©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cystic Fibrosis Transmembrane Conductance Regulator Is Required for Protein Kinase A Activation of an Outwardly Rectified Anion Channel Purified from Bovine Tracheal Epithelia (*)

(Received for publication, August 1, 1994; and in revised form, November 4, 1994)

Biljana Jovov (§) Iskander I. Ismailov Dale J. Benos

From the Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Our laboratory has developed a protocol for the isolation of a 140-kDa protein that forms an anion-selective channel when reconstituted into planar lipid bilayers. Polyclonal antibodies have been raised against the 38-kDa component of this purified protein. This channel has a linear current-voltage relationship and is not activated by protein kinase A (PKA) plus ATP. Using the same antibody and a modified purification protocol (eliminating the ion exchange chromatography steps), we isolated and reconstituted two other anion channels from tracheal membrane vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed four bands migrating at 52, 85, 120, and 174 kDa. Immunoprecipitation experiments with anti-CFTR antibodies indicate that the 174-kDa phosphoprotein was CFTR. Upon incorporation of these isolated proteins into planar bilayers, an anion channel that exhibited a marked outward rectification in symmetrical Cl solutions with a slope conductance of 82 pS at depolarizing voltages was observed. PKA and ATP increased channel activity but only from one side of the bilayer. However, channel activity was unaffected by addition of ATP alone from either side of the membrane. DIDS (100 µM) applied to the opposite side of the bilayer to which PKA and ATP act, blocked channel activity. A linear anion-selective channel with a conductance of 16 pS could be also resolved after inhibition of the outwardly rectified anion channel by DIDS in the presence of PKA and ATP. This small conductance channel was inhibited by 300 µM diphenylamine-2-carboxylic acid. Immunodepletion of the 174-kDa phosphoprotein from the preparation prevented activation of the 82-pS outwardly rectified anion channel by PKA and ATP. However, the PKA-dependent in vitro phosphorylation of the 52-, 85-, and 120-kDa phosphoproteins was unaffected by the absence of CFTR. Our results suggest a direct regulatory relationship between an outwardly rectified anion channel and CFTR.


INTRODUCTION

Maintenance of a fluid layer on the apical surface of alveolar and tracheal epithelial cells primarily involves active transepithelial Cl secretion. The overall rate of transepithelial Cl movement is determined by the activity of Cl channels located in the apical membrane of airway epithelial cells. These channels are regulated by intracellular second messengers such as cAMP and Ca as well as voltage and cell volume(1, 2) . Cytoplasmic addition of either protein kinase A (PKA) (^1)or protein kinase C plus ATP opens secretory Cl channels in patches excised from airway epithelial cells(3, 4, 5) . Malfunction of secretory regulation occurs in cystic fibrosis (CF) such that PKA does not open anion channels from diseased cells, even though depolarization of the patch demonstrates that functionally competent channels are present(5, 6, 7) .

Two recent single channel patch-clamp studies suggest that both outwardly rectified chloride channels (ORCCs) and CFTR Cl channels contribute to cAMP-activated Cl current in normal airway epithelial cells(8, 9) . Furthermore, defective regulation of outwardly rectifying Cl channels by protein kinase A was corrected by insertion of normal copies of CFTR cDNA(8) .

The biophysical properties of ORCC and CFTR Cl channels are very distinct and well established. ORCCs have a nonlinear current-voltage (I/V) relationship with a 20-40-pS single channel conductance at hyperpolarizing voltages and a 60-80-pS conductance at depolarizing voltages(1, 2, 3, 5, 10, 11) . ORCCs are blocked by a wide variety of molecules including DIDS and the calixarenes (12) and have a halide permeability sequence of I > Cl > Br. PKA and protein kinase C can activate these ORCCs (13, 14, 15, 16) . Conversely, the CFTR Cl channel has a linear I/V relationship with an 8-16-pS single channel conductance. Channel activity can be blocked by diphenylamine-2-carboxylic acid (DPC), but not by DIDS. The halide permeability sequence for CFTR is Br > Cl > I(27, 28) .

