Purification and reconstitution of an outwardly rectified Cl 2 channel from tracheal epithelia

Jovov, Biljana, Vadim G. Shlyonsky, Bakhram K. Berdiev, Iskander I. Ismailov, and Dale J. Benos. Purification and reconstitution of an outwardly rectified Cl2 channel from tracheal epithelia. Am. J. Physiol. 275 (Cell Physiol. 44): C449–C458, 1998.—We reported the identification of three outwardly rectified Cl2 channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of anionand cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 μg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 6 9 pS in symmetrical Cl2 solutions, halide selectivity sequence of I2 . Cl2 . Br2, and lack of sensitivity to protein kinase A, Ca21, or dithiothreitol. Using anti-Gai antibodies to precipitate Gai protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gai protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.

protein, G␣ i-2 , inhibits the ORCC, as demonstrated by patch-clamp studies (36) and in planar lipid bilayers (18).Furthermore, in planar lipid bilayers, treatment of the incorporated ORCC with pertussis toxin (PTX) conferred a linearity to the previously rectified I-V curve of the ORCC (18).Some nonphysiological stimuli can also activate the ORCC: exposing silent patches to depolarizing voltages greater than ϩ50 mV (17) or to trypsin (40) will induce activation of previously quiescent ORCCs.
Despite the fairly detailed information concerning the electrophysiological characteristics of this channel, the protein that forms the ORCC and the gene that codes for the protein are unknown.Our laboratory has developed a protocol for the isolation of a 140-kDa protein that forms a Cl Ϫ -selective, Ca 2ϩ /calmodulin kinase II-, DIDS-, and dithiothreitol-sensitive channel when incorporated into planar lipid bilayers (7,31,32).Polyclonal antibodies have been raised against a 38-kDa component of this homopolymeric protein.Using these antibodies and immunopurification as the only step in the purification scheme, we have isolated and reconstituted, in addition to the Ca 2ϩ -activated Cl Ϫ channel, an ORCC and CFTR (18,21,22).Using in vitro phosphorylation of isolated proteins, we have identified potential 115-, 85-, and 52-kDa ORCC candidate proteins (21).
We report the generation of new antibodies raised against the identified ORCC candidate proteins and an associated purification procedure designed to obtain a highly purified 115-kDa protein that acts as an ORCC in bilayers.We also report the effect of G␣ i protein precipitation on the rectification properties of ORCC.

Tracheal Apical Membrane Preparation
Apical membrane vesicles were prepared by differential centrifugation with use of a procedure first described by Langridge-Smith et al. (27) modified as previously described (30).Aliquots of vesicles (average protein concentration 5 mg/ml) were stored in liquid nitrogen until use.
Peripheral proteins were extracted from native membrane vesicles by incubation of vesicles for 30 min in KCl buffer (100 mM KCl, 5 mM Tris-HEPES, and 0.5 mM MgCl 2 ) titrated to pH 10.5 with 0.1 M NaOH.Alkaline-stripped vesicles were recovered by centrifugation at 100,000 g for 60 min.

Solubilization
These prewashed, peripheral protein-extracted apical membrane vesicles (200 µg) were solubilized with 200 µl of different concentrations of the detergents SDS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Triton X-100, and Nonidet NP-40 in the presence of KCl buffer (pH 7.4) and then centrifuged at 100,000 g for 60 min to remove unsolubilized material.Quantitation of solubilized protein was performed using the bicinchoninic acid protein assay.Solubilization of the 115-kDa protein was estimated using Western blotting and quantitative densitometry.Because 2% CHAPS was capable of solubilizing 95% of the 115-kDa protein, this detergent was used in further purification steps.

Protein Separation on DEAE-Cellufine Anion Exchanger
The CHAPS extract (4 ml, 2 mg/ml protein) was incubated with 10 ml of preequilibrated DEAE-Cellufine resin (KCl buffer containing 2% CHAPS) for 60 min with occasional shaking.The resin was washed with 10 ml of the same buffer to remove nonbound proteins.This nonbound fraction contains the 115-kDa protein and was used in the next step of purification.

