Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter
Abstract
Membrane potential (Δψ)-driven and Cl−-dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na+-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Δψ- and Cl−-dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Δψ- and Cl−-dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.
it has been well established that organisms excrete a variety of metabolic waste compounds, drugs, and xenobiotics via transporter-mediated processes to maintain homeostasis. Negatively charged organic compounds are transported by several transporter families, including organic anion transporters (solute carrier family 22, SLC22), organic anion-transporting polypeptides (SLC21), and ATP-dependent multidrug resistance-associated proteins (ATP-binding cassette, subfamily C) (10, 25, 38). These transporters are expressed at high levels in the renal tubular cells and hepatocytes and play critical roles in the pharmacokinetics of anionic compounds. In addition to these transporters, recent molecular biological and biochemical studies indicated that the SLC17 transporter family also functions as an organic anion transporter (3, 9, 13, 18, 22, 23, 26, 32).
SLC17 family consists of nine members and is divided into two subfamilies, namely Na+-phosphate cotransporters (NPTs) and vesicular transporters (Fig. 1A). On the basis of sequence homology, NPTs include NPT1 (SLC17A1), NPT3 (SLC17A2), NPT4 (SLC17A3), and NPT homologue (SLC17A4) (18, 22, 23). NPT1 was the first SLC17 family member identified as an NPT using an oocyte expression system (26, 37). Upon identification, this protein was suggested be an NPT, which is responsible for renal reabsorption of inorganic phosphate (Pi). However, this idea was soon questioned because the affinity for Pi of NPT1 is at the millimolar level, which is lower than that of the native membrane (21). Recently, NPT1 was shown to be a polyspecific exporter of organic anions, in particular urate, and to be responsible for renal urate extrusion (9, 18). The importance of NPT1 for urate homeostasis was confirmed by genetic linkage of NPT1 variant with gout (3). NPT4 and NPT homologues also function as polyspecific exporters of organic anions, such as urate, at the luminal membranes of renal urinary tubules and bile canaliculi (3, 13, 18) and the intestinal brush-border membrane (32), respectively. In contrast, the vesicular transporters include five members, which are vesicular excitatory amino acid transporters (VEAT, SLC17A5), vesicular glutamate transporters (VGLUTs, SLC17A6-8), and vesicular nucleotide transporters (VNUT, SLC17A9) (17, 22, 23, 28). VEAT, VGLUTs, and VNUT are present in secretory vesicles and responsible for vesicular storage of aspartate, glutamate, and nucleotides, respectively, and play essential roles in the respective chemical transmission (22, 23). SLC17A5 protein is also present in lysosomes and plays a role in sialic acid extrusion.
Fig. 1.Δψ-dependent organic anion transport by mouse Na+-phosphate cotransporter 3 (mNPT3). A: members of the solute carrier family 17 (SLC17) anion transporter family and their substrates. VEAT, vesicular excitatory amino acid transporter; VGLUT, vesicular glutamate transporter; VNUT, vesicular nucleotide transporter. B: purified mNPT3 was subjected to SDS-PAGE and visualized by Coomassie brilliant blue staining. Positions of molecular markers are indicated. C: time course of p-aminohippuric acid (PAH) uptake by proteoliposomes containing mNPT3. The reaction was started by adding radiolabeled PAH to a final concentration of 100 μM in the presence (●) or absence (○) of 2 μM valinomycin (Val.). Proteoliposomes were prepared in buffer containing 20 mM MOPS-Tris, pH 7.0, 150 mM sodium acetate, and 5 mM magnesium acetate. The reaction mixture contained 20 mM MOPS-Tris, pH 7.0, 150 mM potassium acetate, 10 mM KCl, and 5 mM magnesium acetate. PAH uptake by proteoliposomes without mNPT3 is shown (□). Values are means ± SE (n = 5). D: energetics of PAH uptake. PAH uptake was measured in the presence or absence of 2 μM valinomycin or 2 μM nigericin (Nig.) (n = 3). E: dose-dependence curve of PAH uptake. The valinomycin-dependent PAH uptake at 1 min was determined at various PAH concentrations (n = 3). F: Cl− dependence of PAH uptake. The uptake was measured after 1 min in the presence or absence of Cl−. Part of the potassium acetate in the reaction mixture was replaced with the indicated concentration of KCl (n = 3). G: effects of DIDS and Evans blue on PAH uptake at 1 min. Assay media contained 10 μM DIDS or 1 μM Evans blue (n = 3). Statistical significance was determined by Student's t-test. ***P < 0.001.
Although the major members of the SLC17 family have been well characterized, the kinetic properties, expression, and localization of NPT3 (SLC17A2) are less well understood. In contrast to the limited expression profile of other NPTs, the human NPT3 gene shows a wide expression profile and is predominantly expressed in heart and muscle, but lesser amounts of mRNA were found in other tissues, such as the brain, placenta, lungs, liver, kidneys, and pancreas on Northern blotting analysis (26, 27). However, the function and expression of NPT3 protein have not been reported.
In the present study, we investigated the transport and expression properties of mouse NPT3 protein (mNPT3). We showed that mNPT3 is a Δψ-driven polyspecific organic anion transporter that interacts with urate and anionic drugs. mNPT3 was expressed in a wide range of tissues, such as hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, and astrocytes around the blood vessels in the cerebrum.