Despite the fairly detailed information concerning the biophysical characteristics of these Cl channels, relatively few biochemical investigations of individually isolated Cl channels have been undertaken. The protein that forms the ORCC is unknown, as are the mechanisms by which CFTR regulates this protein. In this paper we report the immunopurification from bovine tracheal epithelia and functional reconstitution in planar lipid bilayers of an outwardly rectified anion channel (ORAC) and the CFTR Cl channel and demonstrate a direct regulatory relationship between these channels.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP were obtained from Dupont NEN. C500M Cellufine cation exchange resin was from Amicon (Danvers, MA), and a hydrazide-derivatized Acti-Disk was obtained from FMC Corporation (Pine Brook, NJ). A monoclonal CFTR C terminus-specific antibody was obtained from Genzyme Corp. (Natick, MA). Donkey anti-rabbit IgG, conjugated to alkaline phosphatase, was obtained from Jackson ImmunoResearch (West Grove, PA). Peroxide-free Triton X-100 and SM2-Biobeads were from Bio-Rad. Phospholipids were obtained from Avanti Polar Lipids (Birmingham, AL). All other reagents were of analytical grade and were purchased from Sigma, Bio-Rad, or Fisher.

Methods

Tracheal Apical Membrane Preparation

Apical membrane vesicles were prepared by differential centrifugation using a procedure first described by Langridge-Smith et al.(17) , modified as described previously(18) . Aliquots of vesicles (average protein concentration, 5 mg/ml) were stored in liquid nitrogen until use. Extraction of peripheral proteins from native membrane vesicles was achieved 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 10.8 with 0.1 M NaOH. Alkaline-stripped vesicles were recovered by centrifugation at 35,000 times g for 35 min. These peripheral protein-extracted apical membrane vesicles were next solubilized with Triton X-100 (0.08%) in the presence of KCl buffer (at pH 7.4) when used for immunopurification or by Triton X-100 (0.8%) in 10 mM Tris/Mes buffer at pH 6.3 when used for cation exchange purification. Quantitation of solubilized protein was performed using the BCA method.

Protein Separation on CM-Cellufine Cation Exchanger

Prewashed apical membrane vesicles were solubilized with 0.8% Triton X-100 in the presence of 10 mM MES/Tris (pH 6.3) buffer (CM buffer). After removal of nonsolubilized material, the clear supernatant was mixed with CM-Cellufine beads equilibrated with CM buffer. The mixture was incubated on ice for 60 min with occasional shaking. Nonbound proteins were subsequently removed, and the resin was washed twice with CM buffer. Bound proteins were stepwise eluted with 10 mM MES/Tris buffers of the following pH values: 7, 7.5, 8.0, and 8.5. Fractions of 10 ml (from 10 ml of resin) were collected.

Immunopurification of Cl Channel Proteins with alphap38 Antibodies

A polyclonal rabbit antibody (alphap38) generated against a previously purified, reduced 38-kDa anion channel protein (p38; (18) and (19) ) was covalently linked to a hydrazide-activated disk. Purified immune IgG was oxidized by metaperiodate (0.02 M) in the dark at room temperature for 1 h and coupled to the disk by recirculating overnight at room temperature to reach maximum binding capacity (15-20 mg of IgG/disk). Prior to use, the disk was washed extensively with 1 M NaCl, 10 mM Na(2)HPO(4), pH 7.4. Solubilized prewashed apical membrane vesicles or fractions from the cation exchange CM column were diluted in 40 ml of the above buffer and recirculated over the disk for 1 h to allow binding of the protein to the antibody. The disk was then extensively washed with NaCl/phosphate 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 15 fractions contained the most protein and were pooled and concentrated to 100 µl and used for reconstitution in liposomes or for further biochemical characterization. To ensure the specificity of this immunopurification procedure, an equivalent amount of nonimmune IgG was linked to another hydrazide-activated disk, and the same protocol using solubilized bovine tracheal apical membrane vesicles was followed. Eluted material from this nonimmune IgG-bound disk was subjected to in vitro phosphorylation and SDS-PAGE and reconstitution into liposomes for incorporation into planar lipid bilayers.

Polyacrylamide Gels and Western Blots

Protein separation on polyacrylamide gels was performed using the method of Laemmli(20) . Proteins were transferred to nitrocellulose membranes by applying 100 V for 1 h. Blots were washed with TBS buffer (20 mM Tris, pH 7.5, and 500 mM NaCl) for 10 min and then incubated with 2% milk in TBS overnight. Primary antibodies (10 µg/ml) 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 1:5000. Development was performed using the p-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine system according to the manufacturer's instructions. In some experiments, simultaneous autoradiography of the blotted phosphorylated proteins was carried out. Each gel was calibrated by simultaneously running M(r) standards in a parallel lane. The apparent M(r) of the unknown proteins was calculated manually from appropriately constructed log M(r)versus relative mobility (R(F)) curves. The results are expressed as mean value ± 1 S.D.