Protein Separation on CM-Cellufine Cation Exchanger
The nonbound fraction from the previous anion-exchange separation step was combined with 10 ml of a CM-Cellufine resin equilibrated with CM buffer (MES-Tris ϩ 2% CHAPS, pH 6.0) and incubated for 60 min with occasional shaking.The resin was washed with 10 ml of the same buffer to remove nonbound proteins and then eluted stepwise with buffers of higher pH as follows: 6.2, 6.5, 7.5, 7.8, and 8.5.The pH 6.2 and 6.5 fractions contained the 115-kDa protein and were combined and used in the next step of immunopurification.

Immunopurification of Cl Ϫ Channel Proteins With Anti-p115 Antibodies
Highly purified preparation.A polyclonal rabbit antibody (anti-p115) generated against purified 115-kDa protein [isolated using anti-p38 antibodies (6,26)] was covalently linked to a hydrazide-activated gel (Carbolink).Purified immune IgG was oxidized by metaperiodate (0.02 M) in the dark at room temperature for 1 h and coupled to the beads by incubation overnight at room temperature to reach maximum binding capacity (4-5 mg IgG/ml gel).The same amount of preimmune IgG was covalently linked to hydrazide-activated gel, and nonimmune beads were used as a control for the specificity of immunopurification.Before use, the Carbolink beads were washed extensively with MES-Tris buffer ϩ 2% CHAPS, pH 7.4.To obtain the highly purified preparation, the pH 6.2 and 6.5 fractions eluted from the cation-exchange column were combined; pH was adjusted to 7.4, and the sample was incubated with the antibody-linked Carbolink beads for 1 h to allow binding of the protein to the antibody.The column was then extensively washed with MES-Tris buffer before elution with 100 mM glycine, pH 3. Successive 2-ml fractions were collected and immediately neutralized with 100 µl of 1.5 M Tris.The first two fractions that contained the most protein were pooled and concentrated to 100 µl and used for reconstitution in liposomes or for further biochemical characterization.
Partly purified preparation.To obtain the partly purified preparation, apical membrane vesicles were solubilized with 0.8% Triton X-100, and the cation-and anion-exchange steps were omitted, leaving immunopurification as the only step of purification after solubilization of integral membrane proteins.
Partly purified preparation after immunoprecipitation of G␣ i protein(s).Immunoprecipitation of G␣ i protein(s) from partly purified ORCC material was performed using polyclonal antipeptide antibodies (Calbiochem).These antibodies were raised against a COOH-terminal decapeptide found in G␣ i-1 and G␣ i-2 proteins.The immunopurified fractions containing partly purified 115-kDa protein (20 µg) were concentrated to a volume of 200 µl, and radioimmune precipitation buffer (RIPA) was added (50 µl of 5ϫ RIPA) to ensure dissociation of G␣ i protein(s) from the ORCC protein.RIPA buffer was also chosen on the basis of previous experience that immunoprecipitation of CFTR in this buffer did not affect biophysical properties of ORCC (21).Anti-G␣ i-2 antibodies (5 µl) were added and incubated at 4°C for 1 h.Then 20 µl of protein A-Sepharose were added and incubated for 1 h at 4°C.After centrifugation to pellet protein A, the supernatant was collected and reconstituted as previously described ( 6) for bilayer studies.The precipitated G␣ i protein(s) was eluted from the protein A-Sepharose with use of 0.1 M glycine buffer, pH 2.8, and used for coreconstitution experiments with highly purified 115-kDa protein.

PAGE and Western Blots
Protein separation on polyacrylamide gels was performed using the method of Laemmli (26).Proteins were transferred to polyvinylidine difluoride membranes by applying 100 V for 1 h or 30 V overnight.Blots were washed with TBS buffer (20 mM Tris, pH 7.5, and 500 mM NaCl) for 10 min and then incubated with 5% milk in TBS for at least 30 min.Primary antibodies (dilution 1:100 for anti-p115 and 1:500 for anti-G␣ i-2 protein) were dissolved in 1% nonfat dry milk in TBS buffer containing 0.05% Tween 20 (TTBS) and added to the blot for a 2-h incubation.After extensive washing with TTBS, donkey anti-rabbit IgG antibodies conjugated to alkaline phosphatase were added at a dilution of 1:5,000.Development was performed using the nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate system according to the manufacturer's instructions.Each gel was calibrated by simultaneously running relative molecular weight (M r ) standards in a parallel lane.The apparent M r of the unknown proteins were calculated from appropriately constructed log M r -relative mobility curves.