MATERIALS AND METHODS
cDNA.
The mouse SLC17A2 cDNA (accession no. NM_144836) was cloned by PCR. Briefly, mouse SLC17A2 cDNA was amplified using a forward primer 5′- CGGGGGATCCGAATTCATGGATGAGAAGCCTACCAC-3′ and a reverse primer 5′-CCTTGTTCATCTCGAGGAGGCGGGTGAGAGTCCTTT-3′. The PCR product was inserted into the EcoRI and XhoI sites of a modified pET-28a(+) vector (Novagen, Madison, WI) containing two YbeL sequences, as described previously (15). The resultant plasmid, pET-β-mNPT3-β, encodes a cDNA for mouse NPT3 fused with YbeL (β) at both NH2 and COOH termini. Fusion of the function-unknown bacterial α-helical protein YbeL promotes expression of mammalian membrane protein in Escherichia coli (E. coli) (15). Cloning of other SLC17 family transporter cDNAs was described previously (11, 32).
RT-PCR.
Mouse total RNAs from several tissues were purchased from Clontech (Palo Alto, CA) and UNITECH (Chiba, Japan). cDNA was generated from total RNA with a Reverse Transcriptase Kit (Toyobo, Osaka, Japan) using 1 μg of total RNA as the template. The resulting cDNA pool was used for RT-PCR, which was carried out with 400 nmol/l each of specific forward and reverse primers with 5 U/μl of SYBR Premix Ex Taq (TaKaRa BIO, Shiga, Japan). Reactions were performed 35 times with denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. The primer set with 5′-tgggaccagcaatttgtgtga-3′ and 5′-actgataaggaatccggtggta-3′ was used for detection of mNPT3. The primer set used for detection of the housekeeping glyceraldehyde 3-phosphate dehydrogenase gene was as follows: 5′-tgtgtccgtcgtggatctga-3′ and 5′-ttgctgttgaagtcgcaggags-3′.
Expression and purification of mNPT3 protein.
A combinatorial method for overexpressing and purifying membrane proteins using E. coli was used as described previously (15). Briefly, E. coli C43 (DE3) cells harboring the above mentioned plasmid were transformed with expression vectors and grown in TB medium containing 20 μg/ml kanamycin sulfate at 37°C (15). E. coli cells were grown until A600 reached 0.6–0.8; then isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 1 mM, and the culture was incubated for a further 16 h at 18°C. The cells were then harvested by centrifugation and suspended in buffer consisting of 20 mM MOPS-Tris, pH 7.5, 300 mM sucrose, and 1 mM phenylmethylsulfonyl fluoride. The cell suspension was then disrupted by sonication with a TOMY UD200 tip sonifier (OUTPUT4) and centrifuged at 5,900 g at 4°C for 10 min to remove large inclusion bodies and cell debris. The resultant supernatant was carefully collected and centrifuged again at 150,000 g for 1 h at 4°C. The pellet was suspended in buffer consisting of 70 mM Tris·HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, and 15% glycerol with the protein concentration adjusted to 10 mg/ml. The membranes were then treated with 1.5% Fos-choline 14 (Affymetrix, Santa Clara, CA), incubated for 25 min on ice, and then centrifuged at 150,000 g at 4°C for 30 min. The supernatant containing mNPT3 was collected, diluted twice with buffer consisting of 70 mM Tris·HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, 15% glycerol, and 0.05% Fos-choline, and then applied to a column containing 1 ml of nickel-nitrilotriacetic acid (NTA) Superflow resin (Qiagen, Hilden, Germany) equilibrated with buffer consisting of 70 mM Tris·HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, 15% glycerol, and 0.05% Fos-choline. After incubation for 3 h at 4°C, the column was washed with washing buffer consisting of 70 mM Tris·HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, 20% glycerol, and 0.1% N-decyl-β-d-thiomaltopyranoside (DTM) (Affymetrix). The mNPT3 protein was eluted with buffer consisting of 20 mM Tris·HCl, pH 8.0, 300 mM imidazole, 100 mM NaCl, 10 mM KCl, 20% glycerol, and 0.1% DTM and then stored at −80°C without noticeable loss of activity for at least a few months.
Reconstitution.
Reconstitution of the purified mNPT3 into liposomes was carried out by the freeze-thaw method as described previously (11, 12). Briefly, 30 μg of purified mNPT3 protein was mixed with liposomes (0.5 mg of lipid) and frozen at −80°C. The mixture was then thawed quickly by holding the sample tube in hands and diluted 20-fold with reconstitution buffer consisting of 20 mM MOPS-Tris, pH 7.0, 150 mM sodium acetate, 5 mM magnesium acetate, and 0.5 mM dithiothreitol. Proteoliposomes were sedimented by centrifugation at 200,000 g for 1 h at 4°C and suspended in 0.2 ml of reconstitution buffer. For the Na+-driven Pi transport, reconstitution of purified recombinant mNPT3 into liposomes was carried out by the method described previously (12). Briefly, protein was mixed with liposomes (0.5 mg of lipid) and frozen at −80°C. The mixture was the thawed quickly by holding in hands and diluted 20-fold with reconstitution buffer containing 20 mM MOPS-Tris, pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate, and 0.5 mM dithiothreitol. Proteoliposomes were sedimented by centrifugation at 200,000 g for 1 h at 4°C and suspended in 0.2 ml of reconstitution buffer.
