduced into TM2 to both sides of the membrane in the closed and open states, delimited the gate to the same narrow pore region (−2′ to +2′) that houses the selectivity filter (404).

D. Molecular Mechanisms of Desensitization

Desensitization is a property whereby an agonist-gated channel closes in the continued presence of agonist. In general, the rates of onset and recovery from desensitization are important parameters governing the size, decay rate, and frequency of fast synaptic currents (175). Until recently, it was considered that GlyRs desensitized too slowly to influence these parameters (219). However, a recent study using ultra-fast solution exchange identified a very rapid desensitization component (decay time constant ∼5 ms) that is induced by either the clustering of α1-GlyRs (220) or by coexpression of the α1- with the β-subunit (268). This rapid component could conceivably influence the properties of repetitive glycinergic synaptic currents. However, further research is required to clarify the role, if any, of GlyR desensitization at glycinergic synapses.

The location of the desensitization gate in the GlyR is not yet known. However, SCAM evidence from the nAChR suggests that the desensitized state is associated with a crimping of the channel pore between the −2′ and 9′ residues (403). In contrast, a similar approach showed that the activation gate crimps the pore between only the −2′ and +2′ positions.

A growing body of evidence reveals that intracellular domains can profoundly influence GlyR desensitization. A functional comparison of human α3K- and α3L-GlyRs showed that the removal of a 15-amino acid segment from the large intracellular domain dramatically increased the desensitization rate (280). Desensitization rate is also dramatically enhanced by the human startle disease mutations, I244N and P250T (also known as P-2′T), in the α1-GlyR TM1-TM2 loop (237, 334). Other mutations in this region, including W243A, I244A, and A251E (also known as A-1′E), have similar effects (186, 237). The relationship between desensitization rate and the properties of side chains introduced into the P250 position showed that bulky hydrophobic residues yielded the fastest desensitization rates (51). Thus desensitization rate is exquisitely sensitive to structural perturbations in the TM1-TM2 loop. However, in the GlyR at least, such observations are of phenomenological interest only until it can be demonstrated that events that alter desensitization in vivo do so by varying the conformation of this domain.

V. AGONIST BINDING AND RECEPTOR ACTIVATION

A. Introduction

This section considers the molecular basis by which agonists bind to and activate (or gate) the GlyR. In particular, it will consider the following important questions. What is the structure of the binding site and where is it located? What is the molecular basis for binding site selectivity? What structural changes underlie the activation of the receptor? One problem with addressing these questions is that it is difficult to experimentally dissect binding from gating mechanisms as the two processes are tightly coupled (72). To understand how this separation may be achieved, it is necessary to briefly consider the theory of receptor activation.

1. Review of basic receptor theory

In its simplest form, the receptor activation process can be represented as:

where A is the agonist, R is the vacant receptor, AR is occupied but shut, and AR* is occupied and activated. The equilibrium constant for binding (or binding affinity) is given by:
(1)
where koff is the dissociation rate constant (units of s−1) and kon is the association rate constant (units of M−1·s−1). The equilibrium constant for gating (or efficacy) is given by:
(2)
where β is the opening rate constant and α is the closing rate constant (both with units of s−1). With the use of classical receptor theory (along with some simplifying assumptions), it can be shown (72) that the agonist concentration required for a half-maximal response:
(3)
and the maximum fraction of receptors in the activated state (or maximum open probability)
(4)

Equation 3 tells us that the EC50 is a function of both the binding and gating properties of the receptor. Equation 4 tells us that variations in E have no measurable effect on maximum open probability unless they occur within a limited range of ∼0.1–10.

The structural basis of GlyR binding and gating has mainly been investigated using site-directed mutagenesis coupled with functional (i.e., electrophysiological) analysis. If a mutation affects mainly KA, then it is assumed to affect binding, either directly or allosterically. If a mutation affects mainly E, then the mutated residue is assumed to affect the conformational change leading to the open state, either directly or allosterically. Single-channel kinetic analysis can provide reasonably direct estimates of both KA and E. A more convenient, but less precise, method involves measuring relative changes in the peak whole cell current (Imax) activated by partial agonists. Since Imax = inPomax, where i is single-channel conductance and n is number of activated receptors, Imax provides a qualitative measure of changes in E, assuming that i, n, and desensitization rate remain constant.

2. Concerted versus sequential modelsof receptor activation

The above model of receptor activation is an oversimplification because α-GlyRs, as pentameric multimers, contain five agonist binding sites. Two starkly contrasting models have been proposed to describe multisubunit protein allosteric mechanisms. These are the coupled Monod-Wyman-Changeux (MWC) model (60) and the uncoupled or sequential Koshland-Nemethy-Filmer (KNF) model (193). In the simplest version of the MWC model, all the subunits change conformation simultaneously, and in consequence, the receptor can exist in only the closed or entirely activated states. In contrast, in the KNF model, each subunit can independently adopt a specific conformation change depending on the number of bound agonist molecules, leading to a series of intermediate protein conformational states. Extended MWC models that allow for multiple functional states and subunit asymmetry can explain many characteristics of nAChR behavior (60). In particular, two simple observations that support an MWC model over a KNF model are that 1) nAChR single-channel conductance is independent of agonist concentration and 2) spontaneous channel openings are occasionally seen in the absence of agonist. As discussed in the ensuing paragraphs, the information available to date also favors an MWC model for the GlyR.

B. Kinetic Models of Glycine Receptor Gating

The single-channel kinetic properties of GlyRs were first characterized in native receptors from embryonic mouse spinal neurons (374). This study revealed several important features of GlyR kinetic behavior. First, unlike nAChRs, GlyR channel openings did not occur in the absence of agonist. Second, although the single-channel conductance was not constant, it displayed no variation with agonist concentration. The third and perhaps most revealing feature was that there were at least three exponential components in the open period distributions. Short-lived channel bursts (corresponding to the faster exponential components) predominated at low concentrations, whereas the relative frequency of longer lived bursts (slower exponential components) increased at higher concentrations. It was therefore concluded that progressively longer-lived states were associated with increasing numbers of bound glycine molecules and that maximal channel activation therefore required a minimum of three bound glycines (374). Because the molecular identity of the mouse GlyRs is not known, the number of glycine binding sites per receptor is uncertain.

Irrespective of this, the three-open-state model is in broad agreement with several more recent studies on recombinant α1-GlyRs (25, 123, 139, 212, 227). However, one study that examined the equilibrium gating of recombinant α1-GlyRs identified two further burst-length components, namely, a particularly short-lived state that was prominent at low glycine concentrations and a particularly long-lived state that was seen only at the highest tested concentrations (25). The authors interpreted these results by proposing that the shortest to longest lived states correspond to receptors occupied by one to five glycine molecules, respectively. These findings were reconciled into an MWC-like model where any one of five possible liganded states can lead to channel opening. An attractive feature of this model is that α1-GlyRs do indeed possess five glycine-binding sites.

However, this model is controversial because it predicts that mono-liganded openings should add an instantaneous linear component to the onset of the glycine-stimulated response, and experiments involving the rapid application of glycine to both native and recombinant GlyRs have revealed the distinct absence of such a component (128, 139, 218, 227). The shape of the glycine activation response was consistent with a minimum of two bound glycines being required to activate the native GlyR (218) or a minimum of two or three bound glycines for activation of the recombinant α1-GlyRs (128, 139, 227). Thus the GlyR kinetic models generated by analysis of stationary and nonstationary receptor kinetics cannot be resolved at present. Because such models are crucial for understanding and predicting receptor behavior, it is hoped that both stationary and nonstationary kinetic analyses will be included in the development and functional testing of future models.

In contrast to the α1-GlyR, the α2-GlyR can be modeled as possessing only a single open state (246). It also has an extraordinarily slow rate of receptor activation that requires at least two simultaneously bound glycine molecules (246). Mangin et al. (246) successfully modeled these properties by proposing that the single open state was linked to the fully liganded closed state.

Transitions to and from desensitized states have also been modeled in α1- (128, 218, 246) and α2-GlyRs (246). These models depict these states as being accessed from the singly or doubly liganded closed states. Some kinetic studies have ignored the effects of desensitization because it generally occurs at a relatively slow rate.

C. Agonist Binding

1. Agonist affinity and efficacy

Native and recombinant GlyRs are activated by amino acid agonists with the following rank order of potency: glycine > β-alanine > taurine > GABA. In α1-GlyRs expressed in HEK293 cells, glycine, β-alanine, and taurine all behave as full agonists, with glycine exhibiting an EC50 value of 20–50 μM (46, 237). However, when the same α1-GlyRs are expressed in Xenopus oocytes, the EC50 values of all agonists are increased by about an order of magnitude, and the peak magnitudes of saturating β-alanine-, taurine- and GABA-gated currents are reduced relative to those activated by glycine (214). These differences, shown in diagrammatic form in Figure 4, imply variations in GlyR conformation between the two expression systems. A recent detailed study of Xenopus oocyte-expressed α1- and α2-GlyRs showed that the glycine, taurine, and GABA sensitivity varied in parallel from cell to cell over a surprisingly large (10-fold) range (90). In addition, the relative peak magnitudes of taurine- and GABA-gated currents varied according to their EC50 values. The origin of this variability is not known, but such a degree of variability has not been reported in GlyRs expressed in HEK293 cells.

FIG. 4.

FIG. 4.Typical agonist sensitivity profiles of human α1 GlyRs recombinantly expressed in HEK293 cells (top) and Xenopus oocytes (bottom).


The information available to date suggests that α2-, α3-, and α4-GlyRs exhibit similar agonist sensitivities to the α1-GlyR (90, 130, 157, 201, 202, 246). The incorporation of β-subunits has little effect on receptor sensitivity to glycine (154, 298, 268), although its effect on the EC50 values for other agonists has not been determined to date.

A combination of rapid agonist application techniques and equilibrium single-channel kinetic analysis was used by Lewis et al. (227) to estimate the agonist affinities and efficacies of glycine, β-alanine, and taurine on α1-GlyRs expressed in HEK293 cells. The respective KA values were estimated as follows: glycine, ∼160 μM; β-alanine, ∼360 μM; and taurine, ∼460 μM. These differences were found to be entirely due to variations in the agonist dissociation rates. The E values were predicted to be 16, 8.4, and 3.4 for glycine, β-alanine, and taurine, respectively (227). The differences among these values were caused by variations in the channel opening rate, with the channel closing rate remaining constant for all three agonists. Earlier studies on homomeric α1-GlyRs (128) and heteromeric α1β-GlyRs (218) estimated comparable E and KA values for glycine. In contrast, the homomeric α2-GlyR was estimated to have a glycine E value of at least 250 and a KA value near 40,000 μM (246).

2. Agonist binding domains

As discussed in detail in section iiiC, the ligand-binding pocket is formed as a cleft between adjacent subunits. Three loops (domains A, B, and C) form the “principal” ligand-binding surface on the + side of the interface (Fig. 3A) and three β-strands (domains D, E, and F) from the adjacent subunit comprise the “complementary” (or −) face. The residues of AChBP that contribute to these domains are indicated in Figure 2. The human GlyR α1- and β-subunit residues that align with the AChBP ligand-binding site residues are also shown.

Kuhse et al. (202) observed that the GlyR α2*-splice variant subunit had a glycine EC50 that was ∼40 times higher than those of the α2- or α1-subunits. Glycine sensitivity was restored by incorporating the E167G mutation into the α2*-subunit, thus reversing the only nonconserved amino acid between the α2*- and α2-subunits. This residue aligns with G160 in agonist binding domain B of the GlyR α1-subunit (Fig. 2). It was subsequently shown that the double mutant F159Y, Y161F α1-GlyR caused a modest (12-fold) increase in glycine sensitivity, but surprisingly large (121- and 45-fold) increases in the sensitivities to β-alanine and taurine (338). Although EC50 changes alone do not provide evidence for binding interactions, domain B was considered likely to bind glycine since 1) the mutation also dramatically affected GlyR sensitivity to the competitive antagonist strychnine (see below), and 2) this domain had already been identified as an nAChR binding site (reviewed in Ref. 19). The domain has since been implicated in agonist binding in the GABAA and 5-HT3 receptors (14, 354).

The agonist-binding role of the GlyR α1-subunit C domain has been investigated by the Schofield group (309, 381, 382). The GlyR is unusual among LGICs in that its C domain is contained within a second extracellular disulfide loop. It was shown that mutations to the even-numbered residues, L200, Y202, and T204, profoundly affect receptor sensitivity to glycine or strychnine, or both (309, 381, 382). Of particular note, the conservative Y202F mutation caused a drastic increase (480-fold) in the glycine EC50, while having little effect on strychnine sensitivity (309). Due to the differential sensitivity of specific residues to glycine and strychnine and the fact that this domain had been implicated in acetylcholine binding to the nAChR (reviewed in Ref. 19), binding domain C is likely to be involved in glycine binding. However, most mutations to this domain also affected receptor gating, as evidenced by their conversion of taurine from a full into a partial agonist (309).

Residues I93 and N102 in domain A were identified as agonist-binding elements on the grounds that conservative mutations affected agonist EC50 values only, without affecting strychnine sensitivity or the agonist efficacies of β-alanine or taurine (151, 379). The R97G mutation, which induced spontaneous gating in α1-GlyRs (32), may also have exerted a direct or allosteric effect on this site.

The possible agonist-binding roles of the GlyR α-subunit complementary (D, E, and F) domains have not yet been investigated. This constitutes a significant gap in our understanding of glycine binding mechanisms, as it is currently unclear whether bound glycine molecules are coordinated by adjacent subunits.

The possible agonist-binding role of the β-subunit has also received scant attention. A study on Xenopus oocyte-expressed GlyRs found that the incorporation of β-subunits reduced the Hill coefficient for glycine activation from 4.2 in α1-homomers to 2.4 in α1β-heteromers (46). This implied that β-subunits did not contain glycine binding sites. However, not all studies have observed a change in Hill coefficient upon β-subunit incorporation (e.g., Ref. 154). Interestingly, a single-channel study investigating the effect of the β-subunit startle disease mutation G229D found that it disrupted the agonist binding affinity (314). Because G229 lies in loop C (Fig. 2), this effect was most likely mediated by either a direct or allosteric disruption of a β-subunit glycine binding site. Clearly, further experiments are required to clarify the agonist-binding role of the β-subunit.

3. Physical basis of agonist binding

There is abundant evidence that the ACh-nAChR binding reaction is mediated mainly by noncovalent “cation-π” electrostatic interactions (430). In this system, the aromatic side chains of phenylalanine, tyrosine, or tryptophan contribute a negatively charged π surface while the cation is provided by the agonist. In the α1-nAChR subunit, the ACh quaternary nitrogen interacts directly with Y149 by this mechanism (434). The corresponding GlyR α1-subunit residue Y161 is conserved in all GlyR subunits. By analogy with the nAChR, Y161 could conceivably form a cation π interaction with the glycine amine nitrogen.

Glycinergic agonists have been subjected to a number of structure-activity investigations, with the most notable being that of Schmieden and Betz (336). This study tested the agonist and antagonist potencies of a range of α- and β-amino acids. Agonist activity was found exclusively to be a property of those molecules where the amino and acidic moieties existed in a cis-conformation (Fig. 5). One molecule (nipecotic acid) that was locked into a trans-conformation exhibited only antagonist activity. However, β-amino acids (e.g., taurine) that randomly flicker between both conformations displayed both agonist and antagonist activity (Fig. 5). This model predicts a specific antagonist recognition site in a common ligand binding pocket that is accessible only by trans-isomers (336). By binding in the trans-conformation, β-amino acids may either stabilize the closed state or sterically prevent glycine from binding in the pocket, or both. This model also predicts that the partial agonist activity of taurine results from a fraction of molecules binding as antagonists and another fraction binding as agonists at the same receptor. Although attempts to identify a putative antagonist contact site have proven inconclusive to date (151, 339), this interesting model is certainly worthy of further consideration.

FIG. 5.

FIG. 5.Structural comparison of GlyR agonists and antagonists. Ligands (glycine and l-alanine) that display only agonist activity are shown on the left. Nipecotic acid, which displays only antagonist activity, is shown on the right. Taurine, which displays both agonist and antagonist activity, flickers randomly between the two indicated conformations. In the cis-conformation, the distance between amino and acidic groups is similar to that found in glycine. In the trans-conformation, the distance is similar to that seen for nipecotic acid.


D. Structural Changes Accompanying Activation

1. The ligand-binding domain

Functional data suggest that nAChR activation is mediated by global intersubunit movements (74). A recent structural analysis of the nAChR by Unwin et al. (377) has provided more direct support for such a model. This group interpreted low-resolution electron diffraction images of Torpedo nAChRs obtained in both the closed and open states using the AChBP crystal structure as a template. In modeling the structural changes, they divided the α-subunit into inner and outer parts and considered each separately. The inner part (which includes all residues up to the conserved cysteine loop) is so-called because it faces the channel vestibule and contains most of the intersubunit contact points plus agonist-binding domain A. The outer part (which comprises β-sheets 7–10) includes the agonist-binding domains B and C. Upon agonist binding, the outer part was found to undergo an upwards tilt around an axis parallel with the membrane plane. Simultaneously, the inner part rotated ∼15° in a clockwise direction (when viewed from the synapse) around an axis perpendicular to the membrane plane. Being essentially a combination of rigid body movements, the largest residue displacements (up to 2 Å) occurred at the subunit interfaces. The rotation of the inner sheets is viewed as the crucial event in transmitting agonist-binding information to the channel gate (377). The inner sheets contain two loops, the conserved cysteine loop and loop linking the first and second β-sheets (referred to hereafter as loop 2), that are ideally located to interact directly with the TM2-TM3 domain (52, 267). Interactions between these loops and the TM2-TM3 domain appear crucial to the GlyR activation process, and the extensive body of evidence implicating these regions in receptor gating will now be considered.

2. The TM2-TM3 domain

The structural changes initiated in the ligand-binding domain are transmitted to the membrane-spanning domains, where they culminate in a change in conformation of TM2. As stated above, one such contact point is the “TM2-TM3 domain” which extends from R271 to D284 in the GlyR α1-subunit. Although originally thought to comprise an extended extramembranous loop, the Miyazawa TM structure now reveals this domain to comprise the extramembranous portions of the TM2 and TM3 α-helices plus a short connecting loop (267).

A signal transduction role for single residues in this domain was first suggested by studies on the GABACR (204), the GlyR (305), and the nAChR (56). A systematic study subsequently proposed a structural role for the entire α1-GlyR TM2-TM3 domain in the gating process (237). This conclusion was based on the observation that several naturally occurring human disease mutations, plus several more alanine-substitution mutations, scattered throughout this domain acted similarly in reducing the agonist efficacies of taurine and β-alanine relative to glycine. Because β-alanine and taurine retained high potency as antagonists, their agonist binding affinities were considered to be little affected. Thus, because a number of mutations throughout this domain predominantly impaired E, the whole domain was hypothesized to comprise a structural element of the receptor activation pathway (237). A subsequent single-channel study on one of the tested mutants (K276E) supported this interpretation (228). A more direct test of this theory required an investigation into whether the region moves during activation. Accordingly, SCAM was employed to probe for changes in its surface accessibility between the closed and open states. The results did reveal an increased surface accessibility of the NH2-terminal (α-helical) half of the domain in the open state (234), consistent with a conformational change during gating. Several approaches have since been used to implicate various TM2-TM3 residues in the gating of other LGICs including the nAChR (75, 141, 142, 320, 321) and GABAAR (37, 44, 113, 181, 285).

Recent studies have begun to shed light on the specific structural role of this domain. The Auerbach group examined the linear free-energy relationships (LFERs) of nAChRs incorporating mutations in various positions (142). They showed that the energy transition experienced by a TM2-TM3 residue was intermediate between those experienced by residues at the binding site and the activation gate. This was interpreted to mean that the TM2-TM3 domain is positioned midway along the agonist-induced “conformational wave” that proceeds from the binding site to the activation gate. Another study, undertaken on the GABAAR, employed a mutant cycle analysis approach to identify an electrostatic attraction between the negatively charged residue D149 and the positively charged residue K279 (181). D149 lies in the conserved cysteine loop, whereas K279 lies in the TM2-TM3 domain. A cysteine cross-linking approach further supported the idea of a closer association between these residues in the open state (181). Together, the results suggested that channel activation is accompanied by an increased electrostatic attraction between these two residues. However, the electrostatic attraction between the corresponding charged residues (D148 and K276) in the α1-GlyR is weaker, suggesting other interactions may also contribution to channel activation (1, 340).

Structural and functional analyses have also revealed a close interaction between the TM2-TM3 domain and loop 2 of the ligand-binding domain (181, 267). Indeed, the Miyazawa TM structure suggests that one loop two hydrophobic side chain fits into the end of the nAChR TM2 helix like “a pin in a socket.” Charged loop 2 residues have also been implicated in gating the α1-GlyR (1, 340) and the GABAAR (181). Clearly, substantial gaps remain in our understanding of how agonist signals are transduced from the binding site to the activation gate of the GlyR. Although it is possible that different LGIC members employ different coupling mechanisms, a common coupling mechanism for ρ1-GABACRs and α1-GlyRs is suggested by the chimeric studies of Mihic and colleagues (262).