In Vitro Phosphorylation of Immunopurified Cl Channel Proteins with ATP and PKA

Immunopurified proteins dissolved in 30 µl of reaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl(2), 100 µg/ml BSA) were mixed with PKA (300 ng) and 2 nmol of [-P]ATP (3000 Ci/mmol) in the absence or presence of 100 µg of protein kinase inhibitor (Sigma) and incubated for 60 min at 30 °C. The reaction was stopped by adding SDS sample buffer without dithiothreitol. The samples were run on 10% acrylamide gels, and the dried gels were analyzed by autoradiography.

Immunoprecipitation of CFTR

Immunoprecipitation of CFTR from alphap38-immunopurified bovine tracheal proteins was performed as described previously (21) using either a polyclonal (21) or a monoclonal anti-human CFTR antibody (Genzyme). Subsequent labeling and analysis of precipitated proteins with [-P]ATP was done as described previously(21, 22) . Briefly, 200 ng of alphap38-immunopurified anion channel proteins were dissolved in radioimmune precipitation buffer (50 µM Tris, pH 7.5; 550 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) and precleared by incubating for 1 h with nonimmune IgG and an additional 1 h with protein A-Sepharose. The supernatant was then incubated with anti-CFTR antibodies (8 µg) for 1 h and then precipitated with protein A-Sepharose beads. The precipitated proteins were then labeled with [-P]ATP in the presence of 300 ng of the catalytic subunit of protein kinase A (kindly provided by Dr. Gail Johnson, University of Alabama at Birmingham) for 60 min at 30 °C. The beads were washed and the bound proteins solubilized with 30 µl of gel sample buffer and loaded onto a 10% SDS-PAGE gel. The gels were dried and visualized by autoradiography on Kodak X-Omat AR film at -70 °C for appropriate lengths of time (1-72 h).

Planar Lipid Bilayer Experiments

Immunopurified Cl channel proteins were reconstituted into liposomes as described previously(23) . Concentrated protein samples were mixed with a phospholipid mixture (phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine at a ratio of 50:30:20, w/w/w) in the presence of 400 mM KCl, 0.5 mM MgCl(2), and 5 mM Tris/HCl, pH 7.4. The final volume was 600 µl, and the protein:phospholipid ratio was 1:10 (w/w). To remove Triton X-100, samples were mixed with 150 mg of Bio-Beads SM-2 and incubated at room temperature for 45 min followed by incubation at 4 °C overnight. Proteoliposomes were separated from the beads using a 1-ml syringe fitted with a 27-gauge needle.

Planar lipid bilayers, composed of a mixture of 25 mg/ml phosphatidylethanolamine, phosphatidylserine, and oxidized cholesterol in a 2:1:2 (w/w/w) ratio, were painted with a fire-polished glass capillary over a 200-µm hole drilled in a polystyrene chamber, as described previously(24) . Bilayer formation was monitored by the increase in membrane capacitance to a final value of 200-400 picofarads. Artificial liposomes containing anion 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(25) . Steady-state single channel current-voltage (I/V) curves were measured after channel incorporation by applying a known voltage and measuring individual channel current. Single channel open probability (P(o)) was calculated from P(o) = I/nbulleti, where i is the unitary current, I is the mean current, and n is the total number of active channels. I, n, and i were estimated from an all points current amplitude histogram and events list produced by pCLAMP software (version 5.5; Axon Instruments). The dashed line in the figures represents the zero current level.


RESULTS

Immunopurification of Anion Channel Proteins from Bovine Tracheal Epithelia

Experiments reported in this study were performed on immunopurified anion channel proteins from bovine tracheal membrane vesicles. Our laboratory previously developed a biochemical procedure for the isolation of a 140-kDa anion channel protein from tracheal apical membrane vesicles, and antibodies against its component 38-kDa polypeptide were raised. Assuming that anion channels share some structural similarities with each other, we modified our purification procedure to see if we could isolate other Cl channels with our alphap38 antibodies. The purification scheme that we eventually adopted utilized only apical tracheal membrane solubilization and immunoaffinity purification. The previously used purification protocol included a cation exchange step prior to immunopurification. The material obtained using these two different purification procedures was subjected to immunoblot analysis, using polyclonal alphap38 antibodies. The protein purified with the procedure that included cation exchange (Fig. 1, lane 2) contained two bands: a dominant 140-kDa protein and a minor 64-66-kDa band that is actually a reduced form of the 140-kDa protein, as described previously(19, 23) . Because SDS-PAGE was run under nonreducing conditions, the 38-kDa protein was not present. If this material was reduced with dithiothreitol, only the 38-kDa polypeptide could be seen (data not shown; (19) and (23) ). When the cation exchange step was omitted, an additional immunoreactive band at 52 ± 4 kDa was recognized by the antibody (Fig. 1, lane 3). This same 52-kDa protein was phosphorylated in vitro by PKA + ATP, as demonstrated by Western blotting, the phosphorylated proteins, and overlaying the autoradiogram over the immunoblot (data not shown).