Protein Iodination
Protein iodination was carried out using the chloramine-T procedure (28).Highly or partly purified Cl Ϫ channel protein(s) was dissolved in 25 µl of 0.5 mM sodium phosphate buffer, pH 7.5.Carrier-free Na 125 I (500 µCi) and 25 µl of 2 mg/ml chloramine-T were added to purified protein(s) and incubated at room temperature for 60 s.The reaction was stopped by addition of 50 µl of chloramine-T stop buffer.The iodinated Cl Ϫ channel protein(s) was separated from unreacted I Ϫ by passing the mixture over a gel filtration column (G-25 Sephadex).Detection of iodinated protein was carried out by autoradiography after separation of proteins with SDS-PAGE.
Planar lipid bilayers, composed of 25 mg/ml of 2:1 (wt/wt) phosphatidylethanolamine-phosphatidylserine, were painted with a fire-polished glass capillary over a 200-µm hole drilled in a polystyrene chamber, as described previously (19).Bilayer formation was monitored by the increase in membrane capacitance to a final value of 200-300 pF.Artificial liposomes containing Cl Ϫ channel proteins were incorporated into bilayers bathed with symmetrical solutions of 100 mM KCl and 10 mM MOPS adjusted to pH 7.4.In some experiments, KCl was substituted with 100 mM choline chloride or N-methyl-D-glucamine chloride.The observation of a stepwise increase in current was taken as an indication of channel incorporation into the lipid bilayer.Current measurements were performed with a high-gain amplifier circuit based on a design previously described (18).Steady-state single channel I-V curves were measured after channel incorporation by applying a known voltage and measuring individual channel current.

Production of Specific Antibodies for Immunopurification of ORCC
Using in vitro phosphorylation of immunopurified material isolated using anti-p38 antibodies, our laboratory has identified three ORCC candidate proteins (21).Repetitive immunopurification using anti-p38 antibodies was performed to isolate a sufficient amount of identified proteins for rabbit immunization.Immunopurified proteins were precipitated (methanol-chloroform), dissolved in 40 µl of sample buffer, and separated on gel under nonreducing conditions.The separated proteins were blotted onto nitrocellulose paper and stained by Ponceau S. The appropriate protein bands were cut from the nitrocellulose paper, dried in air, and dissolved in 200 µl of DMSO.DMSO-solubilized paper was mixed with an equal volume of complete Freund's adjuvant for the first injection or with incomplete adjuvant for subsequent injections.Approximately 6-8 µg of each protein were injected over a period of 3 mo (6 times at 2-wk intervals).After the last injection the rabbit was bled from an ear vein, and the blood was collected and left to coagulate at room temperature.After coagulation, blood was spun at 10,000 g for 20 min, and the clear serum was divided among vials and lyophilized.IgG purification from serum was performed using immobilized protein A-agarose, as previously described (21).

Western Blot Characterization of New Antibodies (Anti-p115 and Anti-p52)
Immune serums were first tested for specificity on blots of total proteins extracted from bovine tracheal apical membranes (Fig. 1).Tracheal apical membranes were solubilized using 0.8% Triton X-100 in the presence of KCl buffer.The extracts of 50 µg of proteins were mixed with sample buffer, separated on a 10% gel, and immunoblotted.Under these conditions, immune IgGs (anti-p115 and anti-p52) reacted strongly with the original antigens (Fig. 1, A and B, lane 1).Preimmune IgGs showed no reactivity to any of the blotted proteins (Fig. 1, A and B, lane 2).
These antibodies were subsequently used as ligands for immunopurification.Immunopurification was a single step of purification after solubilization of integral membrane proteins in these experiments.Immunopurified proteins were reconstituted and incorporated in planar lipid bilayers to test for channel activity.Only incorporation of immunopurified material obtained using the anti-p115 antibody resulted in ORCC activity.