Transport assay.
Proteoliposomes (1.5 μg total protein/assay) were suspended in 20 mM MOPS-Tris, pH 7.0, 150 mM potassium acetate, 10 mM KCl, and 5 mM magnesium acetate and then incubated for 3 min at 27°C. Valinomycin was added to a final concentration of 2 μM, and the mixture was incubated for a further 2 min. The assay was initiated by addition of 100 μM [glycyl-2-3H] p-aminohippuric acid (PAH) (0.6 MBq/μmol), [8-14C] urate (0.05 MBq/μmol), or [8-14C] salicylate (0.05 MBq/μmol). Aliquots of 130 μl were taken at the times indicated and centrifuged through a Sephadex G-50 (fine) spin column at 760 g for 2 min. Radioactivity in the eluate containing proteoliposomes was measured with a liquid scintillation counter (PerkinElmer, Wellesley, MA). All numerical values are shown as means ± SE (n = 3−6). Under these conditions, inside positive membrane potential of ∼90 mV was generated according to previous study using [14C] thiocyanate distribution assay (11). For Na+-driven Pi transport, the reaction was started by addition of proteoliposomes to the reaction mixture containing 20 mM MOPS-Tris, pH 7.0, 100 mM sodium acetate, 5 mM magnesium acetate, and 100 μM [32P] Na2HPO4 (3.7 MBq/μmol) or 20 mM MOPS-Tris, pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate, and 100 μM [32P] K2HPO4 (3.7 MBq/μmol).
Antibodies.
Site-specific rabbit polyclonal antibodies against mNPT3 were prepared by repeated injection of glutathione-S-transferase fusion polypeptides encoding amino acid residues M1-D53 of mNPT3 (mdekpttrkgsgfcslryalalimhfsnftmitqrvslsiaiiamvnstqhqd). The following antibodies were obtained commercially: anti-CD31 rat monoclonal antibody (Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (Thermo Fisher Scientific, Kanagawa, Japan), anti-P-glycoprotein mouse monoclonal antibody (GeneTex, Los Angeles, CA), Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA), Alexa Fluor 568-labeled anti-mouse IgG (Invitrogen), or Cy3-labeled anti-rat IgG (Amersham, Little Chalfont, Buckinghamshire, UK).
Immunohistochemistry.
Immunohistochemical analysis was performed by indirect immunofluorescence microscopy as described previously (7, 24). All animal procedures and care were approved by the Institutional Animal Care and Use Committee and were carried out according to the guidelines of Okayama University. Briefly, C57BL/6 mice were anesthetized with ether and then perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The organs were isolated, and frozen sections were prepared. After being washed with PBS, the specimens were incubated for 30 min in the same buffer containing 0.1% Triton X-100, followed by PBS containing 2% goat serum and 0.5% bovine serum albumin. The specimens were incubated with anti-mNPT3 (1:200 dilution), anti-CD31 (10 μg/ml), anti-GFAP (0.2 μg/ml), or anti-P-glycoprotein (7.2 μg/ml) antibody diluted with PBS containing 0.5% bovine serum albumin for 1 h at room temperature. For preabsorption treatment, 1 μl of anti-mNPT3 antibody was incubated with buffer containing 20 μg of antigenic polypeptide for 12 h at 4°C and used for immunohistological analysis. Samples were washed four times with PBS and then reacted with the secondary antibody, Alexa Fluor 488-labeled anti-rabbit IgG (2 μg/ml), Alexa Fluor 568-labeled anti-mouse IgG (1 μg/ml), or Cy3-labeled anti-rat IgG (0.5 μg/ml) for 1 h at room temperature. Finally, the immunoreactivity was examined under a confocal laser microscope (FV300; Olympus, Tokyo, Japan).
Preparations.
Liver microsomes, renal brush-border membrane vesicles, and small intestinal membranes were prepared as described previously (20, 32, 33). Total membrane fractions of C57BL/6 brain, placenta, heart, lungs, and thyroid were isolated, suspended in ice-cold 20 mM MOPS-Tris, pH 7.0, containing 0.3 M sucrose, 5 mM EDTA, and protease inhibitors (pepstatin A and leupeptin at 10 μg/ml each), homogenized, and centrifuged at 800 g for 10 min at 4°C to remove organelles and cell debris. The resultant supernatant was carefully collected and centrifuged again at 150,000 g for 1 h at 4°C. The pellet was suspended with the same buffer. Astrocytes were prepared and cultured as described (8).
Heat treatment and deglycosylation of kidney membrane proteins.
Glycosidase treatment was carried out according to the manufacturer's protocol. Briefly, 40 μg of brush-border membrane vesicles from mouse kidney was heated in 30-μl aliquots of a solution containing 0.5% SDS and 40 mM dithiothreitol at 75°C for 15 min. The mixture was then cooled to room temperature. The samples were incubated with N-glycosidase F (3,000 U; New England Biolaboratories, Ipswich, MA) or O-glycosidase (480,000 U; New England Biolaboratories) with Neuraminidase (300 U; New England Biolaboratories), or 1% NP-40 for 1 h at 37°C. The samples were dissociated with SDS sample buffer and analyzed by SDS-PAGE and Western blotting.