3. The TM1-TM2 domain

Systematic mutagenesis of residues in the intracellular TM1-TM2 domain suggested this domain may also be involved in α1-GlyR gating (237). As discussed in section ivD, several mutations in this region also affect desensitization. These observations imply that GlyR gating is very sensitive to structural perturbations in this domain. It seems likely, therefore, that this region might also move during GlyR activation. Further experiments are required to test this hypothesis.

4. The membrane-spanning domains

Investigations into the structural rearrangements of TM2 have revealed two major features that will be considered in turn. The first feature originally stemmed from the observation that the TM2 domain of the Torpedo nAChR incorporated a centrally located kink that appeared to form the channel gate (376). Concurrent SCAM studies (7) also provided evidence for a discontinuity in the α-helix around the central (L9′) position. Such a discontinuity is likely to introduce a degree of conformational flexibility because some of the H-bonds responsible for maintaining the α-helical structure may be broken. This discontinuity may therefore serve as a swivel joint to permit the outer half of TM2 to move asynchronously with the inner half. In other words, gating may involve a backbone rearrangement in the vicinity of L9′. This has indeed been suggested by SCAM studies (7), LFER analysis (83), molecular dynamics simulations (216), and a study that introduced unnatural, backbone-altering mutations into TM2 (104).

The second major feature of TM2 gating also concerns the 9′ position. The muscle nAChR L9′T mutation dramatically affects the desensitization rate, the size of spontaneous leakage currents, and the effects of allosteric modulators (reviewed in Ref. 74), implying complex (possibly global) effects on channel gating mechanisms. Furthermore, mutating the 9′ leucines to small polar residues (serines or threonines) had an equal effect on the ACh dose-response regardless of which subunit was mutated (110, 206). As binding sites exist at only two of the five subunit interfaces, these results implied that neighboring nAChR subunits interact allosterically via their respective L9′ residues. Recent evidence suggests that GlyR α- and β-subunits interact in a similar manner (344). The Miyazawa TM domain structure (267) provides some insight into how these interactions might occur. The structure suggests the existence of hydrophobic bonds between the 9′ and 10′ residues of adjacent subunits. These bonds appear to “balance” the L9′ residues into a fivefold radially symmetrical arrangement that holds the channel closed. It is likely that agonist-induced conformational changes asymmetrically disrupt some of these bonds, leading to a collapse of symmetry and a simultaneous conversion of all TM2 domains to the activated state. Mutations to one or more L9′ residues may achieve a similar effect.

In summary, the main features of agonist-induced TM2 movements are that 1) its midpoint acts as a swivel, and 2) this swivel point mediates interactions between adjacent subunits. Agonist-induced backbone rearrangements at this position may thereby lead to a concerted conformational change at either a centrally located or an intracellularly located gate (376, 404).

Functional evidence suggests that TMs 1, 3, and 4 may also be involved in LGIC gating. Single-channel kinetic analysis of mutations incorporated into each of these domains suggests that some residues may have specific roles in gating the nAChR (47, 89, 104, 388). SCAM analysis has also provided evidence for state-dependent structural rearrangements of TM1 and TM3 in the nAChR and GABAAR, respectively (6, 400402, 432). However, insufficient information is available to date on any LGIC member to form a coherent picture of how these domains contribute to activation. The recently elucidated nAChR TM domain structure (267) now permits the design of more specific experiments to investigate these mechanisms.

5. The β-subunit

The role of the β-subunit in α1β-GlyR gating was recently investigated by incorporating mutations into corresponding positions in α1- and β-subunits and comparing their effects on receptor function (344). Although cysteine-substitution mutations to residues in the NH2-terminal half of the α1-subunit TM2-TM3 loop dramatically impaired gating efficacy (234), the same mutations exerted little effect when incorporated into corresponding positions of the β-subunit (344). Furthermore, although α1-subunit TM2-TM3 loop cysteines were modified by cysteine-specific reagents (234), the corresponding β-subunit cysteines showed no evidence of reactivity (344). These observations suggest structural or functional differences between α1- and β-subunits. However, the incorporation of the L9′T mutation into the β-subunit dramatically increased the glycine sensitivity (344), suggesting an allosteric modulatory effect on the α1-subunit. Thus β-subunit conformational changes do contribute to the activation of the GlyR, although their involvement in this process is significantly different to that of the α1-subunit.

VI. GLYCINE RECEPTOR MODULATION

A. Phosphorylation

Phosphorylation can cause long-term changes in the functional properties of ion channels, and an abundance of evidence implicates such mechanisms in various forms of synaptic plasticity (177). Intracellular signaling pathways determine the phosphorylation state of proteins by coordinating the activities of protein kinases, which induce phosphorylation, and phosphatases, which reverse it. All of the functional phosphorylation sites on LGICs have been mapped to the major intracellular loop (360). As discussed above, LGIC subunits exhibit a low degree of sequence homology in this region, and this underlies the subunit-specific distribution of phosphorylation consensus sites (Fig. 2). It has recently become apparent that clustering and cytoskeletal anchoring proteins can influence the proximity of kinases and phosphatases to the LGIC intracellular domains (226, 360). Thus the ability of an LGIC to be phosphorylated depends not only on its subunit composition but also its proximity to the appropriate enzymes. In addition, kinases and phosphatases may have indirect effects on the GlyR by controlling the phosphorylation state of modulatory proteins. This diversity could lead to tissue-specific differences in the propensity of a given GlyR isoform to be phosphorylated.

1. Protein kinase A

Contrasting effects of cAMP-dependent phosphorylation on GlyR current magnitudes have been reported in neurons from various parts of the brain (219). Although the differences may be related to the phosphorylation of GlyR modulatory proteins (or possibly to tissue-specific or nonspecific effects of pharmacological probes), biochemical experiments strongly suggest that spinal cord GlyR α-subunits themselves are directly phosphorylated in vitro (378). However, most GlyR α-subunit isoforms do not contain protein kinase A (PKA) phosphorylation consensus sequences. The exception to this is the α1ins splice variant which contains an eight-amino acid insert (… SPMLNLFQ… ) in the large intracellular domain, and the first residue of this insert may serve as a phosphorylation site (244). As shown in Figure 2, the β-subunit also contains a PKA consensus site at a different position (T363) in the TM3-TM4 domain (137). However, it is yet to be determined whether the α1ins or the β-subunit sites are directly phosphorylated and, if so, whether phosphorylation induces changes in receptor function. As noted by Legendre (219), it is possible that the contradictory effects of cAMP-dependent phosphorylation may be explained by the differential expression of α1ins- and β-subunits throughout the central nervous system (245). Whether this is true, and whether cAMP-dependent phosphorylation has a physiological role at glycinergic synapses, are important questions for future research.

2. Protein kinase C

Physiologically, protein kinase C (PKC) is activated by increases in intracellular calcium or diacylglycerol. The GlyR α1-subunit contains a PKC phosphorylation consensus sequence at S391 in the TM4 domain (323). Consistent with this, the spinal cord GlyR α-subunit was shown to be phosphorylated by PKC in an in vitro assay (378). Support for a functional role for S391 is provided by the observation that the homologous residue in the GABAAR β-subunit is phosphorylated by PKA and PKC (274). Surprisingly, however, the corresponding residue in the Torpedo nAChR appears inaccessible to the protein surface (267). The GlyR β-subunit also contains a putative PKC phosphorylation consensus site at position 389 in the TM3-TM4 domain (137), although its functional status is yet to be confirmed. Pharmacological manipulations aimed at stimulating or inhibiting PKC have revealed contrasting effects on glycine-activated currents in a variety of neuron types (219). As discussed above, these differential effects may be due to a multitude of causes, including, in some cases, nonspecific effects of pharmacological agents (e.g., Ref. 281). Our current understanding of PKC-dependent phosphorylation is further complicated by the fact that differences have also been observed in supposedly identical recombinant GlyRs. For example, PKC was found to potentiate glycine currents in Xenopus oocyte-expressed α1- and α2-homomeric GlyRs (281). In apparent contrast, PKC activators did not affect current magnitude in α1-GlyRs expressed in either Xenopus oocytes (253) or HEK293 cells (128). However, the later study reported that PKC activation accelerated the onset of and slowed the recovery from desensitization (128). A definitive understanding of PKC-dependent phosphorylation processes in the GlyR will require functional assays involving site-directed mutagenesis of putative phosphorylation sites and biochemical assays to directly determine the receptor phosphorylation state.

Interestingly, PKC activation has been shown to increase the potentiating effects of ethanol in recombinant α1-GlyRs (253). Ethanol was shown to potentiate, inhibit, or have no effect on glycine-activated responses in 35, 45, and 20%, respectively, of a large sample of ventral tegmental area (VTA) neurons (422, 423). In the population of VTA neurons where ethanol induced inhibition, PKC activators seem to compete with ethanol for a common inhibitory site (364). A pharmacological study on those VTA neurons where ethanol induced potentiation, activation of the PKC epsilon isoform was found to increase potentiation magnitude (173). Together, these findings raise the possibility of a specific allosteric linkage between the phosphorylation site and the alcohol binding site.

3. Protein tyrosine kinase

Lavendustin A, a protein tyrosine kinase (PTK) inhibitor, reduced glycine currents in hippocampal and spinal neurons, whereas intracellular application of c-Src, an endogenous tyrosine kinase, increased glycine current magnitudes (57). The current increases, which were mediated by a leftward shift in the glycine EC50, were accompanied by an enhanced desensitization rate (57). Similar effects of c-Src were observed on recombinant α1β-GlyRs expressed in HEK293 cells, but not in recombinant α1-GlyRs expressed in the same cells. Mutation of a putative tyrosine phosphorylation site (Y413F) in the large intracellular loop of the β-subunit (137) abolished the effects of several tyrosine phosphorylation modulators, suggesting this site is functionally phosphorylated (57). Although these experiments provide strong circumstantial evidence for β-subunit phosphorylation, biochemical confirmation is required to eliminate the possibility of allosteric effects induced by the Y413F mutation.

B. Modulators of Possible Physiological Relevance

1. Zinc

A) A PUTATIVE PHYSIOLOGICAL ROLE.

Zinc is concentrated into round clear presynaptic vesicles in the central nervous system and is released into the synaptic cleft by nerve terminal stimulation (20, 120, 164). During synaptic stimulation, zinc is thought to reach a peak external concentration of >100 μM (20, 120, 385). At such concentrations, zinc is able to modulate a wide variety of pre- and postsynaptic ion channels (349). Low (0.01–10 μM) concentrations of zinc potentiate glycinergic currents by increasing the apparent glycine affinity without changing the saturating current magnitude, whereas higher concentrations (>10 μM) inhibit the current by reducing the apparent glycine affinity (213). This pattern of zinc action is seen in native receptors (42, 63, 152, 359, 365) as well as in recombinant α1-, α2-, and α1β-GlyRs (213). In addition to increasing the magnitude of glycinergic IPSCs, low concentrations of zinc have also been shown to prolong their duration and frequency (211, 359), with both effects presumably being due to the increased glycine sensitivity.

The role of zinc has been most thoroughly investigated at the mossy fiber glutaminergic synapse in the hippocampus (120). However, some suggestions have recently emerged that zinc may also have a physiological role at glycinergic synapses. First, an ultrastructural study has found evidence for zinc and glycine colocalization in individual presynaptic terminals in the spinal cord (39). Second, at glycinergic synapses of the intact zebrafish hindbrain, zinc chelators decreased the amplitude, duration, and frequency of glycinergic IPSCs, whereas zinc application had the opposite effect (359). However, it remains to be established whether zinc is coreleased with glycine at concentrations high enough to modulate GlyR function.

It is important to note that even strongly inhibiting zinc concentrations (≥30 μM) causes an initial transient potentiation followed by the slowly developing inhibition (235). The duration of this transient potentiation, which is of the order of 1 s (203, 235), easily exceeds that of a typical glycinergic IPSC. However, zinc inhibition stabilizes much more rapidly in the absence of agonist (235) so that if an inhibiting concentration of zinc reaches the GlyR before glycine does, then only inhibition is seen (211, 235). Together, these observations mean that high zinc concentrations may have opposite effects on glycinergic IPSC magnitude depending on whether the zinc reaches the receptor before or after glycine. This should be considered in models of zinc action on glycinergic synapses.

No other metal ion has convincingly been shown to mimic the biphasic action of zinc. However, the potentiating site is recognized by several metal ions with the potency sequence: zinc > lanthanide > lead > cobalt (96, 203), whereas the inhibitory site exhibits the potency sequence: zinc > copper > nickel (96, 203).

B) MOLECULAR MECHANISM OF ACTION.

In most proteins, zinc ions are coordinated by nitrogen, sulfur, or oxygen atoms found in the side chains of histidine, cysteine, aspartic acid, and glutamatic acid residues (131). Zinc binding sites are usually comprised of two to four residues, and the affinity of a site depends on the number of residues, their relative positions, and the local electrostatic environment (21, 317). Several lines of evidence suggest that the GlyR zinc potentiating and inhibitory binding sites are physically discrete.

C) INHIBITION.

In contrast to the rapid onset of potentiation, zinc inhibition is slow to develop (235). However, as noted above, this inhibition develops much more rapidly in the absence of agonist (235). This result suggests that glycine-induced activation is accompanied by a structural change in this location and that zinc acts by stabilizing the closed conformation. A single-channel study found high (50 μM) zinc concentrations reduced α1-GlyR open probability by reducing mean channel open time and the relative abundance of long channel bursts (212). It concluded that zinc increases the rate at which the channel exits from the open state, supporting the view that zinc stabilizes the closed state.

Zinc inhibition of the recombinant α1-GlyR was found to be selectively abolished by either reducing the pH or by pretreatment with diethylpyrocarbonate, a histidine-specific modifying agent (158). Because both treatments effectively reduce the ability of zinc to bind with histidine imidazole rings, histidines were implicated in the complexation of zinc at its inhibitory site. Mutations to either H107 or H109 were subsequently shown to abolish zinc inhibition, strengthening the case that these residues formed an inhibitory binding site (158). Histidine α-carbonyl oxygen atoms need to be within 13 Å of each other to permit their imidazole rings to coordinate a zinc ion (21). Because the histidines are separated by only one residue, it is certainly feasible that the zinc ions could be coordinated within individual α-subunits. However, the α-carbonyl oxygens of the homologous residues in adjacent AChBP subunits are separated by 7.7 Å (52). With the assumption that this structure is reasonably well conserved in the GlyR, it is plausible that zinc ions could be coordinated by adjacent α-subunits. This possibility was tested by coexpressing α1-subunits containing the H107A mutation with α1-subunits containing the H109A mutation (276). Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the coexpression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild-type α1-homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent α1-subunits, but does not rule out the possibility that zinc may also be coordinated within α1-subunits. No evidence was found for β-subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per heteromeric receptor is sufficient for maximal zinc inhibition (276). This region of the α1-subunit aligns poorly with AChBP due to the existence of three additional α1-subunit residues. Homology models were constructed using several alignments, and only one of these produced a plausible zinc binding site (276). The successful alignment is depicted in Figure 2, and the modeled structure of this site is shown in Figure 3B.

D) POTENTIATION.

ow (5 μM) zinc concentrations increase the open probability of the α1-GlyR by increasing both the opening frequency and the mean burst duration (212). The authors concluded that the channel opening and closing rates were not significantly affected and that zinc acted primarily by slowing the rate of glycine dissociation from the binding site. This suggests an allosteric effect of zinc that improves the fit of glycine to its site. In contrast, zinc had a dual effect on taurine-gated currents. It not only slowed the rate of taurine dissociation from its binding site, but increased the rate at which bound taurine could activate the channel (212). Another group showed that mutations to various residues in the TM1-TM2 and TM2-TM3 domains abolished zinc potentiation of glycine currents, while leaving zinc potentiation of taurine currents intact (235). These results suggest that the allosteric linkage between the zinc potentiating site and the glycine binding site or transduction pathway was selectively disrupted. The results can be reconciled with those of Laube et al. (212) by proposing that the mutations selectively disrupted the ability of zinc to enhance the glycine affinity.

Analysis of chimeric constructs of α1- and β-subunits implicated D80 as a specific determinant of zinc potentiation (213). Indeed, mutations (D80A, D80G) to this residue disrupted zinc potentiation of glycine currents (212, 235), whereas mutations to neighboring aspartic acid residues (D81A, D84A) had no effect (212). However, because D80A did not abolish zinc potentiation of taurine-gated currents (235), its putative role as a zinc binding site must be queried. The potentiating effect of zinc was also abolished by the H109A mutation (158) as well as several mutations in the TM1-TM2 and TM2-TM3 linker domains (235). Unfortunately, this widespread distribution of site-directed mutations that abolish zinc potentiation does not bode well for the use of this approach in identifying the GlyR zinc potentiating site. The lack of zinc voltage dependence and rapid reversibility of the potentiating effect (212, 235) indicate that the potentiating binding site resides in an extracellular domain.

2. Calcium

Calcium current influx through glutamate-activated channels causes a rapid (<100 ms) and transient elevation of glycine current magnitude (124, 411, 412) that may have an important physiological role in modulating the gain of glycinergic transmission. Based on a pharmacological analysis on GlyRs expressed on rat spinal sensory neurons, Xu and colleagues (411, 412) proposed that the effect was mediated by calcium activation of calmodulin-dependent protein kinase II and calcineurin. However, Fucile et al. (124), who examined a similar effect in both cultured spinal neurons and recombinant α1-GlyRs, found it was resistant to a variety of manipulations designed to disrupt phosphorylation, dephosphorylation, and G protein-dependent processes. These differences may relate to the different origins of the neurons studied and could be indicative of multiple mechanisms contributing to this effect. Another recent study, conducted on VTA neurons, found the calcium-dependent potentiation to be antagonized by ethanol (438).

In the effect seen by Fucile et al. (124), single-channel analysis suggested the calcium-dependent factor exerted complex effects on both the glycine binding affinity and the gating rate (124). Because the calcium-induced increase did not reverse in inside-out patches, it was considered likely to be mediated by the calcium-induced removal of a soluble cytoplasmic intermediate from the receptor internal surface. Although the identity of this intermediate remains elusive, it appears to be endogenously expressed in HEK293 cells as well as in spinal neurons (124).

3. pH

Transient increases in extracellular pH occur in response to the activation of anionic LGIC receptors (65). The mechanism is most likely related to the high HCO3 permeability of anionic LGICs. When the pore opens it is likely that HCO3 exit the cell, causing intracellular acidification and extracellular alkalization (65). In recombinant α1- and α1β-GlyRs, the glycine EC50 is significantly increased as the pH is lowered from 7.5 to 6.0 (64). This effect appears to be mediated by a specific interaction with the GlyR extracellular domain as it is abolished by the α1-subunit mutations H109A, T112A, and T112F, but is not affected by other mutations to T112 or by mutations to neighboring negatively charged residues (64). Mutation to the β-subunit residue that corresponds to T112 (i.e., T135A) was also found to reduce proton sensitivity (64). Thus pH sensitivity appears to be specific to the receptor region that houses the zinc inhibitory binding site. However, as threonines are not ionizable, it is unlikely that they directly form the proton acceptor site.

4. Neurosteroids

Neurosteroids are hormones that are synthesized in central nervous system glia and neurons from cholesterol or blood-borne steroidal precursors (24, 357, 372). Although neuroactive steroids produced in the peripheral steroidogenic glands can easily access the brain, it is thought that steroids produced in the central nervous system have important paracrine roles (207). Although the complex behavioral effects of neurosteroids have been attributed primarily to GABAA and NMDA receptors (24, 103, 207, 357), potent neurosteroid actions have been observed on native and recombinant GlyRs. Antagonistic effects of progesterone, its precursor pregnenalone (PREG), and pregnenolone sulfate (PREGS) were first shown on glycine currents in cultured spinal neurons (407, 408). Another early report showed that glycine sensitivity in the rat optic nerve was increased by several corticosteroids at concentrations of 1–10 μM (299). On the other hand, both α1- and α1β-GlyRs were found to be insensitive to the action of 5α-pregnen-3α-ol-20-one (295). Using a more systematic approach, Maksay et al. (243) examined a battery of neurosteroids on recombinant α1-, α2-, α4-, α1β-, and α2β-GlyRs. Both PREGS and dehydroepiandrosterone sulfate (DHEAS) inhibited all of these receptors with inhibitory constant (Ki) values of 2–20 μM, with the compounds showing only a modest degree of subunit specificity. Of particular note, DHEAS inhibited the α2-GlyR with an IC50 of 4.2 μM, whereas its IC50 at the α1β-GlyR was 21.2 μM. PREG caused an ∼25% increase in the α1-GlyR current with an EC50 of 1.4 μM, but had no effect on α1β- or α2-GlyRs (243). On the other hand, progesterone inhibited the α2-GlyR current by 23% with an IC50 of 20 μM, while exerting no effect on the α1- and α1β-GlyRs (243). Given the α2 → α1β subunit switch that occurs after birth in the rat, the differential effects of DHEAS and progesterone on the α2- and α1β-GlyRs may have physiological relevance for neuronal development.