Reconstitution of Partly Purified Material Obtained Using the Anti-p115 Antibody as a Ligand for Purification
Iodination of partly purified proteins isolated using the anti-p115 antibody as a ligand revealed the presence of polypeptides at 115, 40, and 31 kDa (Fig. 2).Reconstitution and incorporation of this partly purified material into planar lipid bilayers resulted in channel activity.Figure 3 shows representative traces of 11 recordings made with 3 individual preparations reveal-Fig. 1. Specificity of anti-p115 and anti-p52 antibodies.Alkalinestripped apical membrane proteins were solubilized with 0.8% Triton X-100, and unsolubilized material was removed.Total solubilized proteins were mixed with sample buffer containing 20 mM dithiothreitol and incubated at room temperature for 10 min.Protein (50 µg/lane) was separated on a 10% gel and immunoblotted with preimmune (lane 2 in A and B) or immune IgG (lane 1, anti-p115 in A and anti-p52 in B).  ing a channel that exhibited marked rectification in symmetrical Cl Ϫ solutions with slope conductances of 91 Ϯ 11 and 31 Ϯ 5 pS at positive and negative potentials, respectively (Fig. 3).Other biophysical properties of the channel were as follows: 1) anion-to-cation selectivity of the channel was 8:1, as determined by measuring the reversal potentials in the presence of a 10-fold KCl gradient; 2) channels could be inhibited by the addition of 100 µM DIDS to only one side (cis) of the bilayer (n ϭ 8); 3) channel activity was independent of Ca 2ϩ concentration up to 100 µM (n ϭ 4); 4) channels exhibited a halide permeability sequence of I Ϫ Ͼ Cl Ϫ Ͼ Br Ϫ ; and 5) the channels were PKA insensitive (n ϭ 5).These results demonstrated that the basic biophysical characteristics of this channel were identical to those previously observed for ORCC immunopurified using anti-p38 antibodies (21,22).PKA insensitivity of reconstituted ORCC can be attributed to the absence of CFTR in this preparation, as demonstrated by lack of 170-kDa protein in autoradiographs of the iodinated partly purified material (Fig. 2).

Development of a Purification Scheme to Increase the Purity and Yield of the 115-kDa Protein
Since we have identified the 115-kDa protein as an ORCC candidate, we have developed a new purification procedure to increase the yield and purity of this polypeptide.To optimize the solubilization of the 115-kDa protein, several detergents were tested for their ability to solubilize integral membrane proteins as well as their ability to solubilize the 115-kDa protein (Table 1).Solubilization of membrane proteins was determined by the bicinchoninic acid protein assay.The amount of integral membrane protein solubilized with 5% SDS was considered as 100% solubilization, and solubilization with other tested detergents was expressed as a percentage of the solubilization achieved  with 5% SDS.Solubilization with 5% SDS was also used as a reference for solubilization of the 115-kDa protein (Table 1).Quantitation of solubilized 115-kDa protein was performed using densitometry scanning of immunoblots of membrane extracts obtained with different detergents (Fig. 4).Inasmuch as we needed to preserve a functional channel protein through the last stage of purification to test for channel activity, only mild