RESULTS
Functional characterization of mNPT3.
To examine the transport properties of mNPT3, protein was overexpressed in E. coli, solubilized, and purified by nickel-NTA affinity column chromatography. The degree of purification was assessed by SDS-PAGE followed by Coomassie brilliant blue staining. The purified mNPT3 corresponded to a polypeptide of 66 kDa, which is the expected size for YbeL (120 amino acids), with conjugation at both NH2 and COOH termini (Fig. 1B) (15).
Purified mNPT3 protein was then reconstituted into liposomes by a freezing/thaw/dilution procedure (12). When Na+-trapped liposomes are suspended in buffer containing 150 mM K+, the addition of valinomycin induces permeation of K+ into liposomes, resulting in the formation of inside-positive Δψ across the liposomal membrane. Under these conditions, we found the time-dependent uptake of PAH in mNPT3-containing proteoliposomes (Fig. 1C). No PAH uptake was observed in the absence of valinomycin. Only the background level of uptake was observed in liposomes lacking mNPT3 (Fig. 1C). In contrast, addition of nigericin, in the presence of K+-established pH gradient (outside-acidic), failed to stimulate PAH uptake (Fig. 1D). These results indicated that inside-positive Δψ drives PAH uptake.
Kinetic analysis of valinomycin-evoked PAH uptake exhibited dose dependence with Km and Vmax values of 0.4 mM and 96.0 nmol·mg−1·min−1, respectively (Fig. 1E). SLC17 family transporters, including NPT1 and NPT homologue, require Cl− for their activity. As shown in Fig. 1F, PAH transport by mNPT3 is also stimulated by a low concentration of Cl−, and excess Cl− did not further increase transport activity. The PAH uptake was inhibited by DIDS and Evans blue, common inhibitors of SLC17 family transporters (Fig. 1G).
We next examined whether mNPT3 possesses Na+/Pi-cotransport activity because members of the SLC17 family may transport Pi upon the imposition of an Na+ gradient (9, 12, 26, 32). As shown in Fig. 2A, mNPT3 took up Pi upon imposition of an Na+ gradient. In the absence of Na+ (i.e., no Na+ gradient), Pi uptake was markedly reduced. In the absence of mNPT3 protein, the liposomes did not accumulate Pi even in the presence of an Na+ gradient. In contrast to PAH uptake, imposition of Δψ did not drive Pi uptake (Fig. 2B). The Na+/Pi-cotransport activity was not inhibited by an excess amount of PAH (Fig. 2C).
Fig. 2.Na+-dependent transport of inorganic phosphate by mNPT3. A: time course of inorganic phosphate (Pi) uptake by proteoliposomes. Proteoliposomes were prepared in buffer containing 20 mM MOPS-Tris, pH 7.0, 100 mM potassium acetate, and 5 mM magnesium acetate. The reaction mixture contained 20 mM MOPS-Tris, pH 7.0, 100 mM sodium acetate (●) or potassium acetate (○), and 5 mM magnesium acetate. The reactions were started by adding reconstituted proteoliposomes to a mixture containing 100 μM [32P] Pi. Pi uptake by proteoliposomes without mNPT3 is shown (□). Values are means ± SE (n = 3). B: Pi uptake was measured in the presence or absence of 2 μM valinomycin as described in Fig. 1C (n = 6). C: effects of 1 mM PAH on Pi uptake at 1 min (n = 6).
Polyspecific nature of mNPT3.
We examined the substrate specificity of mNPT3, as other NPTs exhibit polyspecific organic anion transport activity. As shown in Fig. 3A, cis-inhibition studies indicated that PAH uptake is sensitive to aspirin, salicylate, ibuprofen, and urate, suggesting that mNPT3 probably recognizes these compounds as transport substrates. To validate this suggestion, we measured Δψ-driven uptake of radiolabeled salicylate and found that mNPT3 actually showed uptake of the labeled compound (Fig. 3B). In contrast, mNPT3 did not recognize tetraethylammonium, a typical substrate of organic cation transporters, indicating that its substrate specificity is different from those of cation transporters, such as organic cation transporters or multidrug and toxic compound extrusion transporters (7). As NPTs transport urate and play important roles in urate homeostasis, we investigated whether mNPT3 takes up radiolabeled urate. As expected, mNPT3 transported urate when Δψ (inside-positive) was imposed across the liposomal membrane (Fig. 4A). In the absence of Δψ, the urate uptake was greatly reduced. Only the background level of uptake was observed for liposomes lacking mNPT3. The urate uptake exhibited saturation kinetics with Km and Vmax values of 0.5 mM and 43.7 nmol·mg−1·min−1, respectively (Fig. 4B). The Δψ-dependent urate uptake required Cl− and was inhibited by DIDS and Evans blue (Fig. 4, C and D). The kinetic features of urate transport by mNPT3 are similar to those of PAH uptake and those of urate uptake by NPT1 and NPT homologue (9, 32).