Although the molecular determinants of neurosteroid action at the GlyR have not yet been determined, some progress has been made on the GABAAR. On the basis of a chimeric study on alphalaxone-sensitive and -insensitive GABAAR subunits, a necessary determinant of neurosteroid action was isolated to the NH2-terminal half of TM2 (318). Consistent with this, the V2'S mutation in the GABAAR α1-subunit reduced the potency of PREGS block by 30-fold (11). Particularly since the GlyR 2′ residue is known to affect the potencies of other GlyR modulators (see below), it is a promising candidate as a specific determinant of neurosteroid action in the GlyR.

Finally, it has long been known that that the synthetic steroid RU 5135 displaces [3H]strychnine binding with a remarkably high (5 nM) affinity (48). Because the mechanism of action of this ligand has never been investigated, it is not known whether it shares a similar mode of action to the endogenous neurosteroids.

5. G protein βγ-subunits

The irreversible activation of G proteins by nonhydrolyzable GTP analogs has recently been shown to exert potent effects on the GlyR. These reagents cause both a leftward shift in the glycine EC50 of recombinant α1-GlyRs and a prolongation of glycinergic synaptic currents in cultured spinal neurons (425). These effects are most likely mediated by a direct interaction of G protein βγ-subunits with the GlyR α1-subunit because 1) βγ-subunits coimmunoprecipitate with the GlyR α1-subunit, and 2) addition of βγ-subunits to the intracellular membrane surface reversibly increases GlyR channel activity (425). These results suggest that G protein-coupled receptor activation may be important in vivo for regulating the gain of glycinergic synaptic transmission.

C. Molecular Pharmacology

1. Strychnine and analogs

The plant alkaloid strychnine (Fig. 6) is a highly selective and extremely potent competitive antagonist of glycine, β-alanine, and taurine with a dissociation constant of 5–10 nM (81, 427, 428). Strychnine sensitivity is currently the most definitive means of discriminating glycinergic from GABAergic synaptic currents. Because of its high affinity and specificity, [3H]strychnine has been widely used in radioligand displacement studies to investigate the potencies and allosteric actions of other GlyR ligands (241). Strychnine has been subjected to several structure-activity investigations (162, 239, 249). However, no analog has ever been shown to possess a higher apparent affinity than strychnine. Similarly, no strychnine-like ligands have yet been shown to exert agonist or allosterically enhancing properties. Surprisingly, however, strychnine has been shown to behave as an agonist in modified α7-homomeric nAChRs (286).

FIG. 6.

FIG. 6.Structures of representative compounds that exhibit bioactivity at the GlyR.


Early evidence from GlyR protein modification experiments indicated that the strychnine and glycine binding sites were mutually interactive but not identical (249). This interpretation has also been reached as a result of site-directed mutagenesis experiments. The first of these experiments stemmed from the observation that the α2*-splice variant subunit had a strychnine IC50 that was ∼560 times higher than those of the α2- or α1-subunits (202). Strychnine sensitivity was restored by incorporating the E167G mutation into the α2*-subunit, thus reversing the only nonconserved amino acid between the α2*- and α2-subunits (202). This residue aligns with G160 in the GlyR α1-subunit. As expected, strychnine insensitivity was conferred to the α1-subunit by the reverse G160E mutation (381). Mutation to the adjacent α1-subunit residue Y161A also abolished strychnine sensitivity (381), although the more conservative mutation Y161F did not (338). Photoaffinity labeling experiments localized another crucial strychnine binding element to either Y197 or Y202 of the α-subunit (322). Subsequently, Vandenberg and colleagues (309, 381, 382) identified K200 and Y202 as strychnine binding sites on the basis of functional analysis of α1-GlyRs incorporating site-directed mutations at these positions. Because glycine also binds to common or adjacent residues in both of these strychnine-binding domains (see above), it is reasonable to conclude that strychnine and glycine share overlapping but nonidentical binding sites in principal ligand binding domains B and C. This, of course, provides a structural basis for the competitive antagonist behavior of strychnine (382).

Substances that act purely as competitive antagonists are comparatively rare. Generally, it would be expected that any substance that binds to a receptor would also bias the conformational equilibrium towards a particular state. However, the possible allosteric effects of strychnine have yet to be considered. One way of doing so would be to determine the effects of low strychnine concentrations on modified GlyRs that spontaneously gate in the absence of agonist.

2. Picrotoxin and analogs

Derived from plants of the moonseed family, the convulsant alkaloid picrotoxin is also widely used to discriminate GABAergic from glycinergic currents (348). Picrotoxin (PTX) strongly inhibits GABAARs at 1–10 μM concentrations, whereas GlyRs in vivo are considerably less sensitive. PTX comprises an equimolar mixture of picrotoxinin and picrotin. As shown in Figure 6, these compounds differ only in the structure of the terminal isoprenyl group, which in the case of picrotin is hydrated to remove the double bond. Picrotoxinin potently inhibits the GABAAR, whereas picrotin is generally inactive at this receptor. This behavior correlates well with the relative systemic toxicities of the two substances. PTX inhibition of the GABAAR is use dependent (i.e., inhibition reaches steady-state at a faster rate in the open state) and noncompetitive, and its inhibitory potency is highly sensitive to TM2 mutations (see below). This combination of properties has frequently led to PTX being classified as a channel blocker. However, several lines of evidence (e.g., Refs. 277, 296) have firmly established PTX as an allosteric inhibitor of the GABAAR.

In 1992, Pribilla et al. (298) reported that αβ-heteromeric GlyRs were much less sensitive to PTX inhibition than were α-homomeric GlyRs. This result was independent of which α-subunit (α1, α2, or α3) was investigated. They also showed that inserting the α1-subunit TM2 into the β-subunit bestowed high PTX sensitivity to α1β-GlyRs. These findings were important for two reasons. First, by establishing TM2 as a crucial determinant of PTX sensitivity, they prompted an intensive investigation into the molecular basis of PTX action in various LGIC members (106, 109, 150, 389, 410, 433). Second, they established PTX sensitivity as a standard pharmacological tool for identifying the presence of β-subunits in recombinant and native GlyRs. Although several other compounds also display subunit sensitivity (see below), there is as yet no better tool than PTX for this purpose.

It was subsequently shown that picrotin and picrotoxinin were equally efficacious in inhibiting α1-GlyRs (236). The same study demonstrated that PTX inhibition was not use dependent and that its inhibition was “competitive,” meaning that its potency decreased as agonist concentration increased. Both behaviors are uncharacteristic of channel blockers. An allosteric mode of action was confirmed by the findings that the R19′L and R19′Q startle disease mutations transformed PTX into an allosteric potentiator at low concentrations (<3 μM) and a noncompetitive, slow-onset inhibitor at higher concentrations (236).

A series of studies on the GABAAR, GABACR, and GluClR established the 2′ and 6′ residues as crucial determinants of PTX sensitivity (106, 109, 150, 389, 410, 433). A common feature in all of these studies was that a ring of 6′ threonines was invariably required for high PTX sensitivity. Although all GlyR α-subunits contain a threonine at this position, the β-subunit contains a phenylalanine. As anticipated, a range of mutations to T6′ in the GlyR α1-subunit, including the α→β substitution T6′F, greatly diminished the inhibitory potency of PTX and related compounds (341, 343, 356). In addition, incorporating a threonine into the β-subunit 6′ position restored high PTX sensitivity to the α1β-GlyR (341), although a range of other β-subunit 6′ mutations had no such effect (343). Does this highly specific requirement mean that T6′ is the PTX binding site? Despite a molecular modeling study finding that PTX can fit into this part of the pore and that T6′ hydrogen bonds could plausibly coordinate a PTX molecule (435), it is premature to draw this conclusion.

The PTX-competitive compound α-ethyl-α-methyl-γ-thiobutyrolactone (αEMTBL; Fig. 6) was found to potentiate glycine responses in α1-GlyRs but inhibit them in α3-GlyRs (356). The TM2 domains of these subunits are conserved with the exception of the 2′ residue: in the α1-subunit it is a glycine, but in the α3-subunit it is an alanine. The inhibition seen in the α3-GlyR is abolished by the T6′F mutation, although the potentiation seen in the α1-GlyR is not affected by this mutation (356). Moreover, incorporating the α1 2′ residue into the α3-subunit (via the A2′G mutation) converts αEMTBL inhibition into potentiation. To interpret these results in terms of binding sites, one would have to postulate a site that toggles between inhibitory and potentiating depending on the identity of residues at the 2′ and 6′ positions. The existence of two discrete sites (an inhibitory site in the pore and a potentiating site elsewhere) seems equally implausible. This would require that a mutation that abolishes the PTX or αEMTBL inhibitory sites should simultaneously render functional a distant potentiating site. Thus, although they provide little support for a PTX or αEMTBL binding site at T6′, the above results argue strongly for a close allosteric coupling between the 2′ and 6′ residues.

The 15′ residue has also been implicated into this scheme. When the GlyR α1-subunit S15′ was mutated to a glutamine or asparagine, inhibition became use dependent and noncompetitive (92). This is similar to the effect previously seen with R19′ mutations (236). Because S15′ and R19′ mutations abolished the rapid-onset PTX effect that is also abolished by T6′ mutations, it suggests an allosteric interaction between R19′, S15′, and T6′. Does this imply that S15′ is the PTX-binding site? After all, evidence summarized below strongly implicates S15′ as an alcohol and volatile anesthetic binding site.

The current picture regarding the effects of PTX is complex because mutations to residues at the 2′, 6′, 15′, and 19′ positions can each affect the mode or potency of PTX action. Since all of these mutations have been shown to exert allosteric effects on PTX binding or effector sites, it is uncertain which, if any, is a PTX contact site. It appears that an imaginative approach will be required to convincingly resolve this issue.

Single-channel analysis also reveals complexities in the actions of PTX. In homomeric α-GlyRs, it reduced the predominant conductance state from ∼80 to 40 pS, with the probability of entering the lower conductance state progressively increasing as the PTX concentration was raised from 1 to 30 μM (217). In contrast, 30 μM PTX had no effect on αβ-heteromeric GlyRs, which (perhaps not coincidentally) also exhibited a 40-pS predominant conductance level (217). Higher (100 μM) PTX concentrations induced flickery kinetics in both α- and αβ-GlyRs (217, 426). These observations support the conclusion that PTX acts in an allosteric manner.

In summary, experiments to date have revealed an unusually complex mode of action for PTX. It seems they brought us little closer to formulating testable hypotheses concerning the binding site or mechanism of action of this enigmatic compound.

3. Cyanotriphenylborate

Negatively charged cyanotriphenylborate (CTB; Fig. 6) was chosen as a potential GlyR pore blocker due to its structural similarity with triphenylmethylphosphonium bromide, a classical nAChR pore blocker. CTB was duly shown to act in the predicted manner: its inhibition of the α1-GlyR was potent (IC50 ∼1.3 μM), use dependent, voltage dependent, and noncompetitive (324). Furthermore, it was not a potent inhibitor of the α2-GlyR or α3-GlyRs, and replacing the α1-subunit 2′ residue with the α2-subunit residue (via the G2′A mutation) abolished block. Together, these observations provide a strong case for CTB binding in the pore. However, it is not straightforward to conclude that CTB binds to the 2′ glycine, as it also potently blocked an α2β-GlyR in which the β-subunit TM2 domain had been entirely replaced by that of the α2-subunit. This paradoxical result demonstrates that CTB sensitivity can reside in a receptor containing only alanines at the 2′ position, thereby directly refuting the idea that the CTB binding site requires 2′ glycines. One possibility is that regions apart from the TM2 domain may also contribute to CTB sensitivity (324). Alternatively, β-subunits may serve the same role as α-subunit 2′ glycines in creating a favorable geometry for the binding of CTB at some level in the pore. Progress in characterizing the CTB mechanism of action is currently limited due to its lack of commercial availability.

4. Ginkgolides

Isolated from the leaves, roots, and bark of the Gingko tree, these macrocyclic terpeine compounds are widely used in herbal medicine. They also share common structural features with picrotoxinin (170). Ginkgolide B, which is well known as a platelet activating factor antagonist (Fig. 6), is also a specific and potent blocker (IC50 ∼0.27 μM) of glycine-gated currents in dissociated rat hippocampal pyramidal neurons (192). It is noncompetitive and use dependent, and its inhibitory potency is not affected by the competitive antagonist strychnine. Importantly, when applied externally, its blocking ability increased with cell depolarization, as expected for a negatively charged compound binding in the pore (192). These characteristics establish ginkgolide B as a classical pore blocker. Its molecular determinants of action and subunit specificity have yet to be investigated.

More recently, the effects of several ginkgolides were compared on GlyRs expressed in rat embryonic cortical neuron slices (170). As these GlyRs were insensitive to PTX, they may have comprised predominantly α2β-heteromers. Ginkgolides B, C, and M were found to be more potent than ginkgolide A, ginkgolide J, or bilolabide (a related compound from the same tree). In agreement with the earlier study (192), ginkgolide B inhibited the GlyR in a use-dependent, noncompetitive manner and showed specificity for the GlyR over a GABAAR expressed in the same tissue (170).

5. Tropisetron and other 5-HT3R antagonists

Several 5-HT3R antagonists have potent effects on the GlyR. The most potent of these compounds are those which contain tropeine groups (i.e., esters and amides of 3α-hydroxytropane) and include tropisetron, LY-278,584, zatosetron, and bemesetron. The muscarinic acetylcholine receptor antagonist atropine also belongs in this structural group. Some representative structures are given in Figure 6. This review focuses on tropisetron, the most widely characterized of these compounds.

In 1996, Chesnoy-Marchais found that tropisetron potentiated glycine currents in cultured spinal neurons at low concentrations (0.01–1 μM) but caused inhibition at higher concentrations (66). Its potentiation was mediated by a leftward shift in the glycine dose-response curve. Because the potentiation was additive with the potentiating effects of zinc, ethanol, and propofol (67), tropisetron appeared to act via a novel mechanism. In radioligand binding studies on membrane extracts from the spinal cord and brain stem, tropisetron displaced [3H]strychnine with a Ki of 2 μM (240). In addition, 0.1 μM tropisetron increased the ability of glycine to displace [3H]strychnine (240), suggesting an allosteric enhancing effect on glycine binding. Turning then to an electrophysiological analysis, Maksay et al. (242) subsequently failed to detect tropisetron potentiation in α1- or α2-GlyR homomers or in α1β- or α2β-GlyR heteromers, although the lower apparent affinity inhibitory effect was observed. In apparent contradiction, Supplisson and Chesnoy-Marchais (358) reported tropisetron potentiation in the α1-homomeric and the α1β- and α2β-heteromeric GlyRs, but not in the α2-homomer where only inhibition was seen (358). The discrepancy was convincingly resolved by the demonstration that potentiation required a low (EC10) glycine concentration, whereas higher (EC50) glycine concentrations, as used by Maksay et al. (242), uncovered only inhibition (358). The molecular determinants of tropisetron action have not been elucidated. Because β-subunits are needed to confer tropisetron potentiation to α2-subunits (358), the potentiating binding site may be located at the interface of these subunits.

Tropisetron suppresses glycine-gated currents when applied at high (>10 μM) concentrations to both native neuronal and recombinant GlyRs. At least in the α2-GlyR, it seems to behave in a noncompetitive manner. The tropisetron inhibitory potency is modestly affected (IC50 increased by a factor of 2) by the T112A mutation (242), a mutation that completely abolishes the inhibitory potency of zinc. The increased tropisetron inhibitory potency of the α2-GlyR is not transferred to the α1-GlyR via the A2′G mutation (358), the only TM2 domain residue which is not conserved between these subunits. However, as previously seen for CTB and PTX, it may be misleading to infer locations of binding sites on the basis of site-directed mutations at this position in the pore.

A structure-activity study concluded that the tropeine structure itself was required for potentiation (240). However, atropine, which shares the tropeine moiety, does not increase glycine displacement of strychnine binding, although its inhibitory potency is only marginally weaker than that of tropisetron (240, 242). Thus the tropeine moiety appears to be no guarantee of a potentiating effect. These results are broadly consistent with another structure-activity analysis, which concluded that an aromatic ring, a carbonyl group, and a tropane nitrogen are required for glycinergic potentiation (70).

6. Ivermectin

Ivermectin (22,23-dihydroavermectin B1a) is a naturally occurring macrocyclic lactone (Fig. 6) that is widely used as an antiparasitic agent in agriculture, veterinary practice, and human medicine (98, 319). Although the target of its antiparasitic action is believed to be a GluClR that exists in a number of invertebrate phyla (78, 178), it also has direct activating or potentiating effects on GABAARs and nAChRs (2, 87, 197, 198). At low (0.03 μM) concentrations, ivermectin potentiates subsaturating glycine responses, but at higher (≥ 0.03 μM) concentrations it irreversibly activates recombinant α1- and α1β-GlyRs (342). Because ivermectin-gated currents have a different pharmacology to glycine-gated currents, and glycine binding site mutations do not drastically affect its sensitivity (342), ivermectin appears to activate the GlyR via a novel mechanism. Apart from cesium (352), ivermectin is the only non-amino acid agonist of the GlyR to be identified to date.

7. Alcohols, anesthetics, and inhaled drugs of abuse

Traditionally, anesthetics have been depicted as nonselective agents that act by partitioning into and disordering lipid bilayers. However, in recent years it has become increasingly apparent that specific binding sites on ligand-gated ion channels are among their major molecular targets (36, 117, 196, 260, 418). Because alcohol and some anesthetic effects on the GlyR are observed at pharmacologically relevant concentrations, it is possible that at least part of their acute effects are mediated by this receptor.

Exposure to pharmacologically relevant (50–100 mM) concentrations of ethanol was first shown by Celentano et al. (59) to produce a persistent increase in the glycine sensitivity of spinal neurons. Most subsequent studies on GlyRs natively expressed in neurons have observed similar potentiating effects. Neonatal ventral tegmental area neurons appear to constitute an exception to this rule: ethanol (0.1–10 mM) potentiated, inhibited, or had no effect on glycine-activated responses in 35, 45, and 20%, respectively, of an impressively large sample of these neurons (422, 423).

Because ethanol potentiation is achieved without disordering the membrane lipid bilayer (366), it is likely to be acting via a specific site at the GlyR. When expressed in Xenopus oocytes, α1-GlyRs were more sensitive to ethanol than were α2-GlyRs (251). This increased sensitivity was abolished by incorporating the naturally occurring spasmodic mouse mutation A52S into the α1-subunit (251). However, the α1-subunit ethanol sensitivity was largely lost upon recombinant expression in mammalian Ltk or HEK293 cells (380). Because the GlyR α1-subunit contains phosphorylation consensus sites, the phosphorylation status of the receptor was considered a possible cause of this anomaly. Indeed, PKC-mediated phosphorylation selectively increases ethanol potentiation of Xenopus oocyte-expressed α1-GlyRs while having no effect on the enhancement induced by the inhalation anesthetic halothane or the intravenous anesthetic propofol (253). On the other hand, PKA-mediated phosphorylation was without effect (3).

The inhalation (or volatile) anesthetic isoflurane was first shown by Harrison et al. (155) to potentiate recombinant α2-GlyRs. For both recombinant α1-GlyRs and the native GlyR in medullary neurons, the degree of potentiation increased in the rank order methoxyflurane, sevoflurane < halothane, isoflurane, enflurane, F3 (97, 250). The potentiation was induced primarily by a leftward shift in the glycine EC50 (97). Because the leftward shift is more pronounced at low glycine EC values (97), the effects of alcohols and volatile anesthetics have generally been investigated using glycine concentrations that activate 2–5% of the saturating current magnitude.

In contrast to the GlyR, the GABACR is inhibited by both classes of agents (261). By constructing a series of chimeras between these two receptors, a region of 45 amino acid residues was identified as necessary and sufficient for mediating potentiation by both alcohol and volatile anesthetics (262). Site-directed mutagenesis of the nonconserved residues in this region identified S15′ in the TM2 domain and A288 in the TM3 domain as crucial determinants of alcohol and volatile anesthetic sensitivity (262). Because both residues are located towards the extracellular end of their respective TM segments, it was hypothesized that these two residues faced each other to form a pocket to accommodate an alcohol molecule. Lying within the membrane, this putative water-filled pocket is thought to be inaccessible from the ion channel pore. The existence of such a water-filled pocket lined by TM2 and TM3 domains is supported by SCAM data in the GABAAR and structural evidence from the nAChR (see sect. iiiB).

It was already known that alcohol potentiation of the α1-GlyR increases with the length of the side chain of a series of n-alcohols until a cut-off is reached, after which further increases in molecular size decrease alcohol potency (250). Increasing the size of the amino acid side chain by the S15′Q mutation decreased the cutoff from n = 10–12 to 7, consistent with the expected reduction in the volume of the binding pocket (399, 424). The converse experiment on the GABACR, in which a smaller side chain was introduced at the corresponding position, increased the alcohol size cutoff (399). Using a similar idea, the molecular volume and hydropathy of the side chain at the 288 position were revealed as crucial determinants of volatile anesthetic sensitivity (420). Together, these experiments supported the view that S15′ and A288 might form the binding pocket for both alcohols and volatile anesthetics. Alternatively, the mutations might impose structural changes that perturb the allosteric potentiation mechanisms of these compounds. Indeed, because these mutations affect glycine EC50 values (262, 420, 424), this alternative is a distinct possibility. More direct evidence for these residues forming a binding pocket came from a SCAM-type approach (252). Current flux through the α1-GlyR incorporating the S15′C mutation was irreversibly enhanced by the sulfhydryl-containing anesthetic propanethiol under conditions of oxidation by iodine, or directly by propyl methanethiosulfonate. After modification by either compound, the GlyR could no longer be potentiated by alcohols or anesthetics (252). This constitutes strong evidence that these compounds bind specifically in a pocket lined by S15′.