nondenaturating detergents were employed (CHAPS, Triton X-100, and Nonidet NP-40).The lowest concentration tested for any of these detergents was equal to the critical micellar concentration.To obtain optimal solubilization of the 115-kDa protein, higher concentrations of detergents were also tested (Table 1).Of the detergents screened, 2% CHAPS was the most effective in solubilizing the 115-kDa protein.CHAPS was capable of solubilizing 90 Ϯ 6% of the 115-kDa protein from stripped tracheal membranes, whereas it solubilized only 73 Ϯ 7% of integral membrane proteins.On the other hand, 0.8% Triton X-100 was very effective in solubilizing integral apical membrane proteins (80 Ϯ 8%) but not so effective in solubilizing the 115-kDa protein (60 Ϯ 7%).CHAPS is a mild zwitterionic detergent that contains head groups with positive and negative charges.This class of detergents is more efficient than nonionic detergents in overcoming proteinprotein interactions while causing less protein denaturation than ionic detergents.CHAPS also does not interfere with ion-exchange chromatography and was included in all buffers in the subsequent purification step.
Several chromatographic methods were examined for their ability to purify the 115-kDa protein.The purification scheme that yielded the best results in terms of overall yield and purity was a combination of anionand cation-exchange chromatography followed by immunopurification.First, the separation profile of total solubilized membrane proteins on the anionic exchanger DEAE-Cellufine was tested.The CHAPSsolubilized extract in the presence of 100 mM KCl buffer was applied to the DEAE column, and then elution was carried out using a stepwise gradient of KCl (0.1-0.5 M KCl).Five 10-ml fractions were collected and analyzed by Western blot.Only the first, nonbound fraction contained the 115-kDa protein (Fig. 5, lane 2).As expected, the majority of tracheal membrane proteins (75%) were bound to the DEAE column (negatively charged) under these conditions.The nonbound fraction from the anion-exchange chromatography step was next applied to the cationic exchanger (CM-Cellufine), preequilibrated with MES-Tris buffer at pH 6, and incubated for 1 h.The 115-kDa protein was eluted in pH 6.2 and 6.5 fractions (Fig. 5, lane 3).The pH of these two fractions was adjusted to 7.4 before immunopurification.All buffers contained 2% CHAPS during purification.Protein concentration and yields of the 115-kDa protein during the purification procedure are shown in Table 2.The relative amount of the 115-kDa protein was estimated using densitometry scanning of immunoblots of all fractions during the purification procedure.Densitometry scanning of an amido black-stained blot of the highly purified 115-kDa protein was used to estimate the amount of 115-kDa protein after the final step of purification (Fig. 5C).Because the total amount of protein isolated from 20 Ϯ 3 mg of starting tracheal apical membranes after the final immunopurification step was 8 Ϯ 1 µg and 87% of this amount was 115-kDa protein (determined by densitometry scanning), we calculated that the amount of 115-kDa protein in this fraction was 7.0 Ϯ 0.7 µg.Iodination of this highly purified material revealed a single band migrating at 115 kDa (Fig. 5D).