Figure 1: Western blot analysis of purified anion channel proteins following two different purification procedures. Purified proteins were separated on a 10% SDS gel under nonreduced conditions, transferred to nitrocellulose, and probed with either preimmune IgG or immune (alphap38) IgG. Lane 1, solubilized tracheal apical membrane vesicles were subjected to cation exchange chromatography followed by immunopurification and then probed with preimmune IgG; lane 2, proteins purified using the same procedure as for lane 1 except immune alphap38 antibodies were used; lane 3, solubilized tracheal apical membrane vesicles were subjected to immunopurification without prior cation exchange and probed with immune IgG (alphap38 antibodies); lane 4, proteins purified using same procedure as for lane 3 except probed with preimmune IgG.



In Vitro Phosphorylation of Immunopurified Proteins

Tracheal apical membranes contain at least two Cl channels that can be activated by PKA and ATP: an outwardly rectified Cl channel and CFTR.(8, 9, 12) . To examine whether our immunopurified proteins can be phosphorylated, we subjected immunopurified proteins to in vitro phosphorylation using the purified catalytic subunit of PKA plus ATP. In vitro phosphorylation resulted in incorporation of label in a 52-kDa protein as well as in polypeptides with molecular masses of 85 ± 4, 120 ± 6, and 174 ± 5 kDa (n = 8; Fig. 2, lane 3). Incorporation of P into all of these bands could be prevented by inclusion of a protein kinase inhibitor (Walsh inhibitor) in the reaction mixture (lane 4). The immunopurified channel proteins themselves appeared not to contain any endogenous phosphorylating activity as evidenced by the lack of detectable phosphoproteins in the absence of exogenous PKA (lane 1). PKA did not exhibit autophosphorylation under these reaction conditions (lane 2). The specificity of the immunopurification protocol was confirmed by the lack of any phosphoprotein components from the eluate of a disk to which nonimmune IgG was bound (lane 5).


Figure 2: Autoradiograph of in vitro phosphorylated purified anion channel proteins from bovine tracheal epithelia. The phosphorylation reaction mixture consisted of 100-200 ng of purified Cl channel protein, 30 µl of buffer (50 mM Tris, pH 7.5, and 10 mM MgCl(2)), 300 ng of the catalytic subunit of protein kinase A, and 2 nmol of [-P]ATP. The reaction was carried out at 30 °C for 60 min. Lane 1, all reaction components except the catalytic subunit of the cAMP-dependent protein kinase; lane 2, all reaction components except the immunopurified channel protein; lane 3, all reaction components. Phosphorylated products are present at 52, 85, 120, and 174 kDa. Lane 4, all reaction components plus 100 µg of protein kinase inhibitor. Lane 5, all reaction components, except that immunopurification was performed with nonimmune IgG.



CFTR Antibodies Precipitate a 174-kDa Phosphoprotein from Immunopurified Anion Channel Proteins

As shown above, in vitro phosphorylation revealed the presence of a faint 174-kDa protein in the immunopurified material. To explore further the identity of this protein, we subjected this immunopurified material to precipitation with a monoclonal anti-CFTR antibody. Immunoprecipitation was performed in radioimmune precipitation buffer to ensure dissociation of CFTR from other proteins. The precipitated protein was phosphorylated in vitro and subjected to gel electrophoresis under reducing conditions (Fig. 3, lanes 1 and 2). Autoradiography revealed a phosphoprotein migrating at 174 kDa in both lanes. Identical results were obtained with polyclonal antipeptide CFTR antibodies (data not shown). The proteins remaining after precipitation with anti-CFTR antibodies were phosphorylated in vitro under conditions identical to those before CFTR precipitation. Phosphorylated products were present only at 52, 85, and 120 kDa; the 174-kDa phosphoprotein was removed by the anti-CFTR antibody.