A recent investigation has found that ethanol potentiation of recombinant α1-GlyRs was antagonized by increased atmospheric pressure (85). Because the increased pressure did not affect the actions of glycine, strychnine, or zinc, it is likely to exert a selective effect at the alcohol site. It will be of interest to understand the molecular basis of this phenomenon.

Recently, several commonly abused inhalants were investigated in terms of their action at recombinant α1-GlyRs. Toluene, 1,1,1-trichloroethane, trichloroethylene, and chloroform potentiated α1-GlyRs by reducing the glycine EC50 values (31, 33). Because these compounds exhibited different patterns of sensitivity to S15′ mutations than did ethanol and enflurane, and also showed competition with these compounds, it was proposed that their binding sites shared some overlap with the alcohol/anesthetic binding site (31, 33). However, the possibility that the respective binding sites may interact allosterically cannot yet be ruled out.

The gaseous anesthetics nitrous oxide and xenon weakly potentiate submaximal glycine currents in the α1-GlyR at clinically relevant doses (84, 419). Other anesthetics including propofol, thiopentone, pentobarbitone, alphaxalone (a steroid), etomidate, and ketamine also exert potentiating effects on some GlyR isoforms (35, 84, 250, 295). Because more dramatic effects of all these compounds are seen at other receptors (36, 116, 118, 196, 418), their effects on GlyRs are unlikely to be clinically relevant.

8. Miscellaneous bioactive compounds

Dihydropyridine antagonists of L-type calcium channels also inhibit GlyR currents in spinal cord neurons at micromolar concentrations (69). Their effects are stronger at higher glycine concentrations and increase with time during glycine application, reminiscent of an open channel block mechanism. In addition, low (1–5 μM) concentrations of nitrendipine and nicardipine exert potentiating effects that were additive with those of zinc, implying discrete mechanisms of action (69).

The estrogen receptor modulator tamoxifen has recently been shown to have a particularly dramatic potentiating effect on submaximal glycine responses in cultured spinal neurons (68). A 5 μM concentration caused a 6.6-fold reduction in the glycine EC50. Several controls were used to rule out possible effects of tamoxifen on cell signaling cascades. The magnitude of the potentiation was increased when tamoxifen was applied to the cells before glycine application, although maintenance of the potentiation seen upon glycine application required continued tamoxifen application (68). The potentiation persisted in the presence of 10 μM zinc, implying a discrete site of action. The same study also noted an apparently direct potentiating effect of 4 μM dideoxyforskolin on GlyRs, a finding that may be relevant to studies designed to investigate phosphorylation mechanisms.

Clinical concentrations of the neuroprotective drug riluzole were reported to accelerate the α1β-GlyR desensitization rate while having no effect on the maximal current magnitude (269). Paradoxically, however, the time course of currents activated by brief (2 ms) applications of glycine were prolonged by riluzole (269). Similar effects were also observed on the GABAAR. The prolongation of the simulated synaptic currents may help explain the sedative and anticonvulsant side effects of this drug (95).

Colchicine, a microtubule-depolymerizing reagent, competitively antagonizes glycine-induced currents with IC50 values of 64 and 324 μM for the α1- and the α2-GlyRs, respectively. Its effect is instantaneous and independent of the microtubule depolymerization process (238). Interestingly, colchicine also prevents ethanol potentiation of GABAAR currents (394, 397), which perhaps provides a starting point for investigating its mechanism of action.

The tyrosine kinase inhibitor genistein has been shown to have a direct inhibitory effect on native GlyRs in neurons isolated from the hypothalamus and VTA (165, 437). This effect is not a pharmacological effect on PTK activation as it was effective only from the external membrane surface (165). It inhibits in a noncompetitive and use-dependent manner, suggesting that it binds in the pore.

Reasoning that the pharmacology of the GlyR glycine site may have structural similarities with the glutamate (NMDA) receptor glycine site, Schmieden et al. (337) used a potent NMDA receptor glycine antagonist, 2-carboxy-4-hydroxyquinoline, as a lead compound for the development of novel GlyR antagonists. The most potent compound thus identified, 5,7-dichloro-4-hydroxyquinoline-3-carboxylic acid, inhibited recombinant α1-GlyRs with an IC50 of 20 μM. Although this compound was not active at the NMDA receptor, the results imply some degree of similarity in the glycine pharmacophores of the GlyR and NMDA receptors. Another glutamate (KA/AMPA) receptor antagonist, NBQX, has recently been shown to inhibit recombinant α1- and α2-GlyRs with an IC50 of 4 μM (256).

Effects of ginsenosides, the active ingredients of the Panax ginseng plant, have been investigated on recombinant α1-GlyRs. The most active of these compounds, ginsenoside-Rf, potentiated submaximal glycine-gated currents with an EC50 of 50 μM (282).

Glycine-activated currents in recombinant α1- and α2-GlyRs were both inhibited by 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) with an IC50 of ∼0.5 μM (163). When applied at higher (10 μM) concentrations, PMBA had differential effects on α2- and α1-GlyRs. In the α1-GlyR, its main effect was to impose a rightward shift on the glycine dose-response. However, in the α2-GlyR, it also lowered the Hill coefficient for glycine activation (163). Furthermore, preincubation with PMBA delayed the rate of glycine-gated current activation in the α2-GlyR but not the α1-GlyR. Thus the mode of PMBA binding or activation may differ between the α1- and α2-subunits.

Opioid alkaloids have long been known to exhibit selectivity for the GlyR over the GABAAR (82). The relative efficacy of these compounds in antagonizing glycine-induced currents in intact spinal neurons is thebaine > morphine > codeine (79, 82), whereas thebaine was by far the most potent of a series of opioid agonists in displacing [3H]strychnine binding in spinal cord neurons, with an IC50 of 1 μM (132).

Finally, as reviewed previously (307), a wide range of compounds with structural similarities to various GABAAR ligands have been investigated in terms of their [3H]strychnine displacement potencies. Benzodiazepines and their antagonists were among the most potent compounds thus identified, with IC50 values of ∼1–10 μM (48, 249, 429). Interestingly, a recent report has identified a low-affinity benzodiazepine inhibitory site on α2-containing GlyRs (367).

VII. GLYCINE RECEPTOR CHANNELOPATHIES

A. Human Startle Disease

Human hereditary hyperekplexia, or startle disease, is a rare neurological disorder characterized by an exaggerated response to unexpected stimuli (15, 308). The response is typically accompanied by a temporary but complete muscular rigidity often resulting in an unprotected fall. This behavior is graphically illustrated in a published videotape of the bovine form of the disorder (161). Susceptibility to startle responses is increased by emotional tension, nervousness, fatigue, and the expectation of being frightened. Unprotected falls in turn lead to chronic injuries that are also characteristic of this disorder. Symptoms of this disease are present from birth, with infants also displaying a severe muscular rigidity (or hypertonia) that gradually subsides throughout the first year of life. Some affected infants die suddenly from lapses in respiratory function (129). This disorder has a history of being misdiagnosed as epilepsy, although hyperekplexia is readily distinguished by an absence of fits and a retention of consciousness during startle episodes. The symptoms are successfully treated by benzodiazepines, with clonazepam being the current drug of choice (436).

Hyperekplexia is caused by heritable mutations that reduce the magnitude of glycine-gated chloride currents. As summarized in Table 1, this is achieved by either disrupting GlyR surface expression or by reducing the ability of expressed GlyRs to conduct chloride ions. The disease mutations thereby impair the efficiency of glycinergic neurotransmission in reflex circuits of the spinal cord and brain stem, thus increasing the general level of excitability of motor neurons. Patients are apparently able to cope with this reduced inhibitory tone during normal tasks (perhaps due to developmental compensations, see below), although they are unable to cope with the increased demand required to dampen strong, unexpected excitatory commands.

TABLE 1. GlyR mutations underlying startle syndromes in various species

Mutation and SubunitInheritance ModeEffect on GlyR FunctionReference Nos.
Human forms
α1 P250TAutosomal dominantReduced single-channel conductance, reduced glycine sensitivity, increased desensitization rate50, 334
α1 V260MAutosomal dominantAs yet unknown88
α1 Q266HAutosomal dominantReduced open probability, reduced glycine sensitivity263, 271
α1 S270TAutosomal dominantAs yet unknown210
α1 R271L/QAutosomal dominantReduced glycine sensitivity, reduced single-channel conductance208, 306, 327, 346
α1 K276EAutosomal dominantReduced glycine sensitivity, reduced open probability101, 228, 237
α1 Y279CAutosomal dominant, variable penetranceReduced glycine sensitivity, reduced whole cell current magnitude205, 237, 345
α1 I244NAutosomal recessiveReduced glycine sensitivity, reduced whole cell current magnitude, increased desensitization rate237, 312
α1 Deletion of exons 1-6Autosomal recessivePresumed nonfunctional53
α1 S231RAutosomal recessiveReduced membrane insertion166
α1 Stop codon at Y202Autosomal recessiveReduced surface expression, possible heterozygosity with α1 V147M315
α1 G342SCompound heterozygous?No effect of individual mutation, possible heterozygosity with other mutations315
α1 R252H + α1 R392HCompound heterozygousReduced membrane insertion311, 384
β G229D + β exon 5 lossCompound heterozygousReduced glycine sensitivity, reduced surface expression314
Murine forms
β Line-1 intronic insertionAutosomal recessive (Spastic)Reduced surface expression156, 187, 275
α1 A52SAutosomal recessive (Spasmodic)Reduced glycine sensitivity335
α1 Stop codonAutosomal recessive (Oscillator)Reduced surface expression54
Bovine form
α1 Stop codonAutosomal recessive (Myoclonus)Reduced surface expression147, 294

Genetic linkage analysis of startle disease pedigrees first localized this disorder to the distal portion of chromosome 5q (327), where the GlyR α1-subunit gene is found. It was subsequently shown that an autosomally dominant form of startle disease was due to either the R271L or R271Q substitutions in the human α1-subunit (346). Although numerous other startle mutations have since been identified, the R271 mutations seem to be the most common cause of this disorder. When incorporated into recombinantly expressed GlyRs, these mutations caused a reduced glycine sensitivity and single-channel conductance (208, 306). Other autosomal dominant startle mutations, including Y279C, K276E, Q266H, and P250T, have generally similar effects (Table 1). In addition, the P250T mutation causes an enhanced desensitization rate (334), which may also contribute to the disease phenotype. As discussed previously in this review, each of these mutations acts by impairing the receptor activation mechanism, and closer analysis of the mutation phenotypes has provided valuable insights into the structural basis of GlyR function. The effect of the autosomally dominant V260M and S270T startle disease mutations (88, 210) has yet to be characterized.

Autosomally recessive forms of startle disease have been described in sporadic cases, generally in the offspring of consanguineous parents. One such patient exhibited homozygosity for a point mutation, I244N (312), in the α1-subunit. When incorporated into recombinant α1-homomeric GlyRs, this mutation resulted in a reduced glycine current magnitude, a reduced glycine sensitivity, and an enhanced desensitization rate (237). Two recessive forms of startle disease have also been described that must have resulted in the complete nonexpression of the GlyR α1-subunit. In one case, the mutation caused a homozygous deletion encompassing exons 1–6 (53), whereas the other involved a base pair deletion resulting in a frameshift and a premature stop codon prior to the TM1 domain (315). Despite the complete absence of α1-subunit expression, the symptoms experienced by these patients were no more severe than in those where the α1-subunit function was only mildly impaired. Another recently identified α1-subunit recessive point mutation, S231R, caused a reduced membrane insertion of functional receptors (166).

Compound heterozygous forms of startle disease have also been described. In one case, two distinct recessive α1-subunit mutations, R252H and R392H, caused startle disease when present in different alleles (384). However, patients possessing either single mutant allele were healthy. Consistent with this observation, coexpression of the two mutant subunits resulted in a reduced surface expression of functional GlyRs, whereas the individual mutations had no effect (311). The recessive M147V and G342S α1-subunit mutations have also been identified in patients exhibiting startle disease symptoms (315). These mutations are presumed to result in compound heterozygous forms of startle disease, but if this is the case their partner mutations remain to be identified. Finally, compound heterozygous mutations have recently been identified in the β-subunit. A β-subunit missense mutation (G229D) and a splice site mutation (resulting in the excision of exon 5) occurred simultaneously in a compound heterozygote with a transient startle disease phenotype (314). The G229D mutation alone induced a modest (4-fold) decrease in the glycine sensitivity of recombinant α1β-GlyRs (314), whereas the removal of exon 5 would presumably have precluded the expression of functional β-subunits.

To date, no startle mutations have been identified in the human α2- or α3-subunits. It remains to be determined for any species whether expression of these subunits is upregulated to compensate for the loss of α1-subunit expression or function. The effectiveness of clonazepam in treating startle disease is presumably due to its potentiation of the GABAAR. Indeed, evidence from spastic mice (26) (135) and myoclonic cattle (233) suggests that spinal cord GABAergic neurotransmission may be upregulated during development in compensation for the loss of glycinergic tone.

It would not be surprising if startle syndromes resulted from mutations that disrupt the function of other proteins involved in the formation, maintenance, and function of glycinergic synapses. Indeed, a hereditary mutation in the GlyR clustering protein gephyrin results in a hyperekplexia phenotype in humans (313) and targeted deletion of the glycine transporter subtype 2 gene produces a startle phenotype in mice (133).

B. Murine Startle Syndromes

Three naturally occurring murine startle syndromes have been identified to date (Table 1). The symptoms of each are largely similar to those observed in human hyperekplexia (308). An exception is that mouse startle symptoms commence at around the 20th postnatal day, corresponding to the time when the replacement of α2-homomers by α1β-heteromers is complete. The most thoroughly characterized murine syndrome is the spastic mouse, which was first identified in the 1960s (255). This autosomally recessive disorder is characterized by a reduction in the number of expressed GlyR α1-subunits, although the function of the expressed receptors is normal (26, 28). The spastic phenotype was found to be caused by the insertion of a 7.1 kb Line-1 repeating element into intron 5 of the β-subunit (187, 275). This element leads to aberrant splicing of the β-subunit transcripts resulting in an accumulation of prematurely terminated protein and a concomitant reduction in the surface expression of β-subunits (187, 275). Because β-subunits are required to anchor α1-subunits to synaptic scaffolding proteins, the disease phenotype is presumably caused by a loss in the efficiency of GlyR recruitment to postsynaptic densities. Introduction of low levels of a wild-type β-subunit transgene effectively reverses this disease phenotype (156).

As expected, the magnitudes of glycinergic IPSPs in motor and sensory spinal neurons of spastic mice were significantly reduced relative to wild-type controls (135, 386). However, the same two studies observed contradictory effects on the size of GABAergic IPSPs in the same neurons. Graham et al. (135), who measured the magnitude of spontaneous mini-IPSPs in dorsal horn sensory neurons, reported an increase in GABAergic IPSP magnitude. On the other hand, Von Wegerer et al. (386), who recorded electrically stimulated IPSPs in ventral horn motor neurons, reported a reduction in GABAergic IPSP magnitude. The difference may relate to the identity of the neurons studied or to the different IPSPs (evoked versus spontaneous) measured.

The spasmodic mouse contains the homozygous point mutation A52S in the α1-subunit. This mutation results in a moderate (6-fold) reduction in glycine sensitivity (326, 335). Spinal inhibitory neurotransmission has yet to be characterized in this mouse. An allelic variant of spasmodic, termed oscillator, is the only lethal form of startle disease identified to date. This syndrome is caused by a frameshift mutation in the TM3-TM4 domain resulting in the translation of an incomplete form of the α1-subunit protein (54). With the assumption that the majority of spinal glycinergic synapses comprise α1β-heteromers by postnatal day 20, homozygous oscillator mice should have virtually no glycinergic tone. Consistent with this, membranes isolated from oscillator homozygote spinal cords display a 90% reduction in strychnine binding, indicating a drastic loss in the functional expression of the α1-subunit (54). Surprisingly, however, the magnitude of strychnine-sensitive synaptic currents was found to be reduced by only 50% in dorsal horn sensory neurons of mice homozygous for this mutation (135). It would be of interest to examine the pharmacology of the currents that remain: is it possible that the strychnine-insensitive α2*-subunit is upregulated to compensate for the loss of α1-subunits?

It is noteworthy that lethality does not result from human and bovine startle mutations that act similarly to completely preclude the functional expression of α1-subunits (53, 294, 315). A possible reason for the difference is that the α2 → α1β subunit switch in the mouse occurs after birth resulting in an inability to feed. In humans and cattle, the switch is likely to occur before birth, thereby not affecting the ability to feed, and affording time for the development of synaptic adaptations before parturition.

Two transgenic mice strains have been generated with a view to developing an animal model of human hyperekplexia. The first approach mimicked the spastic mouse mutation by engineering a transposon insertion into the β-subunit (29). Homozygous mice displayed a startle phenotype that resembled human hyperekplexia (30). The second approach incorporated the dominant human α1-subunit R271Q mutation into transgenic mice. Transgenic mice that were heterozygous for the R271Q human startle mutation displayed a pronounced startle phenotype, and mice homozygous for the mutation were not viable (30). Knock-in mice bearing the α1-subunit S267Q mutation (which eliminates the alcohol binding site and reduces peak current magnitude) displayed a reduced sensitivity to alcohol and an oscillator-type phenotype (111, 112). Finally, as noted above, knock-out of the glycine transporter subtype-2 gene produces a lethal oscillator-type phenotype (133).

C. Bovine Myoclonus

A congential recessive startle syndrome, called myoclonus, has been identified in Poll Hereford cattle (160). This disorder was recently shown to be due to a single base pair deletion in the α1-subunit gene, leading to a frameshift and a premature stop codon before TM1 (294). As would be expected, this mutation induced a dramatic reduction in the surface expression of functional GlyRs (147). A similar syndrome may also exist in Peruvian Paso horses (148).

VIII. OUTLOOK

GlyRs have important roles in a variety of physiological processes, especially in mediating inhibitory neurotransmission in the spinal cord and brain stem. Although recent progress in understanding the molecular functional architecture of these receptors has been rapid, many gaps remain in a number of critical areas. The following are considered to be among the most pressing research priorities at the present time.

1) A high-resolution structure of the entire GlyR complex will permit the design of much more precise experiments to understand receptor structure and function. Unfortunately, however, crystal structures are notoriously difficult to obtain for membrane-spanning proteins, and complete structures have yet to be resolved for any LGIC member.

2) It is essential to establish the exposure pattern of residues in TM2 and in the pore selectivity filter of the GlyR α- and β-subunits. This is a prerequisite for understanding the ion-permeation and channel-opening mechanisms. It is also essential to probe the surface electrostatic potential in the pore selectivity filter to further our understanding of the ionic charge discrimination mechanism.

3) Because binding sites are located at subunit interfaces, it is necessary to resolve the GlyR subunit stoichiometry and arrangement so that the number and type of subunit interfaces can be defined. This is an important first step toward resolving the structural and functional basis of ligand binding.

4) There are large gaps in our knowledge of glycine binding mechanisms. In particular, the role of the α-subunit complementary binding domains in coordinating glycine needs to be investigated. This is a prerequisite for the structural modeling of the agonist binding pocket.

5) The role of β-subunit in channel in agonist binding and channel activation has received scant attention. Again, this information is necessary for the structural modeling of ligand binding sites and the understanding of activation mechanisms.

6) The modeling of GlyR kinetics remains controversial. Because kinetic models are crucial for understanding and predicting receptor behavior, it is hoped that future studies will carefully readdress this situation.

7) The diversity of GlyR subtypes at least partially underlies the diversity in glycinergic neurotransmission properties throughout the central nervous system (219). Understanding the GlyR structural variations that underlie this synaptic functional diversity is an important question for future research.

8) The role of α2-homomeric GlyRs in embryonic neurons remains to be clarified.

9) Extrasynaptic GlyRs are present on many central nervous system neurons. In addition, GlyRs have been found in a number of nonneuronal tissues. The physiological roles of these GlyRs need to be investigated in more detail.

10) The therapeutic possibilities of GlyR modulatory agents warrant further investigation. As noted in a recent review (215), the fact that GlyRs are involved in motor reflex circuits and nociceptive sensory pathways suggests that GlyR modulators could have therapeutic potential as analgesics and muscle relaxants. This review describes the effects of a number of molecules with potential to be lead compounds for the development of such therapeutics. Further therapeutic possibilities may emerge as the roles of extrasynaptic and nonneuronal GlyRs are characterized in more detail.

I gratefully acknowledge the patience of my laboratory members during the writing of this review. Professor Peter Barry and the anonymous reviewers are acknowledged for providing valuable insights and helpful suggestions. Dr. Brett Cromer and Prof. Michael Parker are thanked for kindly providing the unpublished image in Figure 3B.

Research in the author's laboratory is funded by the Australian Research Council and the National Health and Medical Research Council of Australia.