Incorporation of Highly Purified 115-kDa Protein
A channel with a linear I-V relationship and unitary conductance of 87 Ϯ 9 pS (n ϭ 10) was recorded in planar lipid bilayers after incorporation of the proteoliposomes containing this highly purified 115-kDa protein (Fig. 6).The halide permeability of the recorded channel was I Ϫ Ͼ Cl Ϫ Ͼ Br Ϫ , and the channel was completely inhibited by 100 µM DIDS (n ϭ 9; data not shown).Identical to the channel observed after incorporation of a partly purified material, the channel formed by highly purified 115-kDa protein was not sensitive to addition of a phosphorylating cocktail (1.85 ng/ml catalytic subunit of PKA ϩ 100 µM ATP) or to Ca 2ϩ (up to 100 µM).Comparison of these characteristics with the properties of a channel recorded from partly purified material (Table 3) suggests that they are essentially identical, except for the I-V curve.

Analysis of Partly and Highly Purified ORCC Proteins for Presence and Role of G␣ i Protein(s)
Previously, using PTX-induced ADP-ribosylation, we demonstrated that a G i protein copurified with the ORCC and that the ORCC was regulated by a G i protein in planar lipid bilayers (23).We also demonstrated that addition of PTX induced a shift in the I-V curve of the ORCC toward linearity (23).Here we tested whether the partly and highly purified 115-kDa protein preparations contained a G i protein.These experiments were performed using commercially available anti-G␣ i protein antibodies specific for G␣ i-1 and G␣ i-2 subunits of heterotrimeric G proteins.Partly and highly purified 115-kDa proteins were separated on an 8% SDS gel under reducing conditions, transferred to a polyvinylidine difluoride membrane, and probed with anti-G␣ i antibodies.As demonstrated by Western blot analysis, the partly purified preparation contained a G␣ i protein (Fig. 7, lane 2), but no G␣ i protein could be detected in the highly purified preparation (Fig. 7, lane 3).Next, we also tested whether precipitation of a G␣ i protein would induce a change from a rectified to a linear I-V curve of the ORCC.In fact, the I-V curves of the channels incorporated into planar lipid bilayers (Fig. 8) after immunoprecipitation of G␣ i protein(s) from a partly purified preparation became linear with a unitary conductance of 85 Ϯ 9 pS (n ϭ 5).This immunoprecipitation of G␣ i protein(s) did not affect any other properties of the channel (halide permeability, inhibition by DIDS, or insensitivity to PKA ϩ ATP, Ca 2ϩ , and dithiothreitol).The attempt to coreconstitute the commercially available recombinant G␣ i-2 protein Fig. 5. Ion-exchange-immunoaffinity purification of 115-kDa protein.B: amido black staining of 115-kDa protein-containing fractions during purification.Fractions 1-3 (100 µl of each) and fraction 4 (2 ml) (total fraction vol ϭ 4 ml) were subjected to SDS-PAGE (8%) under reducing conditions, then fractions were blotted and stained with amido black.A: immunoblot of 115-kDa protein-containing fractions in B during purification.Lane 1, CHAPS extract of apical membrane vesicles; lane 2, nonbound 115-kDa protein-containing fractions from DEAE-Cellufine (CM-Cellufine load); lane 3, 115-kDa protein-containing fractions eluted from CM-Cellufine; lane 4, 115-kDa proteincontaining fractions eluted from immunoaffinity resin (p115); lanes are identical for A and B. C: densitometric scan of an amido black-stained blot of highly purified 115-kDa protein (lane 4 in B).This analysis was used to calculate amount of 115-kDa protein after immunopurification step.D: autoradiograph of SDS-PAGE of highly purified 115-kDa protein labeled with 125 I.  (Calbiochem) with the highly purified 115-kDa protein did not confer rectification to this otherwise linear anion channel.However, rectification was achieved by coreconstituting the highly purified 115-kDa protein with the G␣ i protein immunoprecipitated from bovine tracheal epithelia (Fig. 9).Thus it appears likely that coupling of the specific G␣ i subunit with the channel protein can induce rectification of the I-V curve of this anion channel.

DISCUSSION
We have described the successful production of new antibodies raised against previously identified ORCC candidate proteins and a purification procedure to obtain a high level of homogeneity for one of these proteins (115-kDa protein).The main obstacles for purification of ion channels from epithelia are the lack of specific ligands and the small amount of channel proteins in epithelial cells.Using antibodies previously raised (anti-p38) as a ligand for immunopurification, we purified microgram amounts of the candidate ORCC proteins and raised two new antibodies: anti-p52 and anti-p115.The specificities of the new antibodies were confirmed by Western blotting.The antibodies reacted strongly with the original antigens.Both antibodies were used as ligands for immunopurification, but only incorporation of immunopurified material obtained using the anti-p115 kDa antibody resulted in ORCC Fig. 6.Reconstituted highly purified 115-kDa protein incorporated into planar lipid bilayers.Reconstitution and bilayer conditions are described in Fig.The fact that anti-p38 antibodies were used to isolate the 115-kDa protein suggests a possible epitope similarity between the previously isolated Ca 2ϩ -activated Cl Ϫ channel and the ORCC.Four distinct structural classes of Cl Ϫ channels have been cloned.The first class includes the glycine and GABA receptors (11,35), which belong to a superfamily of ligand-gated receptor channels.The second class includes CFTR (33), which belongs to the gene family of ATP-binding cassette transporters.The third class is the gene family of ClC Cl Ϫ channels, which include eight members so far (34).