Fig. 3.cis-Inhibition and salicylate uptake of mNPT3. A: mNPT3-mediated Δψ-dependent uptake of 100 μM PAH was measured in the absence or presence of the listed compounds at 1 min. The concentration of compounds was 1 mM. The values are the percentages of radiolabeled PAH uptake under control conditions (no test substance added). Control activity (100%) corresponds to 12.4 nmol·mg−1·min−1. Values are means ± SE (n = 6). TEA, tetraethylammonium. B: salicylate uptake was measured in the presence or absence of 2 μM valinomycin (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 4.mNPT3-mediated urate transport. A: time course of urate uptake by proteoliposomes containing mNPT3. Transport was initiated by the addition of either 2 μM valinomycin (●) or DMSO (control) (○). Urate uptake by proteoliposomes without mNPT3 is shown (□). Values are means ± SE (n = 5). B: Δψ-dependent urate uptake at the indicated concentrations of urate at 1 min (n = 3). C: Cl− dependence of Δψ-dependent urate uptake at 2 min was measured under standard conditions except that Cl− concentration was changed as indicated (n = 3). D: effects of DIDS and Evans blue on urate uptake. Δψ-dependent urate uptake at 2 min was measured under standard conditions in the presence or absence of DIDS at 10 μM or Evans blue at 1 μM (n = 3). ***P < 0.001.
Wide expression of mNPT3.
As shown in Fig. 5, RT-PCR analysis revealed that a 148-bp mNPT3-specific transcript was amplified in tissues from liver, kidneys, small intestine, pancreas, brain, placenta, heart, skeletal muscle, lungs, and thyroid, confirming the previous results of Northern blotting analysis (27).
Fig. 5.Gene expression of mNPT3 in various organs. RT-PCR analysis with total RNA from the indicated organs using probes specific to mNPT3 or mouse glyceraldehyde 3-phosphate dehydrogenase (mG3PDH) was performed.
To demonstrate expression of NPT3 at the protein level, we prepared a specific polyclonal antibody against mNPT3. Other SLC17 members were purified, and immunological specificity of the prepared antibody was examined by immunoblotting. As shown in Fig. 6A, the polyclonal antibody detected only mNPT3 among the purified SLC17 family transporters, suggesting its immunological specificity. Anti-mNPT3 recognized immunoreactive protein bands at 66 kDa and 130 kDa, suggesting formation of complexes composed of two or more molecules of purified mNPT3 protein.
Fig. 6.Protein expression of mNPT3 in various organs. A: immunological specificity of anti-mNPT3 antibody. Purified SLC17 transporter proteins (5 μg) were electrophoresed on a 10% polyacrylamide gel and visualized by Coomassie brilliant blue staining (top). In a parallel experiment, these proteins were transferred onto a nitrocellulose sheet and then analyzed with anti-mNPT3 antibody (bottom). B: crude membrane fractions from various organs were analyzed by immunoblotting with anti-mNPT3 antibody (Ab) (top); purified mNPT3 (100 ng) is shown on the left, with membrane proteins from each organ (70 μg) on the right. Bottom: specificity of anti-mNPT3 antibody was examined by Western blotting using antibody preabsorbed with antigenic polypeptide.
Subsequently, immunoblotting with the specific antibody demonstrated the presence of an immunological counterpart with an apparent molecular mass of 68 kDa in crude membrane fractions of the liver, kidneys, placenta, lungs, and thyroid (Fig. 6B). The positions of the observed signals in these tissue samples were slightly higher than the calculated molecular mass of 52 kDa. However, these immunoreactivities disappeared when the preabsorbed antibodies were used, suggesting that migration of mNPT3 protein on SDS-PAGE is altered by glycosylation or complex formation with lipids or other proteins (Fig. 6B). As shown in Fig. 7, neither heat, N-glycosidase F, nor O-glycosidase treatments altered the position of mNPT3 on the Western blot. These results suggested that mNPT3 is not glycosylated in the membrane, and the migration shift of mNPT3 may be attributable to interactions with other compounds or its high Pi value of 8.7. Immunoreactive polypeptide was not detected in the membrane fractions of the small intestine, brain, and heart, indicating that the mNPT3 in these organs is either below the detection limit of our system or is degraded by proteases (Fig. 6B).
Fig. 7.Glycosylation status of mNPT3 in kidney membranes. The kidney membrane fraction (40 μg) was heat treated and then incubated at 37°C in the presence or absence of N-glycosidase F or O-glycosidase as described in materials and methods. Samples were then subjected to Western blotting analysis with anti-mNPT3 antibodies.
We then examined the localization of mNPT3 immunohistochemically. In the liver, mNPT3 was localized to the apical region of the bile duct (Fig. 8A). In the kidneys, mNPT3 immunoreactivity was observed in the apical regions of the proximal and distal convoluted tubules (Fig. 8B). In the placenta, mNPT3 was expressed on the maternal side (brush-border membrane) but not on the fetal side (basolateral membrane) of the syncytiotrophoblast (Fig. 9A). No immunoreactivity was observed in endothelial cells, as shown by immunoreactivity for CD31, a marker of endothelial cells (Fig. 9A). Strong mNPT3 immunoreactivity was also observed in the apical membrane of follicle cells in the thyroid (Fig. 9B) and bronchiolar epithelial cells in the lungs (Fig. 9C). Little immunoreactivity was observed in alveolar cells. No mNPT3 immunological reactivity was detected in the small intestine and heart (data not shown).