REFERENCES

  • 1 Absalom NL, Lewis TM, Kaplan W, Pierce KD, and Schofield PR. Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor. J Biol Chem 278: 50151–50157, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 2 Adelsberger H, Lepier A, and Dudel J. Activation of rat recombinant α1β2γ2S GABAA receptor by the insecticide ivermectin. Eur J Pharmacol 394: 163–170, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 3 Aguayo LG, Tapia JC, and Pancetti FC. Potentiation of the glycine-activated Cl current by ethanol in cultured mouse spinal neurons. J Pharmacol Exp Ther 279: 1116–1122, 1996.
    PubMed | ISI | Google Scholar
  • 4 Ahmadi S, Kotalla C, Guhring H, Takeshima H, Pahl A, and Zeilhofer HU. Modulation of synaptic transmission by nociceptin/orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin/orphanin FQ receptor. Mol Pharmacol 59: 612–618, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 5 Akabas MH. Channel-lining residues in the M3 membrane-spanning segment of the cystic fibrosis transmembrane conductance regulator. Biochemistry 37: 12233–12240, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 6 Akabas MH and Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the α-subunit. Biochemistry 34: 12496–12500, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 7 Akabas MH, Kaufmann C, Archdeacon P, and Karlin A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit. Neuron 13: 919–927, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 8 Akabas MH, Stauffer DA, Xu M, and Karlin A. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258: 307–310, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 9 Akagi H, Hirai K, and Hishinuma F. Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development. FEBS Lett 281: 160–166, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 10 Akagi H, Hirai K, and Hishinuma F. Functional properties of strychnine-sensitive glycine receptors expressed in Xenopus oocytes injected with a single mRNA. Neurosci Res 11: 28–40, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 11 Akk G, Bracamontes J, and Steinbach JH. Pregnenolone sulfate block of GABAA receptors: mechanism and involvement of a residue in the M2 region of the α subunit. J Physiol 532: 673–684, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 12 Ali DW, Drapeau P, and Legendre P. Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo. J Neurophysiol 84: 1726–1736, 2000.
    Link | ISI | Google Scholar
  • 13 Altschuler RA, Betz H, Parakkal MH, Reeks KA, and Wenthold RJ. Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res 369: 316–320, 1986.
    Crossref | PubMed | ISI | Google Scholar
  • 14 Amin J and Weiss DS. GABAA receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital. Nature 366: 565–569, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 15 Andermann F and Andermann E. Startle disorders of man: hyperekplexia, jumping and startle epilepsy. Brain Dev 10: 213–222, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 16 Aprison MH and Werman R. The distribution of glycine in cat spinal cord and roots. Life Sci 4: 2075–2083, 1965.
    Crossref | PubMed | Google Scholar
  • 17 Araki T, Yamano M, Murakami T, Wanaka A, Betz H, and Tohyama M. Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody. Neuroscience 25: 613–624, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 18 Arias HR. Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor. Biochim Biophys Acta 1376: 173–220, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 19 Arias HR. Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. Neurochem Int 36: 595–645, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 20 Assaf SY and Chung SH. Release of endogenous Zn2+ from brain tissue during activity. Nature 308: 734–736, 1984.
    Crossref | PubMed | ISI | Google Scholar
  • 21 Auld DS. Zinc coordination sphere in biochemical zinc sites. Biometals 14: 271–313, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 22 Barrantes FJ, Antollini SS, Blanton MP, and Prieto M. Topography of nicotinic acetylcholine receptor membrane-embedded domains. J Biol Chem 275: 37333–37339, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 23 Basbaum AI. Distribution of glycine receptor immunoreactivity in the spinal cord of the rat: cytochemical evidence for a differential glycinergic control of lamina I and V nociceptive neurons. J Comp Neurol 278: 330–336, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 24 Baulieu EE, Robel P, and Schumacher M. Neurosteroids: beginning of the story. Int Rev Neurobiol 46: 1–32, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 25 Beato M, Groot-Kormelink PJ, Colquhoun D, and Sivilotti LG. Openings of the rat recombinant α1 homomeric glycine receptor as a function of the number of agonist molecules bound. J Gen Physiol 119: 443–466, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 26 Becker CM. Disorders of the inhibitory glycine receptor: the spastic mouse. FASEB J 4: 2767–2774, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 27 Becker CM, Hoch W, and Betz H. Glycine receptor heterogeneity in rat spinal cord during postnatal development. EMBO J 7: 3717–3726, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 28 Becker CM, Schmieden V, Tarroni P, Strasser U, and Betz H. Isoform-selective deficit of glycine receptors in the mouse mutant spastic. Neuron 8: 283–289, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 29 Becker L, Hartenstein B, Schenkel J, Kuhse J, Betz H, and Weiher H. Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease. Eur J Neurosci 12: 27–32, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 30 Becker L, von Wegerer J, Schenkel J, Zeilhofer HU, Swandulla D, and Weiher H. Disease-specific human glycine receptor α1 subunit causes hyperekplexia phenotype and impaired glycine- and GABAA-receptor transmission in transgenic mice. J Neurosci 22: 2505–2512, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 31 Beckstead MJ, Phelan R, and Mihic SJ. Antagonism of inhalant and volatile anesthetic enhancement of glycine receptor function. J Biol Chem 276: 24959–24964, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 32 Beckstead MJ, Phelan R, Trudell JR, Bianchini MJ, and Mihic SJ. Anesthetic and ethanol effects on spontaneously opening glycine receptor channels. J Neurochem 82: 1343–1351, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 33 Beckstead MJ, Weiner JL, Eger EI II, Gong DH, and Mihic SJ. Glycine and γ-aminobutyric acidA receptor function is enhanced by inhaled drugs of abuse. Mol Pharmacol 57: 1199–1205, 2000.
    PubMed | ISI | Google Scholar
  • 34 Beg AA and Jorgensen EM. EXP-1 is an excitatory GABA-gated cation channel. Nat Neurosci 6: 1145–1152, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 35 Belelli D, Pistis M, Peters JA, and Lambert JJ. The interaction of general anaesthetics and neurosteroids with GABAA and glycine receptors. Neurochem Int 34: 447–452, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 36 Belelli I, Pistis I, Peters JA, and Lambert JJ. General anaesthetic action at transmitter-gated inhibitory amino acid receptors. Trends Pharmacol Sci 20: 496–502, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 37 Bera AK, Chatav M, and Akabas MH. GABAA receptor M2-M3 loop secondary structure and changes in accessibility during channel gating. J Biol Chem 277: 43002–43010, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 38 Bianchi MT, Haas KF, and MacDonald RL. Structural determinants of fast desensitization and desensitization-deactivation coupling in GABAA receptors. J Neurosci 21: 1127–1136, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 39 Birinyi A, Parker D, Antal M, and Shupliakov O. Zinc co-localizes with GABA and glycine in synapses in the lamprey spinal cord. J Comp Neurol 433: 208–221, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 40 Blanton MP and Cohen JB. Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications. Biochemistry 33: 2859–2872, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 41 Blanton MP, Dangott LJ, Raja SK, Lala AK, and Cohen JB. Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound [3H]diazofluorene. J Biol Chem 273: 8659–8668, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 42 Bloomenthal AB, Goldwater E, Pritchett DB, and Harrison NL. Biphasic modulation of the strychnine-sensitive glycine receptor by Zn2+. Mol Pharmacol 46: 1156–1159, 1994.
    PubMed | ISI | Google Scholar
  • 43 Bohlhalter S, Mohler H, and Fritschy JM. Inhibitory neurotransmission in rat spinal cord: co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining. Brain Res 642: 59–69, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 44 Boileau AJ and Czajkowski C. Identification of transduction elements for benzodiazepine modulation of the GABAA receptor: three residues are required for allosteric coupling. J Neurosci 19: 10213–10220, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 45 Bormann J, Hamill OP, and Sakmann B. Mechanism of anion permeation through channels gated by glycine and γ-aminobutyric acid in mouse cultured spinal neurones. J Physiol 385: 243–286, 1987.
    Crossref | PubMed | ISI | Google Scholar
  • 46 Bormann J, Rundstrom N, Betz H, and Langosch D. Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers. EMBO J 12: 3729–3737, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 47 Bouzat C, Gumilar F, del Carmen Esandi M, and Sine SM. Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor. Biophys J 82: 1920–1929, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 48 Braestrup C, Nielsen M, and Krogsgaard-Larsen P. Glycine antagonists structurally related to 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol inhibit binding of [3H]strychnine to rat brain membranes. J Neurochem 47: 691–696, 1986.
    PubMed | ISI | Google Scholar
  • 49 Bray C, Son JH, Kumar P, Harris JD, and Meizel S. A role for the human sperm glycine receptor/Cl channel in the acrosome reaction initiated by recombinant ZP3. Biol Reprod 66: 91–97, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 50 Breitinger HG and Becker CM. Statistical coassembly of glycine receptor α1 wildtype and the hyperekplexia mutant α1(P250T) in HEK 293 cells: impaired channel function is not dominant in the recombinant system. Neurosci Lett 331: 21–24, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 51 Breitinger HG, Villmann C, Becker K, and Becker CM. Opposing effects of molecular volume and charge at the hyperekplexia site α1(P250) govern glycine receptor activation and desensitization. J Biol Chem 276: 29657–29663, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 52 Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, and Sixma TK. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411: 269–276, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 53 Brune W, Weber RG, Saul B, von Knebel Doeberitz M, Grond-Ginsbach C, Kellerman K, Meinck HM, and Becker CM. A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors. Am J Hum Genet 58: 989–997, 1996.
    PubMed | ISI | Google Scholar
  • 54 Buckwalter MS, Cook SA, Davisson MT, White WF, and Camper SA. A frameshift mutation in the mouse α1 glycine receptor gene (Glra1) results in progressive neurological symptoms and juvenile death. Hum Mol Genet 3: 2025–2030, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 55 Buttner C, Sadtler S, Leyendecker A, Laube B, Griffon N, Betz H, and Schmalzing G. Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors. J Biol Chem 276: 42978–42985, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 56 Campos-Caro A, Sala S, Ballesta JJ, Vicente-Agullo F, Criado M, and Sala F. A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors. Proc Natl Acad Sci USA 93: 6118–6123, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 57 Caraiscos VB, Mihic SJ, MacDonald JF, and Orser BA. Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human α1β glycine receptors. J Physiol 539: 495–502, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 58 Cascio M, Shenkel S, Grodzicki RL, Sigworth FJ, and Fox RO. Functional reconstitution and characterization of recombinant human α1-glycine receptors. J Biol Chem 276: 20981–20988, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 59 Celentano JJ, Gibbs TT, and Farb DH. Ethanol potentiates GABA- and glycine-induced chloride currents in chick spinal cord neurons. Brain Res 455: 377–380, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 60 Changeux JP and Edelstein SJ. Allosteric receptors after 30 years. Neuron 21: 959–980, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 61 Charnet P, Labarca C, Leonard RJ, Vogelaar NJ, Czyzyk L, Gouin A, Davidson N, and Lester HA. An open-channel blocker interacts with adjacent turns of α-helices in the nicotinic acetylcholine receptor. Neuron 4: 87–95, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 62 Chatterton JE, Awobuluyi M, Premkumar LS, Takahashi H, Talantova M, Shin Y, Cui J, Tu S, Sevarino KA, Nakanishi N, Tong G, Lipton SA, and Zhang D. Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 415: 793–798, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 63 Chattipakorn SC and McMahon LL. Pharmacological characterization of glycine-gated chloride currents recorded in rat hippocampal slices. J Neurophysiol 87: 1515–1525, 2002.
    Link | ISI | Google Scholar
  • 64 Chen Z, Dillon GH, and Huang R. Molecular determinants of proton modulation of glycine receptors. J Biol Chem 279: 876–883, 2004.
    Crossref | PubMed | ISI | Google Scholar
  • 65 Chesler M. Regulation and modulation of pH in the brain. Physiol Rev 83: 1183–1221, 2003.
    Link | ISI | Google Scholar
  • 66 Chesnoy-Marchais D. Potentiation of chloride responses to glycine by three 5-HT3 antagonists in rat spinal neurones. Br J Pharmacol 118: 2115–2125, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 67 Chesnoy-Marchais D. Mode of action of ICS 205,930, a novel type of potentiator of responses to glycine in rat spinal neurones. Br J Pharmacol 126: 801–809, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 68 Chesnoy-Marchais D. Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neurons. Eur J Neurosci 17: 681–691, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 69 Chesnoy-Marchais D and Cathala L. Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons. Eur J Neurosci 13: 2195–2204, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 70 Chesnoy-Marchais D, Levi S, and Acher F. Glycinergic potentiation by some 5-HT3 receptor antagonists: insight into selectivity. Eur J Pharmacol 402: 205–213, 2000.
    PubMed | ISI | Google Scholar
  • 71 Choe S, Stevens CF, and Sullivan JM. Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation. Proc Natl Acad Sci USA 92: 12046–12049, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 72 Colquhoun D. Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125: 924–947, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 73 Corringer PJ, Bertrand S, Galzi JL, Devillers-Thiery A, Changeux JP, and Bertrand D. Mutational analysis of the charge selectivity filter of the α7 nicotinic acetylcholine receptor. Neuron 22: 831–843, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 74 Corringer PJ, Le Novere N, and Changeux JP. Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40: 431–458, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 75 Croxen R, Newland C, Beeson D, Oosterhuis H, Chauplannaz G, Vincent A, and Newsom-Davis J. Mutations in different functional domains of the human muscle acetylcholine receptor α subunit in patients with the slow-channel congenital myasthenic syndrome. Hum Mol Genet 6: 767–774, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 76 Cruz-Martin A, Mercado JL, Rojas LV, McNamee MG, and Lasalde-Dominicci JA. Tryptophan substitutions at lipid-exposed positions of the γ M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor. J Membr Biol 183: 61–70, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 77 Cui J, Ma YP, Lipton SA, and Pan ZH. Glycine receptors and glycinergic synaptic input at the axon terminals of mammalian retinal rod bipolar cells. J Physiol 553: 895–909, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 78 Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LH, Schaeffer JM, and Arena JP. Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371: 707–711, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 79 Curtis DR and Duggan AW. The depression of spinal inhibition by morphine. Agents Actions 1: 14–19, 1969.
    Crossref | PubMed | Google Scholar
  • 80 Curtis DR, Hosli L, and Johnston GA. Inhibition of spinal neurons by glycine. Nature 215: 1502–1503, 1967.
    Crossref | PubMed | ISI | Google Scholar
  • 81 Curtis DR, Hosli L, and Johnston GA. A pharmacological study of the depression of spinal neurones by glycine and related amino acids. Exp Brain Res 6: 1–18, 1968.
    Crossref | PubMed | ISI | Google Scholar
  • 82 Curtis DR, Hosli L, Johnston GA, and Johnston IH. The hyperpolarization of spinal motoneurones by glycine and related amino acids. Exp Brain Res 5: 235–258, 1968.
    Crossref | PubMed | ISI | Google Scholar
  • 83 Cymes GD, Grosman C, and Auerbach A. Structure of the transition state of gating in the acetylcholine receptor channel pore: a phi-value analysis. Biochemistry 41: 5548–5555, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 84 Daniels S and Roberts RJ. Post-synaptic inhibitory mechanisms of anaesthesia: glycine receptors. Toxicol Lett 100–101: 71–76, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 85 Davies DL, Trudell JR, Mihic SJ, Crawford DK, and Alkana RL. Ethanol potentiation of glycine receptors expressed in Xenopus oocytes antagonized by increased atmospheric pressure. Alcohol Clin Exp Res 27: 743–755, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 86 Davies PA, Wang W, Hales TG, and Kirkness EF. A novel class of ligand-gated ion channel is activated by Zn2+. J Biol Chem 278: 712–717, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 87 Dawson GR, Wafford KA, Smith A, Marshall GR, Bayley PJ, Schaeffer JM, Meinke PT, and McKernan RM. Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the γ-aminobutyric acidA receptor. J Pharmacol Exp Ther 295: 1051–1060, 2000.
    PubMed | ISI | Google Scholar
  • 88 Del Giudice EM, Coppola G, Bellini G, Cirillo G, Scuccimarra G, and Pascotto A. A mutation (V260M) in the middle of the M2 pore-lining domain of the glycine receptor causes hereditary hyperekplexia. Eur J Hum Genet 9: 873–876, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 89 De Rosa MJ, Rayes D, Spitzmaul G, and Bouzat C. Nicotinic receptor M3 transmembrane domain: position 8′ contributes to channel gating. Mol Pharmacol 62: 406–414, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 90 De Saint Jan D, David-Watine B, Korn H, and Bregestovski P. Activation of human α1 and α2 homomeric glycine receptors by taurine and GABA. J Physiol 535: 741–755, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 91 Devignot V, Prado de Carvalho L, Bregestovski P, and Goblet C. A novel glycine receptor αZ1 subunit variant in the zebrafish brain. Neuroscience 122: 449–457, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 92 Dibas MI, Gonzales EB, Das P, Bell-Horner CL, and Dillon GH. Identification of a novel residue within the second transmembrane domain that confers use-facilitated block by picrotoxin in glycine α1 receptors. J Biol Chem 277: 9112–9117, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 93 Dieudonne S. Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci USA 92: 1441–1445, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 94 Dingledine R, Borges K, Bowie D, and Traynelis SF. The glutamate receptor ion channels. Pharmacol Rev 51: 7–61, 1999.
    PubMed | ISI | Google Scholar
  • 95 Doble A. The pharmacology and mechanism of action of riluzole. Neurology 47: S233–S241, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 96 Doi A, Kishimoto K, and Ishibashi H. Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat. Brain Res 816: 424–430, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 97 Downie DL, Hall AC, Lieb WR, and Franks NP. Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes. Br J Pharmacol 118: 493–502, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 98 Drameh P, Richards F, Cross C, Etya'ale D, and Kassalow J. Ten years of NGDO action against river blindness. Trends Parasitol 18: 378, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 99 Du JL and Yang XL. Glycinergic synaptic transmission to bullfrog retinal bipolar cells is input-specific. Neuroscience 113: 779–784, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 100 Dumoulin A, Triller A, and Dieudonne S. IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21: 6045–6057, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 101 Elmslie FV, Hutchings SM, Spencer V, Curtis A, Covanis T, Gardiner RM, and Rees M. Analysis of GLRA1 in hereditary and sporadic hyperekplexia: a novel mutation in a family cosegregating for hyperekplexia and spastic paraparesis. J Med Genet 33: 435–436, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 102 Engblom AC, Carlson BX, Olsen RW, Schousboe A, and Kristiansen U. Point mutation in the first transmembrane region of the beta 2 subunit of the γ-aminobutyric acid type A receptor alters desensitization kinetics of γ-aminobutyric acid- and anesthetic-induced channel gating. J Biol Chem 277: 17438–17447, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 103 Engel SR and Grant KA. Neurosteroids and behavior. Int Rev Neurobiol 46: 321–348, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 104 England PM, Zhang Y, Dougherty DA, and Lester HA. Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. Cell 96: 89–98, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 105 Enz R and Bormann J. Expression of glycine receptor subunits and gephyrin in single bipolar cells of the rat retina. Vis Neurosci 12: 501–507, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 106 Etter A, Cully DF, Liu KK, Reiss B, Vassilatis DK, Schaeffer JM, and Arena JP. Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore. J Neurochem 72: 318–326, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 107 Fatima-Shad K and Barry PH. Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. Proc R Soc Lond B Biol Sci 253: 69–75, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 108 Ferragamo MJ, Golding NL, and Oertel D. Synaptic inputs to stellate cells in the ventral cochlear nucleus. J Neurophysiol 79: 51–63, 1998.
    Link | ISI | Google Scholar
  • 109 Ffrench-Constant RH, Rocheleau TA, Steichen JC, and Chalmers AE. A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363: 449–451, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 110 Filatov GN and White MM. The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol Pharmacol 48: 379–384, 1995.
    PubMed | ISI | Google Scholar