Table 3. Biophysical and pharmacological properties of different preparations of 115-kDa proteins reconstituted into planar lipid bilayer
The fourth class is the gene family of Ca 2ϩ -activated Cl Ϫ channels (3).Although they perform similar functions, there is no significant structural homology among these different Cl Ϫ channel classes.Because we successfully raised specific antibodies (anti-p115), we were able to develop a purification procedure to increase the yield and purity of this 115-kDa protein.Quantitative immunoblotting was used to determine optimal solubilization conditions for the 115-kDa protein.Very good solubilization was achieved using the zwitterionic detergent CHAPS.The same detergent was successfully used for the solubilization of the ClC-0 Cl Ϫ channel from Torpedo californica electroplax (23).Another advantage of using CHAPS over nonionic detergents is that CHAPS more efficiently overcomes protein-protein interaction.Detection and estimation of the amount of the 115-kDa protein through the purification procedure were achieved by quantitative immunoblotting.The new purification procedure resulted in a high level of purification and a significant yield.A substantial amount of the 115-kDa protein (25 µg) was purified from 20 mg of tracheal apical membranes.Expressed as a percentage, our results suggest that isolated channel protein represents 1.2‰ of all membrane proteins.A similar ratio of channel protein to total membrane proteins (1.3‰) was found for porin purified from bovine skeletal muscle (39).Recovery of the 115-kDa protein through the purification procedure  http://ajpcell.physiology.org/was 28 Ϯ 8%, which was comparable to recovery of CFTR purified from Chinese hamster ovary cells (29).
The highly purified protein was successfully reconstituted into planar lipid bilayers.The incorporated channel had a linear I-V relationship as opposed to an outwardly rectified I-V relationship, which was previously observed with the partly purified preparation.Precipitation of G␣ i protein from the partly purified preparation resulted in a shift of the outwardly rectified I-V curve to a linear profile.In addition, coreconstitution of G␣ i protein (precipitated from bovine tracheal epithelia) with highly purified 115-kDa protein induced rectification of an otherwise linear anion channel, suggesting that channel rectification was produced by interaction between G␣ i protein(s) and the channel.We previously demonstrated that treatment of ORCC by PTX in planar lipid bilayers (uncoupling a G␣ i protein from ORCC) induced a shift from a rectified to a linear I-V curve profile.Most studies of G i protein regulation of ion channels have been performed using the patchclamp technique.Many of these studies demonstrated that G proteins can affect gating of ion channels (20,24,25,36), but it has not been shown that rectification properties of a channel depend on interaction with G proteins.Using three independent techniques to uncouple a G i protein from the channel (precipitation with antibodies, PTX treatment, or purification of this 115-kDa protein), we have confirmed that uncoupling of the channel protein from G i protein induces a shift from a rectified to a linear I-V curve of the ORCC.This information will be valuable for the interpretation of expression studies of this ORCC clone when available.According to our results, it can be expected that rectification of the I-V curve of an ORCC clone will depend on the presence and coexpression of a G␣ i protein in the expression system.
In summary, we have developed a specific ligand (anti-p115) and purification procedure for isolation of an ORCC from bovine tracheal epithelia.The isolated 115-kDa protein was functionally reconstituted into planar lipid bilayers.The reconstituted channel protein had a rectified I-V relationship only in the presence of a G␣ i protein.

Fig. 3 .
Fig.3.Reconstituted partly purified (anti-p115) outwardly rectifying Cl Ϫ channel (ORCC) proteins incorporated into planar lipid bilayers.Partly purified ORCC proteins were reconstituted into artificial liposomes and then incorporated into planar lipid bilayers.Bilayers were composed of 2:1 phosphatidylethanolamine-phosphatidylserine.Final lipid concentration was 25 mg/ml.Bilayer was bathed with symmetrical solutions of 100 mM KCl and 10 mM MOPS (pH 7.4).Dashed line, zero-current level.Records were filtered at 100 Hz.A: single channel recording at holding potentials of Ϯ100 mV.B: plot of single channel current-voltage (I-V) relationship of incorporated channels.

Fig. 7 .
Fig.7.Western blot analysis of partly and highly purified 115-kDa protein probed with anti-G␣ i polyclonal antibodies.Solubilized apical membrane proteins (100 µg, lane 1), partly purified preparation obtained from 20 mg of alkali-stripped apical membrane proteins (lane 2), and highly purified 115-kDa protein obtained from same amount of alkali-stripped apical membrane proteins (lane 3) were separated on a 10% SDS gel under reducing conditions, transferred to polyvinylidine difluoride membrane, and probed with polyclonal anti-G␣ i antibodies (1:500 dilution).

Fig. 8 .
Fig. 8. Reconstituted partly purified (anti-p115) Cl Ϫ channel proteins after precipitation of G␣ i protein incorporated into planar lipid bilayers.For reconstitution and bilayer conditions see Fig. 3 legend.Dashed lines, zerocurrent level.A: single channel recording at holding potentials of Ϯ80 mV.B: plot of single channel I-V relationship of incorporated channels.