Fig. 8.Indirect immunofluorescence microscopy revealed localization of mNPT3 in the liver and kidney. A: liver. BD, bile duct; IV, interlobular vein. B: kidney. PCT, proximal convoluted tubule; DCT, distal convoluted tubule; GL, glomerulus. Bars = 20 μm. Insets: background signal with preabsorbed (Abs.) anti-mNPT3 antibody.
Fig. 9.Localization of mNPT3 in the placenta, thyroid, and lung. A: placenta. Specimens were doubly immunostained for mNPT3 (left) and CD31 (middle). A merged image of mNPT3 and CD31 is shown on the right. B: thyroid. C: lung. Bars = 20 μm. Insets: background labeling is shown with preabsorbed anti-mNPT3 antibody.
We then investigated expression of mNPT3 in the brain. Although no immunoreactivity was observed in membranes from the brain on immunoblotting, as shown in Fig. 6B, gene expression detected by RT-PCR analysis suggested that mNPT3 is expressed in the brain at a low level. We found the presence of mNPT3 immunoreactivity around blood vessels in the brain (Fig. 10A). Double-labeling immunofluorescence microscopy showed that the mNPT3 immunoreactivity was colocalized with GFAP, a marker for astrocytes, but not with P-glycoprotein and CD31, markers for endothelial cells (Fig. 10A). These results suggest that mNPT3 is expressed in the astrocytes located around the blood vessels. To examine the presence of mNPT3 in astrocytes in more detail, astrocytes were isolated from mouse brain and cultured. A 148-bp mNPT3-specific transcript was amplified by RT-PCR from primary cultured astrocytes indicating expression of mNPT3 (Fig. 10B). Furthermore, immunoblotting analysis detected the presence of an NPT3 counterpart with an apparent molecular mass of 68 kDa (Fig. 10C). Immunohistochemical analysis indicated that mNPT3 immunoreactivity was localized in the plasma membrane of primary cultured astrocytes (Fig. 10D). These results suggested that NPT3 is localized in the plasma membrane of astrocytes in the brain.
Fig. 10.Expression of mNPT3 in the brain. A: brain. Specimens were doubly immunostained for mNPT3 (left) and glial fibrillary acidic protein (GFAP), P-glycoprotein (P-gp), or CD31 (middle). Merged images of mNPT3 and GFAP, P-gp, or CD31 are shown on the right. Bars = 20 μm. Inset: background signal with preabsorbed anti-mNPT3 antibody. B: gene expression of mNPT3 and mG3PDH in primary cultured mouse astrocytes. RT-PCR analysis with total RNA from primary cultured astrocytes was performed. C: crude membrane fraction from primary cultured mouse astrocytes (20 μg) was analyzed by Western blotting with anti-mNPT3 antibody. D: indirect immunofluorescence microscopy revealed that mNPT3 was localized in plasma membrane of the primary cultured mouse astrocytes. Specimens were doubly immunostained for mNPT3 (left) and GFAP (middle). Merged image of mNPT3 and GFAP is shown on the right. Bars = 20 μm. Inset: background signal with preabsorbed anti-mNPT3 antibody.
DISCUSSION
In the present study, we investigated the transport properties, expression, and localization of mNPT3, the last SLC17 anion transporter member remaining to be characterized.
We showed that mNPT3 can transport PAH and urate. The uptake of PAH and urate was at the expense of Δψ but not ΔpH with the requirement of a low Cl− concentration and was inhibited by typical inhibitors of the SLC17 family, DIDS and Evans blue. Kinetic parameters of the uptake of PAH and urate are similar to those of NPT1, NPT4, and NPT homologue. cis-Inhibition studies showed that mNPT3-mediated PAH uptake was strongly inhibited by various hydrophilic anionic drugs, such as aspirin, salicylate, and ibuprofen. Furthermore, mNPT3 also shows Na+-dependent Pi uptake. These results clearly showed that mNPT3 shares the same transport properties of these other NPTs, strongly suggesting that mNPT3 interacts with these compounds, probably as a transport substrate, and functions as a polyspecific organic anion transporter (Fig. 11).
Fig. 11.Transport model of mNPT3. mNPT3 acts as a Δψ- and Cl−-dependent polyspecific organic anion transporter and also transports Pi in an Na+-dependent manner.
It is interesting to note that mNPT3 requires Cl−. Our previous study showed that Cl− is an allosteric regulator of SLC17 transporters and not a transport substrate (9, 11, 17, 28). The cytosolic concentration of Cl− is 10–20 mM (11). Thus SLC17 family transporters maintain full activity under physiological conditions. However, metabolic intermediates of amino acids and lipids inhibit SLC17 transporters by competing with Cl− (11). This suggests that Cl− and metabolic intermediates act as physiological regulators. The significance of such regulation of mNPT3 is not clear at present and is currently under investigation in our laboratory. Another interesting feature of mNPT3 is that it shows multiple transport functions. A previous kinetic study of VGLUT showed that Na+-dependent Pi uptake and membrane potential-driven anion transport activities differ in driving force, substrate, inhibitor sensitivity, and essential residues (12). These observations indicated that VGLUT has two independent transport machineries within the molecule. As PAH transport is not inhibited by excess Pi, and Pi transport is not inhibited by excess PAH, mNPT3 may have a similar transport system to VGLUT.