  • 111 Findlay GS, Phelan R, Roberts MT, Homanics GE, Bergeson SE, Lopreato GF, Mihic SJ, Blednov YA, and Harris RA. Glycine receptor knock-in mice and hyperekplexia-like phenotypes: comparisons with the null mutant. J Neurosci 23: 8051–8059, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 112 Findlay GS, Wick MJ, Mascia MP, Wallace D, Miller GW, Harris RA, and Blednov YA. Transgenic expression of a mutant glycine receptor decreases alcohol sensitivity of mice. J Pharmacol Exp Ther 300: 526–534, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 113 Fisher JL and MacDonald RL. The role of an α subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations. J Neurosci 18: 2944–2953, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 114 Flint AC, Liu X, and Kriegstein AR. Nonsynaptic glycine receptor activation during early neocortical development. Neuron 20: 43–53, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 115 Florman HM, Arnoult C, Kazam IG, Li C, and O'Toole CM. A perspective on the control of mammalian fertilization by egg-activated ion channels in sperm: a tale of two channels. Biol Reprod 59: 12–16, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 116 Franks NP, Dickinson R, de Sousa SL, Hall AC, and Lieb WR. How does xenon produce anaesthesia? Nature 396: 324, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 117 Franks NP and Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 367: 607–614, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 118 Franks NP and Lieb WR. A serious target for laughing gas. Nat Med 4: 383–384, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 119 Frech MJ, Perez-Leon J, Wassle H, and Backus KH. Characterization of the spontaneous synaptic activity of amacrine cells in the mouse retina. J Neurophysiol 86: 1632–1643, 2001.
    Link | ISI | Google Scholar
  • 120 Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 31: 145–238, 1989.
    Crossref | PubMed | ISI | Google Scholar
  • 121 Friauf E, Hammerschmidt B, and Kirsch J. Development of adult-type inhibitory glycine receptors in the central auditory system of rats. J Comp Neurol 385: 117–134, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 122 Froh M, Thurman RG, and Wheeler MD. Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes. Am J Physiol Gastrointest Liver Physiol 283: G856–G863, 2002.
    Link | ISI | Google Scholar
  • 123 Fucile S, De Saint Jan D, David-Watine B, Korn H, and Bregestovski P. Comparison of glycine and GABA actions on the zebrafish homomeric glycine receptor. J Physiol 517: 369–383, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 124 Fucile S, De Saint Jan D, de Carvalho LP, and Bregestovski P. Fast potentiation of glycine receptor channels of intracellular calcium in neurons and transfected cells. Neuron 28: 571–583, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 125 Fujita M, Sato K, Sato M, Inoue T, Kozuka T, and Tohyama M. Regional distribution of the cells expressing glycine receptor beta subunit mRNA in the rat brain. Brain Res 560: 23–37, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 126 Galzi JL, Devillers-Thiery A, Hussy N, Bertrand S, Changeux JP, and Bertrand D. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 359: 500–505, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 127 Geiman EJ, Zheng W, Fritschy JM, and Alvarez FJ. Glycine and GABAA receptor subunits on Renshaw cells: relationship with presynaptic neurotransmitters and postsynaptic gephyrin clusters. J Comp Neurol 444: 275–289, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 128 Gentet LJ and Clements JD. Binding site stoichiometry and the effects of phosphorylation on human α1 homomeric glycine receptors. J Physiol 544: 97–106, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 129 Giacoia GP and Ryan SG. Hyperekplexia associated with apnea and sudden infant death syndrome. Arch Pediatr Adolesc Med 148: 540–543, 1994.
    Crossref | PubMed | Google Scholar
  • 130 Gisselmann G, Pusch H, Hovemann BT, and Hatt H. Two cDNAs coding for histamine-gated ion channels in D. melanogaster. Nat Neurosci 5: 11–12, 2002.
    Crossref | Google Scholar
  • 131 Glusker JP. Structural aspects of metal liganding to functional groups in proteins. Adv Protein Chem 42: 1–76, 1991.
    Crossref | PubMed | Google Scholar
  • 132 Goldinger A, Muller WE, and Wollert U. Inhibition of glycine and GABA receptor binding by several opiate agonists and antagonists. Gen Pharmacol 12: 477–479, 1981.
    Crossref | PubMed | Google Scholar
  • 133 Gomeza J, Ohno K, Hulsmann S, Armsen W, Eulenburg V, Richter DW, Laube B, and Betz H. Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40: 797–806, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 134 Gorne-Tschelnokow U, Strecker A, Kaduk C, Naumann D, and Hucho F. The transmembrane domains of the nicotinic acetylcholine receptor contain α-helical and beta structures. EMBO J 13: 338–341, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 135 Graham BA, Schofield PR, Sah P, and Callister RJ. Altered inhibitory synaptic transmission in superficial dorsal horn neurones in spastic and oscillator mice. J Physiol 551: 905–916, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 136 Greferath U, Brandstatter JH, Wassle H, Kirsch J, Kuhse J, and Grunert U. Differential expression of glycine receptor subunits in the retina of the rat: a study using immunohistochemistry and in situ hybridization. Vis Neurosci 11: 721–729, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 137 Grenningloh G, Pribilla I, Prior P, Multhaup G, Beyreuther K, Taleb O, and Betz H. Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor. Neuron 4: 963–970, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 138 Grenningloh G, Rienitz A, Schmitt B, Methfessel C, Zensen M, Beyreuther K, Gundelfinger ED, and Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 328: 215–220, 1987.
    Crossref | PubMed | ISI | Google Scholar
  • 139 Grewer C. Investigation of the α1-glycine receptor channel-opening kinetics in the submillisecond time domain. Biophys J 77: 727–738, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 140 Griffon N, Buttner C, Nicke A, Kuhse J, Schmalzing G, and Betz H. Molecular determinants of glycine receptor subunit assembly. EMBO J 18: 4711–4721, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 141 Grosman C, Salamone FN, Sine SM, and Auerbach A. The extracellular linker of muscle acetylcholine receptor channels is a gating control element. J Gen Physiol 116: 327–340, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 142 Grosman C, Zhou M, and Auerbach A. Mapping the conformational wave of acetylcholine receptor channel gating. Nature 403: 773–776, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 143 Grunert U. Distribution of GABA and glycine receptors on bipolar and ganglion cells in the mammalian retina. Microsc Res Tech 50: 130–140, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 144 Grunert U and Ghosh KK. Midget and parasol ganglion cells of the primate retina express the α1 subunit of the glycine receptor. Vis Neurosci 16: 957–966, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 145 Grunert U and Wassle H. Immunocytochemical localization of glycine receptors in the mammalian retina. J Comp Neurol 335: 523–537, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 146 Grunert U and Wassle H. Glycine receptors in the rod pathway of the macaque monkey retina. Vis Neurosci 13: 101–115, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 147 Gundlach AL, Dodd PR, Grabara CS, Watson WE, Johnston GA, Harper PA, Dennis JA, and Healy PJ. Deficit of spinal cord glycine/strychnine receptors in inherited myoclonus of Poll Hereford calves. Science 241: 1807–1810, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 148 Gundlach AL, Kortz G, Burazin TC, Madigan J, and Higgins RJ. Deficit of inhibitory glycine receptors in spinal cord from Peruvian Pasos: evidence for an equine form of inherited myoclonus. Brain Res 628: 263–270, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 149 Gunthorpe MJ and Lummis SC. Conversion of the ion selectivity of the 5-HT3a receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily. J Biol Chem 276: 10977–10983, 2001.
    Crossref | ISI | Google Scholar
  • 150 Gurley D, Amin J, Ross PC, Weiss DS, and White G. Point mutations in the M2 region of the α, β, or γ subunit of the GABAA channel that abolish block by picrotoxin. Receptors Channels 3: 13–20, 1995.
    PubMed | Google Scholar
  • 151 Han NL, Haddrill JL, and Lynch JW. Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the β-amino acid agonist, taurine. J Neurochem 79: 636–647, 2001.
    PubMed | ISI | Google Scholar
  • 152 Han Y and Wu SM. Modulation of glycine receptors in retinal ganglion cells by zinc. Proc Natl Acad Sci USA 96: 3234–3238, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 153 Han Y, Zhang J, and Slaughter MM. Partition of transient and sustained inhibitory glycinergic input to retinal ganglion cells. J Neurosci 17: 3392–3400, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 154 Handford CA, Lynch JW, Baker E, Webb GC, Ford JH, Sutherland GR, and Schofield PR. The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes. Brain Res 35: 211–219, 1996.
    Crossref | Google Scholar
  • 155 Harrison NL, Kugler JL, Jones MV, Greenblatt EP, and Pritchett DB. Positive modulation of human γ-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane. Mol Pharmacol 44: 628–632, 1993.
    PubMed | ISI | Google Scholar
  • 156 Hartenstein B, Schenkel J, Kuhse J, Besenbeck B, Kling C, Becker CM, Betz H, and Weiher H. Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice. EMBO J 15: 1275–1282, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 157 Harvey RJ, Schmieden V, Von Holst A, Laube B, Rohrer H, and Betz H. Glycine receptors containing the α4 subunit in the embryonic sympathetic nervous system, spinal cord and male genital ridge. Eur J Neurosci 12: 994–1001, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 158 Harvey RJ, Thomas P, James CH, Wilderspin A, and Smart TG. Identification of an inhibitory Zn2+ binding site on the human glycine receptor α1 subunit. J Physiol 520: 53–64, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 159 Haverkamp S, Muller U, Harvey K, Harvey RJ, Betz H, and Wassle H. Diversity of glycine receptors in the mouse retina: localization of the α3 subunit. J Comp Neurol 465: 524–539, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 160 Healy PJ, Harper PA, and Dennis JA. Diagnosis of neuraxial oedema in calves. Aust Vet J 63: 95–96, 1986.
    Crossref | PubMed | ISI | Google Scholar
  • 161 Healy PJ, Pierce KD, Dennis JD, Windsor PA, and Schofield PR. Bovine myoclonus: model of human hyperekplexia (startle disease). Movement Disorders 17: 743–747, 2002.
    Crossref | Google Scholar
  • 162 Hershenson FM, Prodan KA, Kochman RL, Bloss JL, and Mackerer CR. Synthesis of beta-spiro[pyrrolidinoindolines], their binding to the glycine receptor, and in vivo biological acitivity. J Med Chem 20: 1448–1451, 1977.
    Crossref | PubMed | ISI | Google Scholar
  • 163 Hosie AM, Akagi H, Ishida M, and Shinozaki H. Actions of 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) on cloned glycine receptors. Br J Pharmacol 126: 1230–1236, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 164 Howell GA, Welch MG, and Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308: 736–738, 1984.
    Crossref | PubMed | ISI | Google Scholar
  • 165 Huang RQ and Dillon GH. Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor. Neuropharmacology 39: 2195–2204, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 166 Humeny A, Bonk T, Becker K, Jafari-Boroujerdi M, Stephani U, Reuter K, and Becker CM. A novel recessive hyperekplexia allele GLRA1 (S231R): genotyping by MALDI-TOF mass spectrometry and functional characterisation as a determinant of cellular glycine receptor trafficking. Eur J Hum Genet 10: 188–196, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 167 Ikejima K, Qu W, Stachlewitz RF, and Thurman RG. Kupffer cells contain a glycine-gated chloride channel. Am J Physiol Gastrointest Liver Physiol 272: G1581–G1586, 1997.
    Link | ISI | Google Scholar
  • 168 Imoto K, Busch C, Sakmann B, Mishina M, Konno T, Nakai J, Bujo H, Mori Y, Fukuda K, and Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335: 645–648, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 169 Imoto K, Methfessel C, Sakmann B, Mishina M, Mori Y, Konno T, Fukuda K, Kurasaki M, Bujo H, Fujita Y, and Numa S. Location of a δ-subunit region determining ion transport through the acetylcholine receptor channel. Nature 324: 670–674, 1986.
    Crossref | PubMed | ISI | Google Scholar
  • 170 Ivic L, Sands TT, Fishkin N, Nakanishi K, Kriegstein AR, and Stromgaard K. Terpene trilactones from Ginkgo biloba are antagonists of cortical glycine and GABAA receptors. J Biol Chem 278: 49279–49285, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 171 Jenkins A, Andreasen A, Trudell JR, and Harrison NL. Tryptophan scanning mutagenesis in TM4 of the GABAA receptor α1 subunit: implications for modulation by inhaled anesthetics and ion channel structure. Neuropharmacology 43: 669–678, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 172 Jeong HJ, Jang IS, Moorhouse AJ, and Akaike N. Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons. J Physiol 550: 373–383, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 173 Jiang ZL and Ye JH. Protein kinase C epsilon is involved in ethanol potentiation of glycine-gated Cl current in rat neurons of ventral tegmental area. Neuropharmacology 44: 493–502, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 174 Jonas P, Bischofberger J, and Sandkuhler J. Corelease of two fast neurotransmitters at a central synapse. Science 281: 419–424, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 175 Jones MV and Westbrook GL. The impact of receptor desensitization on fast synaptic transmission. Trends Neurosci 19: 96–101, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 176 Kalloniatis M and Marc RE. Interplexiform cells of the goldfish retina. J Comp Neurol 297: 340–358, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 177 Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294: 1030–1038, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 178 Kane NS, Hirschberg B, Qian S, Hunt D, Thomas B, Brochu R, Ludmerer SW, Zheng Y, Smith M, Arena JP, Cohen CJ, Schmatz D, Warmke J, and Cully DF. Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin. Proc Natl Acad Sci USA 97: 13949–13954, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 179 Karlin A. Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3: 102–114, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 180 Karlin A and Akabas MH. Substituted-cysteine accessibility method. Methods Enzymol 293: 123–145, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 181 Kash TL, Jenkins A, Kelley JC, Trudell JR, and Harrison NL. Coupling of agonist binding to channel gating in the GABAA receptor. Nature 421: 272–275, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 182 Kazuyoshi K. Glycine receptors and glycinergic synaptic transmission in the deep cerebellar nuclei of the rat: a patch-clamp study. J Neurophysiol 90: 3490–3500, 2003.
    Link | ISI | Google Scholar
  • 183 Kelley SP, Dunlop JI, Kirkness EF, Lambert JJ, and Peters JA. A cytoplasmic region determines single-channel conductance in 5-HT3 receptors. Nature 424: 321–324, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 184 Kelly D, Zhong Z, Wheeler MD, Li X, Froh M, Schemmer P, Yin M, Bunzendaul H, Bradford B, and Lemasters JJ. l-Glycine: a novel anti-inflammatory, immunomodulatory, and cytoprotective agent. Curr Opin Clin Nutr Metab Care 6: 229–240, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 185 Keramidas A, Moorhouse AJ, French CR, Schofield PR, and Barry PH. M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective. Biophys J 79: 247–259, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 186 Keramidas A, Moorhouse AJ, Pierce KD, Schofield PR, and Barry PH. Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity. J Gen Physiol 119: 393–410, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 187 Kingsmore SF, Giros B, Suh D, Bieniarz M, Caron MG, and Seldin MF. Glycine receptor beta-subunit gene mutation in spastic mouse associated with LINE-1 element insertion. Nat Genet 7: 136–141, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 188 Kirsch J and Betz H. The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton. J Neurosci 15: 4148–4156, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 189 Kneussel M and Betz H. Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends Neurosci 23: 429–435, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 190 Kneussel M and Betz H. Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations. J Physiol 525: 1–9, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 191 Kneussel M, Hermann A, Kirsch J, and Betz H. Hydrophobic interactions mediate binding of the glycine receptor β-subunit to gephyrin. J Neurochem 72: 1323–1326, 1999.
    PubMed | ISI | Google Scholar
  • 192 Kondratskaya EL, Lishko PV, Chatterjee SS, and Krishtal OA. BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel. Neurochem Int 40: 647–653, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 193 Koshland DE Jr, Nemethy G, and Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5: 365–385, 1966.
    Crossref | PubMed | ISI | Google Scholar
  • 194 Kotak VC, Korada S, Schwartz IR, and Sanes DH. A developmental shift from GABAergic to glycinergic transmission in the central auditory system. J Neurosci 18: 4646–4655, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 195 Koulen P, Sassoe-Pognetto M, Grunert U, and Wassle H. Selective clustering of GABAA and glycine receptors in the mammalian retina. J Neurosci 16: 2127–2140, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 196 Krasowski MD and Harrison NL. General anaesthetic actions on ligand-gated ion channels. Cell Mol Life Sci 55: 1278–1303, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 197 Krause RM, Buisson B, Bertrand S, Corringer PJ, Galzi JL, Changeux JP, and Bertrand D. Ivermectin: a positive allosteric effector of the α7 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 53: 283–294, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 198 Krusek J and Zemkova H. Effect of ivermectin on γ-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones. Eur J Pharmacol 259: 121–128, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 199 Kuhse J, Kuryatov A, Maulet Y, Malosio ML, Schmieden V, and Betz H. Alternative splicing generates two isoforms of the α2 subunit of the inhibitory glycine receptor. FEBS Lett 283: 73–77, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 200 Kuhse J, Laube B, Magalei D, and Betz H. Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron 11: 1049–1056, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 201 Kuhse J, Schmieden V, and Betz H. Identification and functional expression of a novel ligand binding subunit of the inhibitory glycine receptor. J Biol Chem 265: 22317–22320, 1990.
    PubMed | ISI | Google Scholar
  • 202 Kuhse J, Schmieden V, and Betz H. A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron 5: 867–873, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 203 Kumamoto E and Murata Y. Glycine current in rat septal cholinergic neuron in culture: monophasic positive modulation by Zn2+. J Neurophysiol 76: 227–241, 1996.
    Link | ISI | Google Scholar
  • 204 Kusama T, Wang JB, Spivak CE, and Uhl GR. Mutagenesis of the GABA rho1 receptor alters agonist affinity and channel gating. Neuroreport 5: 1209–1212, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 205 Kwok JB, Raskin S, Morgan G, Antoniuk SA, Bruk I, and Schofield PR. Mutations in the glycine receptor α1 subunit (GLRA1) gene in hereditary hyperekplexia pedigrees: evidence for non-penetrance of mutation Y279C. J Med Genet 38: E17, 2001.
    Crossref | PubMed | Google Scholar
  • 206 Labarca C, Nowak MW, Zhang H, Tang L, Deshpande P, and Lester HA. Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature 376: 514–516, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 207 Lambert JJ, Harney SC, Belelli D, and Peters JA. Neurosteroid modulation of recombinant and synaptic GABAA receptors. Int Rev Neurobiol 46: 177–205, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 208 Langosch D, Laube B, Rundstrom N, Schmieden V, Bormann J, and Betz H. Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia. EMBO J 13: 4223–4228, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 209 Langosch D, Thomas L, and Betz H. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc Natl Acad Sci USA 85: 7394–7398, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 210 Lapunzina P, Sanchez JM, Cabrera M, Moreno A, Delicado A, De Torres ML, Mori AM, Quero J, and Lopez Pajares I. Hyperekplexia (Startle disease): a novel mutation (S270T) in the M2 domain of the GLRA1 gene and a molecular review of the disorder. Mol Diagn 7: 125–128, 2003.
    PubMed | Google Scholar
  • 211 Laube B. Potentiation of inhibitory glycinergic neurotransmission by Zn2+: a synergistic interplay between presynaptic P2X2 and postsynaptic glycine receptors. Eur J Neurosci 16: 1025–1036, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 212 Laube B, Kuhse J, and Betz H. Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors. J Physiol 522: 215–230, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 213 Laube B, Kuhse J, Rundstrom N, Kirsch J, Schmieden V, and Betz H. Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors. J Physiol 483: 613–619, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 214 Laube B, Langosch D, Betz H, and Schmieden V. Hyperekplexia mutations of the glycine receptor unmask the inhibitory subsite for beta-amino-acids. Neuroreport 6: 897–900, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 215 Laube B, Maksay G, Schemm R, and Betz H. Modulation of glycine receptor function: a novel approach for therapeutic intervention at inhibitory synapses? Trends Pharmacol Sci 23: 519–527, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 216 Law RJ, Forrest LR, Ranatunga KM, La Rocca P, Tieleman DP, and Sansom MS. Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles. Proteins 39: 47–55, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 217 Legendre P. Pharmacological evidence for two types of postsynaptic glycinergic receptors on the Mauthner cell of 52-h-old zebrafish larvae. J Neurophysiol 77: 2400–2415, 1997.
    Link | ISI | Google Scholar
  • 218 Legendre P. A reluctant gating mode of glycine receptor channels determines the time course of inhibitory miniature synaptic events in zebrafish hindbrain neurons. J Neurosci 18: 2856–2870, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 219 Legendre P. The glycinergic inhibitory synapse. Cell Mol Life Sci 58: 760–793, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 220 Legendre P, Muller E, Badiu CI, Meier J, Vannier C, and Triller A. Desensitization of homomeric α1 glycine receptor increases with receptor density. Mol Pharmacol 62: 817–827, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 221 Leite JF, Amoscato AA, and Cascio M. Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor. J Biol Chem 275: 13683–13689, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 222 Leite JF and Cascio M. Probing the topology of the glycine receptor by chemical modification coupled to mass spectrometry. Biochemistry 41: 6140–6148, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 223 Leite JF and Cascio M. Structure of ligand-gated ion channels: critical assessment of biochemical data supports novel topology. Mol Cell Neurosci 17: 777–792, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 224 Leonard RJ, Labarca CG, Charnet P, Davidson N, and Lester HA. Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor. Science 242: 1578–1581, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 225 Lester HA. The permeation pathway of neurotransmitter-gated ion channels. Annu Rev Biophys Biomol Struct 21: 267–292, 1992.