Figure 3: Autoradiograph of CFTR immunoprecipitation and in vitro phosphorylation of immunopurified anion channel proteins after CFTR precipitation. Purified anion channel proteins (100-200 ng) were incubated with monoclonal anti-CFTR antibody (Genzyme) and then precipitated with poly(A)-Sepharose beads. Precipitated protein was phosphorylated in the presence of 300 ng of catalytic subunit of PKA and 2 nmol of [-P]ATP for 60 min at 30 °C. Proteins that remained after the CFTR precipitation were phosphorylated in vitro under conditions identical to those described in the legend to Fig. 1. Lane 1, protein precipitated with monoclonal anti-CFTR antibody (SDS-PAGE run under reducing conditions). A 174-kDa phosphoprotein is present. Lane 2, phosphoprotein remaining after CFTR immunodepletion. Phosphorylated products are present at 52, 85, and 120 kDa. The 174-kDa phosphoprotein was removed by the anti-CFTR antibody.



Reconstitution of Immunopurified Anion Channel Protein

The proteins isolated by our modified immunopurification protocol contained an abundance of immunoreactive 140-kDa protein, as shown in Fig. 1. This 140-kDa protein, when reconstituted into planar bilayers, forms 30-40-pS anion channels that are activated by Ca(23, 29) . Therefore, when the entire complex of immunoaffinity-purified anion channel proteins obtained by the protocol described above was reconstituted into artificial liposomes and then incorporated into planar lipid bilayers, two different anion channels were routinely seen. Incorporated channels were recorded at different holding potentials in symmetrical 100 mM KCl solution at pH 7.0. Typical traces of channel activity recorded at +100 mV are shown in Fig. 4. Only in a small percentage of the total number of successful trials (<5%, n = 42) were the channels seen in the same record (Fig. 4A). One channel had a conductance of 32 ± 3 pS (Fig. 4B), whereas the other had a conductance of 82 ± 9 pS (Fig. 4C) at +100 mV. The frequency of appearance of either channel was approximately equal. The sidedness of channel incorporations was random. Nevertheless, the larger conductance channel displayed current rectification (see below), and the smaller conductance channel did not. One or two channels, oriented in the same direction, were usually recorded. Both large and small conductance channels were inhibited by DIDS, but only the smaller conductance channel could be activated by Ca (data not shown; see (29) ). Moreover, if the proteoliposomes were stored at 4 °C for several hours or for longer than 4 weeks at -80 °C, the 32-pS channel was no longer seen. This latter observation is consistent with our previous findings that the 140-kDa protein was unstable following reconstitution, with the concomitant loss of associated channel activity(23) . Hence, all additional bilayer studies were performed on stored proteoliposomes so as to eliminate any contributions of the Ca-activated anion channel to the recorded currents.


Figure 4: Immunopurified anion channels incorporated into planar lipid bilayers. Immunoaffinity purified anion channel proteins were reconstituted into artificial liposomes and then incorporated into planar lipid bilayers. Bilayers were composed of a mixture of phosphatidylethanolamine, phosphatidylserine, and oxidized cholesterol in a 2:1:2 ratio. The final lipid concentration was 25 mg/ml. The bilayer was bathed with symmetrical solutions of 100 mM KCl and 10 mM MOPS (pH 7.0). The dashed line represents the zero current level. The records were filtered at 100 Hz. A, simultaneous presence of a small (30 pS) and large (80 pS) conductance anion channel in the bilayer. B, small conductance anion channel. C, large conductance anion channel.



Fig. 5shows typical current traces of channel activity recorded at ±100 mV following incorporation of these stored proteoliposomes into planar bilayers. The channels were anion-selective, the ratio of cation to anion permeability being 0.11 ± 0.03 at a 10-fold KCl gradient (n = 3), and had a single channel conductance of 82 ± 9 pS at +100 mV and 35 ± 4 pS at -100 mV. Current through the open channel was greater at positive versus negative applied potentials, but single channel open probability (P(o)) was independent of voltage. Under these conditions, P(o) averaged 0.41 ± 0.07. Thus, this anion channel had kinetic characteristics similar to those were previously described in patch clamp studies of airway epithelia(3, 5, 10, 26) , including outward rectification (see Fig. 8).