Consistent with the results of Northern blotting analysis in humans, we found that mNPT3 is expressed in various tissues at the protein level throughout the body in mice. mNPT3 was localized on the bile ducts of the liver and luminal membranes of the urinary tubules in the kidneys, suggesting that mNPT3 is involved in organic anion and urate excretion from these organs similar to other NPTs. It is noteworthy that mNPT3 is also expressed in organs and tissues that are not related to extrusion of organic anions from the body. In particular, mNPT3 is strongly expressed and localized on the maternal side of the syncytiotrophoblast in the placenta. The placenta is a barrier for the fetus against the potential toxicity of drugs, xenobiotics, and metabolites. The expression and localization of breast cancer resistance protein (ABCG2) and P-glycoprotein in the placenta are similar to those of NPT3 (16). mNPT3 in the placenta may functionally coordinate with these proteins to control pregnancy-related compounds and toxic compounds.
mNPT3 is also expressed at the apical membrane of follicle cells in the thyroid and bronchiole epithelial cells in the lungs. As the most important functions of follicle cells in the thyroid are the synthesis and secretion of thyroid hormones (THs), we speculated that mNPT3 may be involved in the secretory processes of THs, where T3 and T4 are produced in lysosomes and somehow released from the follicle cells (34). However, this is unlikely because mNPT3 is not localized at the basolateral membrane where THs are released, and our preliminary biochemical study indicated that proteoliposomes containing mNPT3 did not take up radiolabeled T3 upon imposing Δψ (data not shown). In the lungs, we showed that mNPT3 is expressed in the apical region of the bronchiole epithelial cells, whereas little expression is observed in alveolar epithelial cells that are involved in gas exchange and secretion of surfactants. As the bronchiole epithelial cells are the site for egress of xenobiotics and possess various types of drug transporter, it is likely that mNPT3 is also involved in excretion of drugs in this organ (2, 6). The physiological substrates and functional significance of mNPT3 in these cells are not yet known, and further studies are necessary.
Similar to other NPTs, we showed that mNPT3 also transports urate. Urate is the product of purine metabolism and acts as a natural antioxidant with neuroprotective properties (14, 35). An elevated serum urate level is associated with several metabolic disorders, such as gout and kidney stones in humans (1). Hence, keeping the appropriate serum urate level is important for maintenance of human health. It has been reported that two-thirds of total urate is excreted from the kidneys, and the remaining urate is excreted from the intestine and liver (30, 31). NPT1, NPT4, and NPT homologue participate in urate extrusion in these organs. Although urate synthesized in various cells should be extruded, a transporter for egress of urate remains unknown. As mNPT3 is present in various tissues, it is reasonable to suggest that mNPT3 may participate in urate extrusion in organs other than the kidneys, liver, and intestine. In the brain, a recent epidemiological study indicated that high plasma urate concentrations decrease the risk of Parkinson's disease because urate acts as a potent antioxidant to protect neurons (36). In vitro and in vivo studies confirmed the neuroprotective effect of urate in dopaminergic neurons (5, 35). Moreover, protection of dopaminergic cells by urate requires its accumulation in astrocytes (4). In this respect, it is noteworthy that mNPT3 is localized on astrocytes besides the blood-brain barrier. It is possible that mNPT3 may regulate the secretion of urate from astrocytes and is involved in neuroprotection by urate. This possibility is now under investigation in our laboratory.
In conclusion, we presented evidence that mNPT3 is expressed in a wide range of organs and acts as a Δψ- and Cl−-dependent polyspecific organic anion transporter. Unlike other NPTs, NPT3 is localized in various tissues that are not related to direct extrusion of anionic drugs from the body, suggesting that mNPT3 contributes to the dynamics of metabolic and xenobiotic organic anions in various organs.
GRANTS
This work was supported in part by a Grant-in-Aid from the
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: N.T., N.J., T.M., M.H., H.O., and Y.M. conception and design of research; N.T., N.J., T.M., M.H., H.O., and Y.M. performed experiments; N.T., N.J., and H.O. analyzed data; N.T., N.J., T.M., M.H., H.O., and Y.M. interpreted results of experiments; N.J., H.O., and Y.M. prepared figures; N.J., T.M., M.H., H.O., and Y.M. drafted manuscript; N.J., T.M., M.H., H.O., and Y.M. edited and revised manuscript; Y.M. approved final version of manuscript.
REFERENCES
- 1. . Hyperuricemia and associated disease. Rheum Dis Clin North Am 32: 275–293, 2006.
Crossref | PubMed | ISI | Google Scholar - 2. . Drug transporters in the lung-do they play a role in the biopharmaceutics of inhaled drugs? J Pharm Sci 99: 2240–2255, 2010.
Crossref | PubMed | ISI | Google Scholar - 3. . NPT1/SLC17A1 is a renal urate exporter in human and its common gain-of-function variant decreases the risk of renal underexcretion gout. Arthritis Rheum 67: 281–287, 2015.
Crossref | Google Scholar - 4. . Protection of dopaminergic cells by urate requires its accumulation in astrocytes. J Neurochem 123: 172–181, 2012.
Crossref | PubMed | ISI | Google Scholar - 5. . Urate and its transgenic depletion modulate neuronal vulnerability in a cellular model of Parkinson's disease. PLoS One 7: e37331, 2012.
Crossref | PubMed | ISI | Google Scholar - 6. . Drug transport and metabolism characteristics of the human air way epithelial cell line Calu-3. J Control Release 87: 131–138, 2003.