    Crossref | PubMed | Google Scholar
  • 226 Levitan IB. Modulation of ion channels by protein phosphorylation. How the brain works. Adv Second Messenger Phosphoprotein Res 33: 3–22, 1999.
    Crossref | PubMed | Google Scholar
  • 227 Lewis TM, Schofield PR, and McClellan AM. Kinetic determinants of agonist action at the recombinant human glycine receptor. J Physiol 549: 361–374, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 228 Lewis TM, Sivilotti LG, Colquhoun D, Gardiner RM, Schoepfer R, and Rees M. Properties of human glycine receptors containing the hyperekplexia mutation α1(K276E), expressed in Xenopus oocytes. J Physiol 507: 25–40, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 229 Li Y, Wu LJ, Legendre P, and Xu TL. Asymmetric cross-inhibition between GABAA and glycine receptors in rat spinal dorsal horn neurons. J Biol Chem 278: 38637–38645, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 230 Lim R, Alvarez FJ, and Walmsley B. GABA mediates presynaptic inhibition at glycinergic synapses in a rat auditory brainstem nucleus. J Physiol 525: 447–459, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 231 Lin B, Martin PR, Solomon SG, and Grunert U. Distribution of glycine receptor subunits on primate retinal ganglion cells: a quantitative analysis. Eur J Neurosci 12: 4155–4170, 2000.
    PubMed | ISI | Google Scholar
  • 232 Llanos MN, Ronco AM, Aguirre MC, and Meizel S. Hamster sperm glycine receptor: evidence for its presence and involvement in the acrosome reaction. Mol Reprod Dev 58: 205–215, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 233 Lummis SC, Gundlach AL, Johnston GA, Harper PA, and Dodd PR. Increased γ-aminobutyric acid receptor function in the cerebral cortex of myoclonic calves with an hereditary deficit in glycine/strychnine receptors. J Neurochem 55: 421–426, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 234 Lynch JW, Han NL, Haddrill J, Pierce KD, and Schofield PR. The surface accessibility of the glycine receptor M2-M3 loop is increased in the channel open state. J Neurosci 21: 2589–2599, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 235 Lynch JW, Jacques P, Pierce KD, and Schofield PR. Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways. J Neurochem 71: 2159–2168, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 236 Lynch JW, Rajendra S, Barry PH, and Schofield PR. Mutations affecting the glycine receptor agonist transduction mechanism convert the competitive antagonist, picrotoxin, into an allosteric potentiator. J Biol Chem 270: 13799–13806, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 237 Lynch JW, Rajendra S, Pierce KD, Handford CA, Barry PH, and Schofield PR. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO J 16: 110–120, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 238 Machu TK. Colchicine competitively antagonizes glycine receptors expressed in Xenopus oocytes. Neuropharmacology 37: 391–396, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 239 Mackerer CR, Kochman RL, Shen TF, and Hershenson FM. The binding of strychnine and strychnine analogs to synaptic membranes of rat brainstem and spinal cord. J Pharmacol Exp Ther 201: 326–331, 1977.
    PubMed | ISI | Google Scholar
  • 240 Maksay G. Bidirectional allosteric modulation of strychnine-sensitive glycine receptors by tropeines and 5-HT3 serotonin receptor ligands. Neuropharmacology 37: 1633–1641, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 241 Maksay G and Biro T. Dual cooperative allosteric modulation of binding to ionotropic glycine receptors. Neuropharmacology 43: 1087–1098, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 242 Maksay G, Laube B, and Betz H. Selective blocking effects of tropisetron and atropine on recombinant glycine receptors. J Neurochem 73: 802–806, 1999.
    PubMed | ISI | Google Scholar
  • 243 Maksay G, Thompson SA, and Wafford KA. Allosteric modulators affect the efficacy of partial agonists for recombinant GABAA receptors. Br J Pharmacol 129: 1794–1800, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 244 Malosio ML, Grenningloh G, Kuhse J, Schmieden V, Schmitt B, Prior P, and Betz H. Alternative splicing generates two variants of the α1 subunit of the inhibitory glycine receptor. J Biol Chem 266: 2048–2053, 1991.
    PubMed | ISI | Google Scholar
  • 245 Malosio ML, Marqueze-Pouey B, Kuhse J, and Betz H. Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. EMBO J 10: 2401–2409, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 246 Mangin JM, Baloul M, Prado De Carvalho L, Rogister B, Rigo JM, and Legendre P. Kinetics properties of the α2 homo-oligomeric glycine receptor impairs a proper synaptic functioning. J Physiol 553: 369–386, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 247 Mangin JM, Guyon A, Eugene D, Paupardin-Tritsch D, and Legendre P. Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra. J Physiol 542: 685–697, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 248 Marchetti C, Pagnotta S, Donato R, and Nistri A. Inhibition of spinal or hypoglossal motoneurons of the newborn rat by glycine or GABA. Eur J Neurosci 15: 975–983, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 249 Marvizon JC, Vazquez J, Garcia Calvo M, Mayor F Jr, Ruiz Gomez A, Valdivieso F, and Benavides J. The glycine receptor: pharmacological studies and mathematical modeling of the allosteric interaction between the glycine- and strychnine-binding sites. Mol Pharmacol 30: 590–597, 1986.
    PubMed | ISI | Google Scholar
  • 250 Mascia MP, Machu TK, and Harris RA. Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics. Br J Pharmacol 119: 1331–1336, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 251 Mascia MP, Mihic SJ, Valenzuela CF, Schofield PR, and Harris RA. A single amino acid determines differences in ethanol actions on strychnine-sensitive glycine receptors. Mol Pharmacol 50: 402–406, 1996.
    PubMed | ISI | Google Scholar
  • 252 Mascia MP, Trudell JR, and Harris RA. Specific binding sites for alcohols and anesthetics on ligand-gated ion channels. Proc Natl Acad Sci USA 97: 9305–9310, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 253 Mascia MP, Wick MJ, Martinez LD, and Harris RA. Enhancement of glycine receptor function by ethanol: role of phosphorylation. Br J Pharmacol 125: 263–270, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 254 Matzenbach B, Maulet Y, Sefton L, Courtier B, Avner P, Guenet JL, and Betz H. Structural analysis of mouse glycine receptor α subunit genes. Identification and chromosomal localization of a novel variant. J Biol Chem 269: 2607–2612, 1994.
    PubMed | ISI | Google Scholar
  • 255 Meier H and Chai CK. Spastic, an hereditary neurological mutation in the mouse characterized by vertebral arthropathy and leptomeningeal cyst formation. Exp Med Surg 28: 24–38, 1970.
    PubMed | Google Scholar
  • 256 Meier J and Schmieden V. Inhibition of α-subunit glycine receptors by quinoxalines. Neuroreport 14: 1507–1510, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 257 Meizel S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol Reprod 56: 569–574, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 258 Melendrez CS and Meizel S. Immunochemical identification of the glycine receptor/Cl channel in porcine sperm. Biochem Biophys Res Commun 223: 675–678, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 259 Meyer G, Kirsch J, Betz H, and Langosch D. Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron 15: 563–572, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 260 Mihic SJ. Acute effects of ethanol on GABAA and glycine receptor function. Neurochem Int 35: 115–123, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 261 Mihic SJ and Harris RA. Inhibition of rho1 receptor GABAergic currents by alcohols and volatile anesthetics. J Pharmacol Exp Ther 277: 411–416, 1996.
    PubMed | ISI | Google Scholar
  • 262 Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, and Harrison NL. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature 389: 385–389, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 263 Milani N, Dalpra L, del Prete A, Zanini R, and Larizza L. A novel mutation (Gln266→His) in the α1 subunit of the inhibitory glycine-receptor gene (GLRA1) in hereditary hyperekplexia. Am J Hum Genet 58: 420–422, 1996.
    PubMed | ISI | Google Scholar
  • 264 Milani N, Mulhardt C, Weber RG, Lichter P, Kioschis P, Poustka A, and Becker CM. The human glycine receptor beta subunit gene (GLRB): structure, refined chromosomal localization, and population polymorphism. Genomics 50: 341–345, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 265 Miller C. Genetic manipulation of ion channels: a new approach to structure and mechanism. Neuron 2: 1195–1205, 1989.
    Crossref | PubMed | ISI | Google Scholar
  • 266 Miyazawa A, Fujiyoshi Y, Stowell M, and Unwin N. Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall. J Mol Biol 288: 765–786, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 267 Miyazawa A, Fujiyoshi Y, and Unwin N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 424: 949–955, 2003.
    Google Scholar
  • 268 Mohammadi B, Krampfl K, Cetinkaya C, Moschref H, Grosskreutz J, Dengler R, and Bufler J. Kinetic analysis of recombinant mammalian α1 and α1β glycine receptor channels. Eur Biophys J 32: 529–536, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 269 Mohammadi B, Krampfl K, Moschref H, Dengler R, and Bufler J. Interaction of the neuroprotective drug riluzole with GABAA and glycine receptor channels. Eur J Pharmacol 415: 135–140, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 270 Monaghan D. Glycine modulation of NMDA receptors: autoradiographic studies. In: Glycine Neurotransmission, edited by Otterson OP and Storm-Mathisen J. New York: Wiley, 1990.
    Google Scholar
  • 271 Moorhouse AJ, Jacques P, Barry PH, and Schofield PR. The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating. Mol Pharmacol 55: 386–395, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 272 Moorhouse AJ, Keramidas A, Zaykin A, Schofield PR, and Barry PH. Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels. J Gen Physiol 119: 411–425, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 273 Mori M, Gahwiler BH, and Gerber U. Beta-alanine and taurine as endogenous agonists at glycine receptors in rat hippocampus in vitro. J Physiol 539: 191–200, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 274 Moss SJ, Smart TG, Blackstone CD, and Huganir RL. Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation. Science 257: 661–665, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 275 Mulhardt C, Fischer M, Gass P, Simon-Chazottes D, Guenet JL, Kuhse J, Betz H, and Becker CM. The spastic mouse: aberrant splicing of glycine receptor beta subunit mRNA caused by intronic insertion of L1 element. Neuron 13: 1003–1015, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 276 Nevin ST, Cromer BA, Haddrill Jl J, Morton CJ, Parker MW, and Lynch JW. Insights into the structural basis for zinc inhibition of the glycine receptor. J Biol Chem 278: 28985–28992, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 277 Newland CF and Cull-Candy SG. On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol 447: 191–213, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 278 Nguyen L, Malgrange B, Belachew S, Rogister B, Rocher V, Moonen G, and Rigo JM. Functional glycine receptors are expressed by postnatal nestin-positive neural stem/progenitor cells. Eur J Neurosci 15: 1299–1305, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 279 Nicoll RA, Malenka RC, and Kauer JA. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol Rev 70: 513–565, 1990.
    Link | ISI | Google Scholar
  • 280 Nikolic Z, Laube B, Weber RG, Lichter P, Kioschis P, Poustka A, Mulhardt C, and Becker CM. The human glycine receptor subunit α3. Glra3 gene structure, chromosomal localization, and functional characterization of alternative transcripts. J Biol Chem 273: 19708–19714, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 281 Nishizaki T and Ikeuchi Y. Activation of endogenous protein kinase C enhances currents through α1 and α2 glycine receptor channels. Brain Res 687: 214–216, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 282 Noh JH, Choi S, Lee JH, Betz H, Kim JI, Park CS, Lee SM, and Nah SY. Effects of ginsenosides on glycine receptor α1 channels expressed in Xenopus oocytes. Mol Cells 15: 34–39, 2003.
    PubMed | ISI | Google Scholar
  • 283 O'Brien JA and Berger AJ. Cotransmission of GABA and glycine to brain stem motoneurons. J Neurophysiol 82: 1638–1641, 1999.
    Link | ISI | Google Scholar
  • 284 O'Mara M, Barry PH, and Chung SH. A model of the glycine receptor deduced from Brownian dynamics studies. Proc Natl Acad Sci USA 100: 4310–4315, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 285 O'Shea SM and Harrison NL. Arg-274 and Leu-277 of the γ-aminobutyric acid type A receptor α2 subunit define agonist efficacy and potency. J Biol Chem 275: 22764–22768, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 286 Palma E, Fucile S, Barabino B, Miledi R, and Eusebi F. Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains. Proc Natl Acad Sci USA 96: 13421–13426, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 287 Pan ZH, Zhang D, Zhang X, and Lipton SA. Evidence for coassembly of mutant GABAC ρ1 with GABAA γ2S, glycine α1 and glycine α2 receptor subunits in vitro. Eur J Neurosci 12: 3137–3145, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 288 Panicker S, Cruz H, Arrabit C, and Slesinger PA. Evidence for a centrally located gate in the pore of a serotonin-gated ion channel. J Neurosci 22: 1629–1639, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 289 Pascual JM and Karlin A. State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the α subunit. J Gen Physiol 111: 717–739, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 290 Pawson T. Protein modules and signalling networks. Nature 373: 573–580, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 291 Pfeiffer F and Betz H. Solubilization of the glycine receptor from rat spinal cord. Brain Res 226: 273–279, 1981.
    Crossref | PubMed | ISI | Google Scholar
  • 292 Pfeiffer F, Graham D, and Betz H. Purification by affinity chromatography of the glycine receptor of rat spinal cord. J Biol Chem 257: 9389–9393, 1982.
    PubMed | ISI | Google Scholar
  • 293 Piechotta K, Weth F, Harvey RJ, and Friauf E. Localization of rat glycine receptor α1 and α2 subunit transcripts in the developing auditory brainstem. J Comp Neurol 438: 336–352, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 294 Pierce KD, Handford CA, Morris R, Vafa B, Dennis JA, Healy PJ, and Schofield PR. A nonsense mutation in the α1 subunit of the inhibitory glycine receptor associated with bovine myoclonus. Mol Cell Neurosci 17: 354–363, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 295 Pistis M, Belelli D, Peters JA, and Lambert JJ. The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study. Br J Pharmacol 122: 1707–1719, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 296 Porter NM, Angelotti TP, Twyman RE, and MacDonald RL. Kinetic properties of α1 β1 γ-aminobutyric acidA receptor channels expressed in Chinese hamster ovary cells: regulation by pentobarbital and picrotoxin. Mol Pharmacol 42: 872–881, 1992.
    PubMed | ISI | Google Scholar
  • 297 Pourcho RG. Neurotransmitters in the retina. Curr Eye Res 15: 797–803, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 298 Pribilla I, Takagi T, Langosch D, Bormann J, and Betz H. The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels. EMBO J 11: 4305–4311, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 299 Prince RJ and Simmonds MA. Steroid modulation of the strychnine-sensitive glycine receptor. Neuropharmacology 31: 201–205, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 300 Prior P, Schmitt B, Grenningloh G, Pribilla I, Multhaup G, Beyreuther K, Maulet Y, Werner P, Langosch D, Kirsch J, and Betz H. Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8: 1161–1170, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 301 Probst A, Cortes R, and Palacios JM. The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [3H]strychnine. Neuroscience 17: 11–35, 1986.
    Crossref | PubMed | ISI | Google Scholar
  • 302 Protti DA, Gerschenfeld HM, and Llano I. GABAergic and glycinergic IPSCs in ganglion cells of rat retinal slices. J Neurosci 17: 6075–6085, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 303 Qian H, Ripps H, Schuette E, and Chappell RL. Responses of small- and large-field bipolar cells to GABA and glycine. Brain Res 893: 273–277, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 304 Qu W, Ikejima K, Zhong Z, Waalkes MP, and Thurman RG. Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells. Am J Physiol Gastrointest Liver Physiol 283: G1249–G1256, 2002.
    Link | ISI | Google Scholar
  • 305 Rajendra S, Lynch JW, Pierce KD, French CR, Barry PH, and Schofield PR. Mutation of an arginine residue in the human glycine receptor transforms β-alanine and taurine from agonists into competitive antagonists. Neuron 14: 169–175, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 306 Rajendra S, Lynch JW, Pierce KD, French CR, Barry PH, and Schofield PR. Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Biol Chem 269: 18739–18742, 1994.
    PubMed | ISI | Google Scholar
  • 307 Rajendra S, Lynch JW, and Schofield PR. The glycine receptor. Pharmacol Ther 73: 121–146, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 308 Rajendra S and Schofield PR. Molecular mechanisms of inherited startle syndromes. Trends Neurosci 18: 80–82, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 309 Rajendra S, Vandenberg RJ, Pierce KD, Cunningham AM, French PW, Barry PH, and Schofield PR. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element. EMBO J 14: 2987–2998, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 310 Rampon C, Luppi PH, Fort P, Peyron C, and Jouvet M. Distribution of glycine-immunoreactive cell bodies and fibers in the rat brain. Neuroscience 75: 737–755, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 311 Rea R, Tijssen MA, Herd C, Frants RR, and Kullmann DM. Functional characterization of compound heterozygosity for GlyRα1 mutations in the startle disease hyperekplexia. Eur J Neurosci 16: 186–196, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 312 Rees MI, Andrew M, Jawad S, and Owen MJ. Evidence for recessive as well as dominant forms of startle disease (hyperekplexia) caused by mutations in the α1 subunit of the inhibitory glycine receptor. Hum Mol Genet 3: 2175–2179, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 313 Rees MI, Harvey K, Ward H, White JH, Evans L, Duguid IC, Hsu CC, Coleman SL, Miller J, Baer K, Waldvogel HJ, Gibbon F, Smart TG, Owen MJ, Harvey RJ, and Snell RG. Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia. J Biol Chem 278: 24688–24696, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 314 Rees MI, Lewis TM, Kwok JB, Mortier GR, Govaert P, Snell RG, Schofield PR, and Owen MJ. Hyperekplexia associated with compound heterozygote mutations in the β-subunit of the human inhibitory glycine receptor (GLRB). Hum Mol Genet 11: 853–860, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 315 Rees MI, Lewis TM, Vafa B, Ferrie C, Corry P, Muntoni F, Jungbluth H, Stephenson JB, Kerr M, Snell RG, Schofield PR, and Owen MJ. Compound heterozygosity and nonsense mutations in the α1-subunit of the inhibitory glycine receptor in hyperekplexia. Hum Genet 109: 267–270, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 316 Reeves DC, Goren EN, Akabas MH, and Lummis SC. Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis. J Biol Chem 276: 42035–42042, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 317 Regan L. Protein design: novel metal-binding sites. Trends Biochem Sci 20: 280–285, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 318 Rick CE, Ye Q, Finn SE, and Harrison NL. Neurosteroids act on the GABAA receptor at sites on the N-terminal side of the middle of TM2. Neuroreport 9: 379–383, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 319 Roos MH, Kwa MS, Veenstra JG, Kooyman FN, and Boersema JH. Molecular aspects of drug resistance in parasitic helminths. Pharmacol Ther 60: 331–336, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 320 Rovira JC, Ballesta JJ, Vicente-Agullo F, Campos-Caro A, Criado M, Sala F, and Sala S. A residue in the middle of the M2-M3 loop of the β4 subunit specifically affects gating of neuronal nicotinic receptors. FEBS Lett 433: 89–92, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 321 Rovira JC, Vicente-Agullo F, Campos-Caro A, Criado M, Sala F, Sala S, and Ballesta JJ. Gating of α3β4 neuronal nicotinic receptor can be controlled by the loop M2-M3 of both α3 and β4 subunits. Pflügers Arch 439: 86–92, 1999.
    PubMed | ISI | Google Scholar
  • 322 Ruiz-Gomez A, Morato E, Garcia-Calvo M, Valdivieso F, and Mayor F Jr. Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor. Biochemistry 29: 7033–7040, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 323 Ruiz-Gomez A, Vaello ML, Valdivieso F, and Mayor F Jr. Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C. J Biol Chem 266: 559–566, 1991.
    PubMed | ISI | Google Scholar
  • 324 Rundstrom N, Schmieden V, Betz H, Bormann J, and Langosch D. Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels. Proc Natl Acad Sci USA 91: 8950–8954, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 325 Russier M, Kopysova IL, Ankri N, Ferrand N, and Debanne D. GABA and glycine co-release optimizes functional inhibition in rat brainstem motoneurons in vitro. J Physiol 541: 123–137, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 326 Ryan SG, Buckwalter MS, Lynch JW, Handford CA, Segura L, Shiang R, Wasmuth JJ, Camper SA, Schofield P, and O'Connell P. A missense mutation in the gene encoding the α1 subunit of the inhibitory glycine receptor in the spasmodic mouse. Nat Genet 7: 131–135, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 327 Ryan SG, Dixon MJ, Nigro MA, Kelts KA, Markand ON, Terry JC, Shiang R, Wasmuth JJ, and O'Connell P. Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q. Am J Hum Genet 51: 1334–1343, 1992.
    PubMed | ISI | Google Scholar