Figure 5: Reconstituted immunopurified anion channel proteins: single channel characteristics, effect of phosphorylation by PKA + ATP, and effect of DIDS. Top traces, single channel recordings at holding potentials of ±100 mV (two channels were present in the bilayer). Middle traces, single channel recordings at holding potentials of ±100 mV after addition of ATP (100 µM) and the catalytic subunit of PKA (1.85 ng/ml). Note the appearance of small, well resolved current steps at both positive and negative voltages. Bottom traces, single channel recording at holding potentials of ±100 mV after addition of 100 µM DIDS added to the opposite side of bilayer from which PKA was active. DIDS completely blocked the outwardly rectified anion channel and made it possible to resolve a second small anion channel with a unitary conductance of 16 pS.




Figure 8: I/V plot of immunopurified and reconstituted anion channels before and after activation by PKA and ATP. A, I/V plots of mean current of purified anion channels reconstituted from two different preparations (with or without CFTR) under control conditions, after phosphorylation with PKA + ATP, and after phosphorylation and addition of DIDS. Conditions are indicated by the symbols defined on the figure. B, I/V plots of single channel current of purified anion channels recorded under conditions identical to those indicated in A. For both A and B, symbols indicate mean values and error bars indicate ± S.E. (n = 8). In B, errors are within the size of the symbol.



Inside-out patch clamp of apical membranes of tracheal epithelial cells showed that the native ORCC could be activated from the cytoplasmic side by PKA and ATP(13, 14, 15, 16) . Consequently, we tested whether this purified outwardly rectified anion channel could be activated by PKA plus ATP. As can be seen in Fig. 5, addition of 100 µM ATP and the catalytic subunit of PKA to the incorporated channel increased channel activity at the same holding potential. PKA plus ATP activation could be achieved from only one side of the bilayer. However, because the orientation of the incorporated channel was random, we generally added PKA and ATP to both sides of the bilayer. Addition of PKA or ATP alone to either or both sides of the bilayer produced no change in channel activity (data not shown). Inspection of the current records after PKA + ATP activation revealed the appearance of another small conductance channel. Because of the observation of the presence of a phosphoprotein at 174 kDa in our preparation ( Fig. 2and 3), we suspected that this small conductance channel might be CFTR. To test this hypothesis, we applied the inhibitor DIDS (100 µM) to the bilayer solution with the expectation that DIDS would inhibit the large conductance outwardly rectified channel but leave the smaller one unaffected. This prediction was borne out by experiment (Fig. 5). During the first 2 min after the addition of DIDS, bursts of outwardly rectified anion channel activity were sometimes observed as shown in Fig. 4, but after this time they were never again seen. The small anion channel had a unitary conductance of 16 pS and a linear current/voltage relationship (see Fig. 8). This small, linear channel was found to be sensitive to DPC, a known blocker of CFTR (Fig. 6). Increasing concentrations of DPC were added to the presumptive extracellular side of the incorporated channel. Doses of 200 µM of DPC inhibited approximately 50% of channel activity. When 300 µM DPC was added, channel activity was completely blocked.


Figure 6: Effect of DPC on small anion channel remains after inhibition of the outwardly recitified anion channel by DIDS. Conditions were the same as for Fig. 5. Dashed lines represent the zero current level. The record was filtered at 100 Hz, and only the current traces at +100 mV are shown. Top trace, single channel recording of small anion channel resolved after inhibition of outwardly rectified anion channel by DIDS. Two current levels were observed, indicating the presence of two identical anion channels in the membrane (Control). Middle trace, single channel recording after addition of 200 µM DPC on the presumptive extracellular side of the incorporated channel. Bottom trace, single channel recording after addition of 300 µM of DPC to the presumptive extracellular side of the incorporated channel.