Crossref | PubMed | ISI | Google Scholar - 7. . Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations. Am J Physiol Cell Physiol 291: C678–C686, 2006.
Link | ISI | Google Scholar - 8. . Identification of a mammalian vesicular polyamine transporter. Sci Rep 6836: 1–9, 2014.
Google Scholar - 9. . Type 1 sodium-dependent phosphate transporter (SLC17A1 protein) is a Cl−-dependent urate exporter. J Biol Chem 285: 26107–26113, 2010.
Crossref | PubMed | ISI | Google Scholar - 10. . Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58: 944–958, 2000.
Crossref | PubMed | ISI | Google Scholar - 11. . Metabolic control of vesicular glutamate transport and release. Neuron 68: 99–112, 2010.
Crossref | PubMed | ISI | Google Scholar - 12. . Vesicular glutamate transporter contains two independent transport machineries. J Biol Chem 281: 39499–39506, 2006.
Crossref | PubMed | ISI | Google Scholar - 13. . Human sodium phosphate transporter 4 (hNPT4/SLC17A3) as a common renal secretory pathway for drugs and urate. J Biol Chem 285: 35123–35132, 2010.
Crossref | PubMed | ISI | Google Scholar - 14. . Altered uric acid levels and disease states. J Pharmacol Exp Ther 324: 1–7, 2008.
Crossref | PubMed | ISI | Google Scholar - 15. . Combinatorial method for overexpression of membrane proteins in Escherichia coli. J Biol Chem 285: 23548–23556, 2010.
Crossref | PubMed | ISI | Google Scholar - 16. . BCRP/ABCG2 in the placenta: expression, function, and regulation. Pharm Res 25: 1244–1255, 2008.
Crossref | PubMed | ISI | Google Scholar - 17. . Identification of a vesicular aspartate transporter. Proc Natl Acad Sci USA 105: 11720–11724, 2008.
Crossref | PubMed | ISI | Google Scholar - 18. . Type 1 sodium-dependent phosphate transporter acts as a membrane potential-driven urate exporter. Curr Mol Pharmacol 6: 88–94, 2013.
Crossref | PubMed | Google Scholar - 19. . One-step purification of Escherichia coli H+-ATPase (FoF1) and its reconstitution into liposomes with neurotransmitter transporters. J Biol Chem 266: 22141–22146, 1991.
PubMed | ISI | Google Scholar - 20. . H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures. J Biol Chem 264: 18445–18450, 1989.
PubMed | ISI | Google Scholar - 21. . Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80: 1373–1409, 2000.
Link | ISI | Google Scholar - 22. . Vesicular neurotransmitter transporter: bioenergetics and regulation of glutamate transport. Biochemistry 50: 5558–5565, 2011.
Crossref | PubMed | ISI | Google Scholar - 23. . Vesicular neurotransmitter transporters: an approach for studying transporters with purified proteins. Physiology 28: 39–50, 2013.
Link | ISI | Google Scholar - 24. . A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci USA 102: 17923–17928, 2005.
Crossref | PubMed | ISI | Google Scholar - 25. . Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev 73: 765–796, 1993.
Link | ISI | Google Scholar - 26. . Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflügers Arch 447: 629–635, 2004.
Crossref | PubMed | ISI | Google Scholar - 27. . A 1.1-Mb transcript map of the hereditary hemochromatosis locus. Genome Res 7: 441–456, 1997.
Crossref | PubMed | ISI | Google Scholar - 28. . Identification of a vesicular nucleotide transporter. Proc Natl Acad Sci USA 105: 5683–5686, 2008.
Crossref | PubMed | ISI | Google Scholar - 29. . A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502–514, 1973.
Crossref | PubMed | ISI | Google Scholar - 30. . Role of the intestinal tract in the elimination of uric acid. Arthritis Rheum 8: 694–706, 1965.
Crossref | PubMed | Google Scholar - 31. . Origin and extra renal elimination of uric acid in man. Nephron 14: 7–20, 1975.
Crossref | PubMed | ISI | Google Scholar - 32. . A NPT homologue (SLC17A4 protein) is an intestinal organic anion exporter. Am J Physiol Cell Physiol 302: C1652–C1660, 2012.
Link | ISI | Google Scholar - 33. . Heterogeneity of sodium-dependent d-glucose transport sites along the proximal tubule: evidence from vesicle studies. Am J Physiol Renal Fluid Electrolyte Physiol 242: F406–F414, 1982.
Link | ISI | Google Scholar - 34. . Up to date with human thyroglobulin. J Endocrinol 170: 307–321, 2001.
Crossref | PubMed | ISI | Google Scholar - 35. . Protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats. Zhonghua Yi Xue Za Zhi 90: 1362–1365, 2010.
PubMed | Google Scholar - 36. . Plasma urate and risk of Parkinson's disease. Am J Epidemiol 166: 561–567, 2007.
Crossref | PubMed | ISI | Google Scholar - 37. . Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc Natl Acad Sci USA 88: 9608–9612, 1991.
Crossref | PubMed | ISI | Google Scholar - 38. . Molecular and cellular physiology of renal organic and anion transport. Physiol Rev 84: 987–1049, 2004.
Link | ISI | Google Scholar