  • 328 Sadtler S, Laube B, Lashub A, Nicke A, Betz H, and Schmalzing G. A basic cluster determines topology of the cytoplasmic M3-M4 loop of the glycine receptor α1 subunit. J Biol Chem 278: 16782–16790, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 329 Sassoe-Pognetto M, Kirsch J, Grunert U, Greferath U, Fritschy JM, Mohler H, Betz H, and Wassle H. Colocalization of gephyrin and GABAA-receptor subunits in the rat retina. J Comp Neurol 357: 1–14, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 330 Sassoe-Pognetto M, Wassle H, and Grunert U. Glycinergic synapses in the rod pathway of the rat retina: cone bipolar cells express the α1 subunit of the glycine receptor. J Neurosci 14: 5131–5146, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 331 Sato K, Zhang JH, Saika T, Sato M, Tada K, and Tohyama M. Localization of glycine receptor α1 subunit mRNA-containing neurons in the rat brain: an analysis using in situ hybridization histochemistry. Neuroscience 43: 381–395, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 332 Sato Y, Son JH, and Meizel S. The mouse sperm glycine receptor/chloride channel: cellular localization and involvement in the acrosome reaction initiated by glycine. J Androl 21: 99–106, 2000.
    PubMed | Google Scholar
  • 333 Sato Y, Son JH, Tucker RP, and Meizel S. The zona pellucida-initiated acrosome reaction: defect due to mutations in the sperm glycine receptor/Cl channel. Dev Biol 227: 211–218, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 334 Saul B, Kuner T, Sobetzko D, Brune W, Hanefeld F, Meinck HM, and Becker CM. Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating. J Neurosci 19: 869–877, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 335 Saul B, Schmieden V, Kling C, Mulhardt C, Gass P, Kuhse J, and Becker CM. Point mutation of glycine receptor α1 subunit in the spasmodic mouse affects agonist responses. FEBS Lett 350: 71–76, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 336 Schmieden V and Betz H. Pharmacology of the inhibitory glycine receptor: agonist and antagonist actions of amino acids and piperidine carboxylic acid compounds. Mol Pharmacol 48: 919–927, 1995.
    PubMed | ISI | Google Scholar
  • 337 Schmieden V, Jezequel S, and Betz H. Novel antagonists of the inhibitory glycine receptor derived from quinolinic acid compounds. Mol Pharmacol 50: 1200–1206, 1996.
    PubMed | ISI | Google Scholar
  • 338 Schmieden V, Kuhse J, and Betz H. Mutation of glycine receptor subunit creates beta-alanine receptor responsive to GABA. Science 262: 256–258, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 339 Schmieden V, Kuhse J, and Betz H. A novel domain of the inhibitory glycine receptor determining antagonist efficacies: further evidence for partial agonism resulting from self-inhibition. Mol Pharmacol 56: 464–472, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 340 Schofield CM, Jenkins A, and Harrison NL. A highly conserved aspartic acid residue in the signature disulfide loop of the α1 subunit is a determinant of gating in the glycine receptor. J Biol Chem 278: 34079–34083, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 341 Shan Q, Haddrill JL, and Lynch JW. A single beta subunit M2 domain residue controls the picrotoxin sensitivity of αβ heteromeric glycine receptor chloride channels. J Neurochem 76: 1109–1120, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 342 Shan Q, Haddrill JL, and Lynch JW. Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J Biol Chem 276: 12556–12564, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 343 Shan Q, Haddrill JL, and Lynch JW. Comparative surface accessibility of a pore-lining threonine residue (T6′) in the glycine and GABAA receptors. J Biol Chem 277: 44845–44853, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 344 Shan Q, Nevin ST, Haddrill JL, and Lynch JW. Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors. J Neurochem 86: 498–507, 2003.
    PubMed | ISI | Google Scholar
  • 345 Shiang R, Ryan SG, Zhu YZ, Fielder TJ, Allen RJ, Fryer A, Yamashita S, O'Connell P, and Wasmuth JJ. Mutational analysis of familial and sporadic hyperekplexia. Ann Neurol 38: 85–91, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 346 Shiang R, Ryan SG, Zhu YZ, Hahn AF, O'Connell P, and Wasmuth JJ. Mutations in the α1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat Genet 5: 351–358, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 347 Singer JH, Talley EM, Bayliss DA, and Berger AJ. Development of glycinergic synaptic transmission to rat brain stem motoneurons. J Neurophysiol 80: 2608–2620, 1998.
    Link | ISI | Google Scholar
  • 348 Sivilotti L and Nistri A. GABA receptor mechanisms in the central nervous system. Prog Neurobiol 36: 35–92, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 349 Smart TG, Xie X, and Krishek BJ. Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Prog Neurobiol 42: 393–341, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 350 Smit AB, Syed NI, Schaap D, van Minnen J, Klumperman J, Kits KS, Lodder H, van der Schors RC, van Elk R, Sorgedrager B, Brejc K, Sixma TK, and Geraerts WP. A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411: 261–268, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 351 Smith AJ, Owens S, and Forsythe ID. Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive. J Physiol 529: 681–698, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 352 Smith SM and McBurney RN. Caesium ions: a glycine-activated channel agonist in rat spinal cord neurones grown in cell culture. Br J Pharmacol 96: 940–948, 1989.
    Crossref | PubMed | ISI | Google Scholar
  • 353 Spencer RH and Rees DC. The α-helix and the organization and gating of channels. Annu Rev Biophys Biomol Struct 31: 207–233, 2002.
    Crossref | PubMed | Google Scholar
  • 354 Spier AD and Lummis SC. The role of tryptophan residues in the 5-hydroxytryptamine3 receptor ligand binding domain. J Biol Chem 275: 5620–5625, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 355 Stein V and Nicoll RA. GABA generates excitement. Neuron 37: 375–378, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 356 Steinbach JH, Bracamontes J, Yu L, Zhang P, and Covey DF. Subunit-specific action of an anticonvulsant thiobutyrolactone on recombinant glycine receptors involves a residue in the M2 membrane-spanning region. Mol Pharmacol 58: 11–17, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 357 Stoffel-Wagner B. Neurosteroid metabolism in the human brain. Eur J Endocrinol 145: 669–679, 2001.
    Crossref | ISI | Google Scholar
  • 358 Supplisson S and Chesnoy-Marchais D. Glycine receptor beta subunits play a critical role in potentiation of glycine responses by ICS-205,930. Mol Pharmacol 58: 763–770, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 359 Suwa H, Saint-Amant L, Triller A, Drapeau P, and Legendre P. High-affinity zinc potentiation of inhibitory postsynaptic glycinergic currents in the zebrafish hindbrain. J Neurophysiol 85: 912–925, 2001.
    Link | ISI | Google Scholar
  • 360 Swope SL, Moss SI, Raymond LA, and Huganir RL. Regulation of ligand-gated ion channels by protein phosphorylation. Adv Second Messenger Phosphoprotein Res 33: 49–78, 1999.
    Crossref | PubMed | Google Scholar
  • 361 Takahashi T, Momiyama A, Hirai K, Hishinuma F, and Akagi H. Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels. Neuron 9: 1155–1161, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 362 Taleb O and Betz H. Expression of the human glycine receptor α1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density. EMBO J 13: 1318–1324, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 363 Tamamizu S, Todd AP, and McNamee MG. Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine. Cell Mol Neurobiol 15: 427–438, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 364 Tao L and Ye JH. Protein kinase C modulation of ethanol inhibition of glycine-activated current in dissociated neurons of rat ventral tegmental area. J Pharmacol Exp Ther 300: 967–975, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 365 Tapia JC and Aguayo LG. Changes in the properties of developing glycine receptors in cultured mouse spinal neurons. Synapse 28: 185–194, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 366 Tapia JC, Aguilar LF, Sotomayor CP, and Aguayo LG. Ethanol affects the function of a neurotransmitter receptor protein without altering the membrane lipid phase. Eur J Pharmacol 354: 239–244, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 367 Thio LL, Shanmugam A, Isenberg K, and Yamada K. Benzodiazepines block α2-containing inhibitory glycine receptors in embryonic mouse hippocampal neurons. J Neurophysiol 90: 89–99, 2003.
    Link | ISI | Google Scholar
  • 368 Tian N, Hwang TN, and Copenhagen DR. Analysis of excitatory and inhibitory spontaneous synaptic activity in mouse retinal ganglion cells. J Neurophysiol 80: 1327–1340, 1998.
    Link | ISI | Google Scholar
  • 369 Todd AJ, Watt C, Spike RC, and Sieghart W. Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord. J Neurosci 16: 974–982, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 370 Triller A, Cluzeaud F, and Korn H. γ-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses. J Cell Biol 104: 947–956, 1987.
    Crossref | PubMed | ISI | Google Scholar
  • 371 Triller A, Cluzeaud F, Pfeiffer F, Betz H, and Korn H. Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J Cell Biol 101: 683–688, 1985.
    Crossref | PubMed | ISI | Google Scholar
  • 372 Tsutsui K, Ukena K, Usui M, Sakamoto H, and Takase M. Novel brain function: biosynthesis and actions of neurosteroids in neurons. Neurosci Res 36: 261–273, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 373 Turecek R and Trussell LO. Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse. Nature 411: 587–590, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 374 Twyman RE and MacDonald RL. Kinetic properties of the glycine receptor main- and sub-conductance states of mouse spinal cord neurones in culture. J Physiol 435: 303–331, 1991.
    Crossref | PubMed | ISI | Google Scholar
  • 375 Umemiya M and Berger AJ. Presynaptic inhibition by serotonin of glycinergic inhibitory synaptic currents in the rat brain stem. J Neurophysiol 73: 1192–1201, 1995.
    Link | ISI | Google Scholar
  • 376 Unwin N. Acetylcholine receptor channel imaged in the open state. Nature 373: 37–43, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 377 Unwin N, Miyazawa A, Li J, and Fujiyoshi Y. Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the α subunits. J Mol Biol 319: 1165–1176, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 378 Vaello ML, Ruiz-Gomez A, Lerma J, and Mayor F Jr. Modulation of inhibitory glycine receptors by phosphorylation by protein kinase C and cAMP-dependent protein kinase. J Biol Chem 269: 2002–2008, 1994.
    PubMed | ISI | Google Scholar
  • 379 Vafa B, Lewis TM, Cunningham AM, Jacques P, Lynch JW, and Schofield PR. Identification of a new ligand binding domain in the α1 subunit of the inhibitory glycine receptor. J Neurochem 73: 2158–2166, 1999.
    PubMed | ISI | Google Scholar
  • 380 Valenzuela CF, Cardoso RA, Wick MJ, Weiner JL, Dunwiddie TV, and Harris RA. Effects of ethanol on recombinant glycine receptors expressed in mammalian cell lines. Alcohol Clin Exp Res 22: 1132–1136, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 381 Vandenberg RJ, French CR, Barry PH, Shine J, and Schofield PR. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor α subunit form the strychnine-binding site. Proc Natl Acad Sci USA 89: 1765–1769, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 382 Vandenberg RJ, Handford CA, and Schofield PR. Distinct agonist- and antagonist-binding sites on the glycine receptor. Neuron 9: 491–496, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 383 Van den Pol AN and Gorcs T. Glycine and glycine receptor immunoreactivity in brain and spinal cord. J Neurosci 8: 472–492, 1988.
    Crossref | PubMed | ISI | Google Scholar
  • 384 Vergouwe MN, Tijssen MA, Peters AC, Wielaard R, and Frants RR. Hyperekplexia phenotype due to compound heterozygosity for GLRA1 gene mutations. Ann Neurol 46: 634–638, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 385 Vogt K, Mellor J, Tong G, and Nicoll R. The actions of synaptically released zinc at hippocampal mossy fiber synapses. Neuron 26: 187–196, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 386 Von Wegerer J, Becker K, Glockenhammer D, Becker CM, Zeilhofer HU, and Swandulla D. Spinal inhibitory synaptic transmission in the glycine receptor mouse mutant spastic. Neurosci Lett 345: 45–48, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 387 Wang F and Imoto K. Pore size and negative charge as structural determinants of permeability in the Torpedo nicotinic acetylcholine receptor channel. Proc R Soc Lond B Biol Sci 250: 11–17, 1992.
    Crossref | ISI | Google Scholar
  • 388 Wang HL, Milone M, Ohno K, Shen XM, Tsujino A, Batocchi AP, Tonali P, Brengman J, Engel AG, and Sine SM. Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating. Nat Neurosci 2: 226–233, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 389 Wang TL, Hackam AS, Guggino WB, and Cutting GR. A single amino acid in γ-aminobutyric acid ρ1 receptors affects competitive and noncompetitive components of picrotoxin inhibition. Proc Natl Acad Sci USA 92: 11751–11755, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 390 Wassle H and Boycott BB. Functional architecture of the mammalian retina. Physiol Rev 71: 447–480, 1991.
    Link | ISI | Google Scholar
  • 391 Wassle H, Koulen P, Brandstatter JH, Fletcher EL, and Becker CM. Glycine and GABA receptors in the mammalian retina. Vision Res 38: 1411–1430, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 392 Watanabe E and Akagi H. Distribution patterns of mRNAs encoding glycine receptor channels in the developing rat spinal cord. Neurosci Res 23: 377–382, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 393 Weaver CD, Partridge JG, Yao TL, Moates JM, Magnuson MA, and Verdoorn TA. Activation of glycine and glutamate receptors increases intracellular calcium in cells derived from the endocrine pancreas. Mol Pharmacol 54: 639–646, 1998.
    PubMed | ISI | Google Scholar
  • 394 Weiner JL, Buhler AV, Whatley VJ, Harris RA, and Dunwiddie TV. Colchicine is a competitive antagonist at human recombinant γ-aminobutyric acidA receptors. J Pharmacol Exp Ther 284: 95–102, 1998.
    PubMed | ISI | Google Scholar
  • 395 Werman R, Davidoff RA, and Aprison MH. Inhibition of motoneurones by iontophoresis of glycine. Nature 214: 681–683, 1967.
    Crossref | PubMed | ISI | Google Scholar
  • 396 Werman R, Davidoff RA, and Aprison MH. Inhibition of glycine on spinal neurons in the cat. J Neurophysiol 31: 81–95, 1968.
    Link | ISI | Google Scholar
  • 397 Whatley VJ, Brozowski SJ, Hadingham KL, Whiting PJ, and Harris RA. Microtubule depolymerization inhibits ethanol-induced enhancement of GABAA responses in stably transfected cells. J Neurochem 66: 1318–1321, 1996.
    PubMed | ISI | Google Scholar
  • 398 Wheeler M, Stachlewitz RF, Yamashina S, Ikejima K, Morrow AL, and Thurman RG. Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J 14: 476–484, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 399 Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye Q, Harrison NL, and Harris RA. Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 95: 6504–6509, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 400 Williams DB and Akabas MH. γ-Aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in γ-aminobutyric acid type A receptors. Biophys J 77: 2563–2574, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 401 Williams DB and Akabas MH. Evidence for distinct conformations of the two α1 subunits in diazepam-bound GABAA receptors. Neuropharmacology 41: 539–545, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 402 Williams DB and Akabas MH. Structural evidence that propofol stabilizes different GABAA receptor states at potentiating and activating concentrations. J Neurosci 22: 7417–7424, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 403 Wilson G and Karlin A. Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method. Proc Natl Acad Sci USA 98: 1241–1248, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 404 Wilson GG and Karlin A. The location of the gate in the acetylcholine receptor channel. Neuron 20: 1269–1281, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 405 Wilson GG, Pascual JM, Brooijmans N, Murray D, and Karlin A. The intrinsic electrostatic potential and the intermediate ring of charge in the acetylcholine receptor channel. J Gen Physiol 115: 93–106, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 406 Wotring VE, Miller TS, and Weiss DS. Mutations at the GABA receptor selectivity filter: a possible role for effective charges. J Physiol 548: 527–540, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 407 Wu FS, Chen SC, and Tsai JJ. Competitive inhibition of the glycine-induced current by pregnenolone sulfate in cultured chick spinal cord neurons. Brain Res 750: 318–320, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 408 Wu FS, Gibbs TT, and Farb DH. Inverse modulation of γ-aminobutyric acid- and glycine-induced currents by progesterone. Mol Pharmacol 37: 597–602, 1990.
    PubMed | ISI | Google Scholar
  • 409 Xu M and Akabas MH. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor α1 subunit. J Gen Physiol 107: 195–205, 1996.
    Crossref | PubMed | ISI | Google Scholar
  • 410 Xu M, Covey DF, and Akabas MH. Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants. Biophys J 69: 1858–1867, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 411 Xu TL, Dong XP, and Wang DS. N-methyl-d-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons. Eur J Neurosci 12: 1647–1653, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 412 Xu TL, Li JS, Jin YH, and Akaike N. Modulation of the glycine response by Ca2+-permeable AMPA receptors in rat spinal neurones. J Physiol 514: 701–711, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 413 Xue H, Shi H, Tsang SY, Zheng H, Savva CG, Sun J, and Holzenburg A. A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABAA counterpart. J Mol Biol 312: 915–920, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 414 Yadid G, Goldstein DS, Pacak K, Kopin IJ, and Golomb E. Functional α3-glycine receptors in rat adrenal. Eur J Pharmacol 288: 399–401, 1995.
    Crossref | PubMed | ISI | Google Scholar
  • 415 Yadid G, Maor G, Youdim MB, Silberman M, and Zinder O. Autoradiographic localization of strychnine-sensitive glycine receptor in bovine adrenal medulla. Neurochem Res 18: 1051–1055, 1993.
    Crossref | PubMed | ISI | Google Scholar
  • 416 Yadid G, Youdim MB, and Zinder O. High-affinity strychnine binding to adrenal medulla chromaffin cell membranes. Eur J Pharmacol 175: 365–366, 1990.
    Crossref | PubMed | ISI | Google Scholar
  • 417 Yadid G, Youdim MB, and Zinder O. Preferential release of epinephrine by glycine from adrenal chromaffin cells. Eur J Pharmacol 221: 389–391, 1992.
    Crossref | PubMed | ISI | Google Scholar
  • 418 Yamakura T, Bertaccini E, Trudell JR, and Harris RA. Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol 41: 23–51, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 419 Yamakura T and Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol. Anesthesiology 93: 1095–1101, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 420 Yamakura T, Mihic SJ, and Harris RA. Amino acid volume and hydropathy of a transmembrane site determine glycine and anesthetic sensitivity of glycine receptors. J Biol Chem 274: 23006–23012, 1999.
    Crossref | PubMed | ISI | Google Scholar
  • 421 Yamashita M, Ueno T, Akaike N, and Ikemoto Y. Modulation of miniature inhibitory postsynaptic currents by isoflurane in rat dissociated neurons with glycinergic synaptic boutons. Eur J Pharmacol 431: 269–276, 2001.
    Crossref | PubMed | ISI | Google Scholar
  • 422 Ye JH, Tao L, Ren J, Schaefer R, Krnjevic K, Liu PL, Schiller DA, and McArdle JJ. Ethanol potentiation of glycine-induced responses in dissociated neurons of rat ventral tegmental area. J Pharmacol Exp Ther 296: 77–83, 2001.
    PubMed | ISI | Google Scholar
  • 423 Ye JH, Tao L, Zhu L, Krnjevic K, and McArdle JJ. Ethanol inhibition of glycine-activated responses in neurons of ventral tegmental area of neonatal rats. J Neurophysiol 86: 2426–2434, 2001.
    Link | ISI | Google Scholar
  • 424 Ye Q, Koltchine VV, Mihic SJ, Mascia MP, Wick MJ, Finn SE, Harrison NL, and Harris RA. Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position α267. J Biol Chem 273: 3314–3319, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 425 Yevenes GE, Peoples RW, Tapia JC, Parodi J, Soto X, Olate J, and Aguayo LG. Modulation of glycine-activated ion channel function by G-protein βγ subunits. Nat Neurosci 6: 819–824, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 426 Yoon KW, Wotring VE, and Fuse T. Multiple picrotoxinin effect on glycine channels in rat hippocampal neurons. Neuroscience 87: 807–815, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 427 Young AB and Snyder SH. Strychnine binding associated with glycine receptors of the central nervous system. Proc Natl Acad Sci USA 70: 2832–2836, 1973.
    Crossref | PubMed | ISI | Google Scholar
  • 428 Young AB and Snyder SH. The glycine synaptic receptor: evidence that strychnine binding is associated with the ionic conductance mechanism. Proc Natl Acad Sci USA 71: 4002–4005, 1974.
    Crossref | PubMed | ISI | Google Scholar
  • 429 Young AB, Zukin SR, and Snyder SH. Interaction of benzodiazepines with central nervous glycine receptors: possible mechanism of action. Proc Natl Acad Sci USA 71: 2246–2250, 1974.
    Crossref | PubMed | ISI | Google Scholar
  • 430 Zacharias N and Dougherty DA. Cation-π interactions in ligand recognition and catalysis. Trends Pharmacol Sci 23: 281–287, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 431 Zarbin MA, Wamsley JK, and Kuhar MJ. Glycine receptor: light microscopic autoradiographic localization with [3H]strychnine. J Neurosci 1: 532–547, 1981.
    Crossref | PubMed | ISI | Google Scholar
  • 432 Zhang H and Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit. Biochemistry 36: 15856–15864, 1997.
    Crossref | PubMed | ISI | Google Scholar
  • 433 Zhang HG, French-Constant RH, and Jackson MB. A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms. J Physiol 479: 65–75, 1994.
    Crossref | PubMed | ISI | Google Scholar
  • 434 Zhong W, Gallivan JP, Zhang Y, Li L, Lester HA, and Dougherty DA. From ab initio quantum mechanics to molecular neurobiology: a cation-π binding site in the nicotinic receptor. Proc Natl Acad Sci USA 95: 12088–12093, 1998.
    Crossref | PubMed | ISI | Google Scholar
  • 435 Zhorov BS and Bregestovski PD. Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships. Biophys J 78: 1786–1803, 2000.
    Crossref | PubMed | ISI | Google Scholar
  • 436 Zhou L, Chillag KL, and Nigro MA. Hyperekplexia: a treatable neurogenetic disease. Brain Dev 24: 669–674, 2002.
    Crossref | PubMed | ISI | Google Scholar
  • 437 Zhu L, Jiang ZL, Krnjevic K, Wang FS, and Ye JH. Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area. Neuropharmacology 45: 270–280, 2003.
    Crossref | PubMed | ISI | Google Scholar
  • 438 Zhu L, Krnjevic K, Jiang Z, McArdle JJ, and Ye JH. Ethanol suppresses fast potentiation of glycine currents by glutamate. J Pharmacol Exp Ther 302: 1193–1200, 2002.
    Crossref | PubMed | ISI | Google Scholar

AUTHOR NOTES

  • Address for reprint requests and other correspondence:J. W. Lynch, School of Biomedical Sciences, Univ. of Queensland, Brisbane QLD 4072, Australia (E-mail: )