Reconstitution of Immunopurified Anion Channel Proteins after CFTR Precipitation

We have thus far demonstrated that our immunopurified anion channel preparation contains two PKA-sensitive anion channels: CFTR and an outwardly rectified anion channel. We decided to exploit the simultaneous bilayer reconstitution of both of these channels to test directly whether CFTR was indeed involved in modulating the activity of outwardly rectified anion channels. Our strategy was to immunodeplete our anion channel preparation of CFTR and, subsequent to incorporation into bilayers, determine whether the outwardly rectified anion channels are present and whether they could still be activated by PKA and ATP. The results are presented in Fig. 7. Essentially, a protocol identical to that used in the experiment of Fig. 4was employed. As can be seen in the traces labeled Control at the top of the figure, typical outwardly rectifying anion channels were seen. For this particular experiment, two channels were present in the bilayer. Unitary conductance (82 pS at +100 mV), open probability (0.37 ± 0.06), and mean current (see Fig. 8) for this channel were indistinguishable from those of outwardly rectifying channels observed prior to immunoprecipitation of CFTR (cf. Fig. 5). However, addition of the catalytic subunit of PKA together with 100 µM ATP to either side of the incorporated outwardly rectified anion channel at holding potentials of ±100 mV did not increase channel activity, in contrast to the observation of increased channel activity produced by PKA + ATP-induced phosphorylation in the presence of CFTR. Single channel P(o) after addition of ATP and PKA was 0.39 ± 0.07 at ±100 mV, values indistinguishable from controls. Furthermore, when 100 µM DIDS was added to the presumptive extracellular side of the incorporated channel (with or without previously added ATP and PKA), a complete block of channel activity was seen, i.e. no small, linear anion channels were present. This experiment has been performed a total of eight times with identical results. This finding confirms that CFTR was successfully immunoprecipitated and was not present in the CFTR-depleted preparation. As a control, nonimmune IgG was substituted for anti-CFTR antibodies in the above immunoprecipitation protocol. Following reconstitution and incorporation of this material into bilayers, the outwardly rectifying anion channel could still be activated by PKA + ATP, and CFTR channels could also be seen. These results suggest a direct regulatory relationship between CFTR and this outwardly rectified anion channel in bilayers.


Figure 7: Reconstituted immunopurified anion channel proteins after CFTR precipitation: single channel characteristic and effect of PKA and DIDS. Immunopurification and reconstitution of purified protein was the same as described in the legend of Fig. 3except that immunopurified protein was immunodepleted of CFTR with anti-CFTR antibodies. Incorporated channels were recorded following a procedure identical to that described for Fig. 5. Dashed lines represent the zero current level. The record was filtered at 100 Hz. Top traces, single channel recording at holding potentials of ±100 mV (two channels were present in the bilayer). Middle traces, single channel recording at holding potentials of 100 mV after addition of ATP (100 µM) and the catalytic subunit of PKA (1.85 ng/ml) to both sides. Note that PKA + ATP did not alter channel activity. Bottom traces, single channel recording at holding potentials of ±100 mV after addition of 100 µM DIDS. Channel activity was completely blocked by DIDS. The small anion channel was not present in this preparation.



Fig. 8presents summary I/V curves for the immunopurified anion channels recorded under control conditions with (open circles) and without (open triangles and diamonds) CFTR and after phosphorylation with PKA plus ATP (solid circles and squares). Fig. 8A shows the mean current versus voltage curves, whereas Fig. 8B displays the single open channel I/V relations. It is apparent that PKA-induced phosphorylation activates an outwardly rectified anion channel (with no change in single channel current), but only when CFTR is present in the bilayer. After phosphorylation but in the presence of DIDS, only a small, linear conductance remained in the bilayer (solid squares).


DISCUSSION

Our results demonstrate that we have immunopurified from bovine tracheal epithelia and functionally reconstituted into planar lipid bilayers an outwardly rectified anion channel and the CFTR Cl channel. The purified material contained four phosphoproteins with apparent molecular masses of 52, 85, 120, and 174 kDa. We demonstrated that the 174-kDa protein is CFTR by precipitation of this protein with anti-CFTR antibodies. In bilayers, the CFTR channel was activated by PKA + ATP, had a linear I/V curve, was inhibited by DPC, and was insensitive to DIDS, properties similar to those reported for CFTR(27, 28) . Western blot analysis of the immunopurified material revealed that the 52-kDa protein was recognized by alphap38 antibodies. The biophysical characteristics of the isolated and reconstituted anion channels were very similar if not identical to the characteristics of similar channels recorded in patch clamp studies from airway epithelia (i.e. activation by PKA + ATP, conductance, I/V properties, inhibitor sensitivities, etc.), suggesting that the isolated channels were well preserved. Because an antibody (alphap38) that was raised against another anion channel, namely a 30-40-pS, linear, Ca-activated anion channel(19, 29) , was used for immunopurification, we also observed this channel in bilayers. However, because this channel is extremely labile, its appearance in the bilayer could be controlled by preparing proteoliposomes and storing them at 4 °C overnight or longer. Under these conditions, the Ca-activated anion channel was never observed (in over 50 experiments) after incorporation of the stored immunopurified material into the bilayer. Hence, only the outwardly rectified and CFTR anion channels could be seen in the bilayer.

As both channels were present in the same preparation, it was possible to explore the proposed regulatory relationship between the two channels. The outwardly rectified anion channel was not affected by voltage or [Ca] or by ATP from either side of the membrane. This finding is in contrast to previous reports that ORCCs are activated by ATP from the extracellular side of the apical membrane (30, 31, 32, 33) .

We demonstrate that the reconstituted outwardly rectified anion channel was activated by PKA and ATP only when CFTR was present in immunopurified material. This finding is in agreement with three recent patch clamp studies(8, 9, 12) . Egan et al.(8) showed that transfection of CF airway epithelial cells with the wild-type CF gene corrected cAMP regulation of Cl secretion, induced the appearance of low conductance Cl channels attributable to CFTR, and permitted PKA to activate ORCC. Gabriel et al.(9) demonstrated that PKA regulation of ORCC was defective in nasal epithelial cells isolated from a transgenic CF (-/-) mouse in which both CFTR mRNA and protein were absent. Third, in whole-cell studies of normal human tracheal epithelial cells and cell lines of CF tracheal cells complemented with the normal CF gene, Schwiebert et al.(12) concluded that both CFTR and ORCC contribute to whole-cell anion channel current and that CFTR is necessary and required for cAMP regulation of ORCC. At least two possibilities exist to account for these observations. First, because mutations in CFTR lead to diminished CFTR trafficking to the apical plasma membrane(34, 35) , the absence of membrane CFTR would prohibit cAMP activation of ORCC. This hypothesis is consistent with the results presented in this paper. Second, assuming some mutated CFTR does get to the membrane, a mutation in the CF gene may alter the activation process of the ORCC, interfering with the ability of kinase A to phosphorylate the channel. Alternatively, defective CFTR may interfere with the channel gating mechanism that controls PKA-induced phosphorylation-mediated entry of the channel into its open state.

Our study on purified and reconstituted anion channels resolves several questions concerning the relationship between CFTR and outwardly rectified anion channels. We demonstrated that in vitro phosphorylation of the isolated proteins was not affected by CFTR precipitation but that the outwardly rectified anion channel could not be activated by PKA and ATP in the absence of CFTR. The biophysical properties of the outwardly rectified anion channel were independent of the presence of CFTR in the absence of PKA and ATP, indicating that CFTR itself does not affect channel conduction or gating. This observation is consistent with earlier patch clamp studies where it was possible to record ORCCs in CF (-/-) mouse cells and CF human trachea in cells activated by sustained strong depolarization(12) . Our study suggests that a direct interaction between CFTR and outwardly rectified anion channels is required for PKA activation, although it does not exclude the possibility that intermediate steps may still be involved.

In summary, we have isolated and reconstituted an outwardly rectified anion channel and the CFTR Cl channel from bovine tracheal epithelia. These isolated channels were functionally very well preserved as demonstrated by the similar biophysical characteristics of reconstituted channels with the corresponding channels recorded in patch clamp studies of native airway epithelia. We also demonstrated that the outwardly rectified anion channel can be activated by PKA and ATP. Functional channel activation by the protein kinase A was possible only when CFTR was contained in the immunopurified material in spite of the fact that there was no difference in phosphoprotein distribution. After precipitation of CFTR, PKA and ATP did not activate these outwardly rectified anion channels in bilayers. This result suggests that the mechanism of regulation of these outwardly rectified anion channels by CFTR involves an interaction between the two channels. In vitro phosphorylation of purified proteins by PKA and ATP was unaffected by CFTR precipitation, suggesting that CFTR does not play a role in phosphorylation of protein(s) that forms these conductive pathways. The purification of an outwardly rectified anion channel now raises the possibility of direct biochemical and molecular studies on this important family of anion channels, particularly important in light of our finding that CFTR can modulate PKA-mediated regulation of this channel.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK42017. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowship funds provided by the Cystic Fibrosis Foundation.

(^1)
The abbreviations used are: PKA, protein kinase A; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; S, siemens; CF, cystic fibrosis; ORCC, outwardly rectified chloride channel; DPC, diphenylamine-2-carboxylic acid; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; CFTR, cystic fibrosis transmembrane conductance regulator.


ACKNOWLEDGEMENTS

We thank Dr. Catherine Fuller for helpful discussions and for critiquing the manuscript several times during its preparation. We thank Deborah Keeton for excellent technical assistance and Charlae Starr and Ann Harter for prompt and courteous secretarial help. We greatly appreciate the gift of purified catalytic subunit of protein kinase A from Dr. Gail Johnson.


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