©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Epithelial Sodium Channels by the Cystic Fibrosis Transmembrane Conductance Regulator (*)

(Received for publication, October 19, 1995; and in revised form, December 22, 1995)

Iskander I. Ismailov (1) Mouhamed S. Awayda (1)(§) Biljana Jovov (1)(¶) Bakhram K. Berdiev (1) Catherine M. Fuller (1) John R. Dedman (2) Marcia A. Kaetzel (2) Dale J. Benos (1)(**)

From the  (1)Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005 and the (2)Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cystic fibrosis airway epithelia exhibit enhanced Na reabsorption in parallel with diminished Cl secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na channel (alpha,beta,-rENaC) or an immunopurified bovine renal Na channel protein complex. The single channel open probability (P(o)) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na channel P(o) following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na current. This interaction between CFTR and Na channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na channel activity, thus accounting, at least in part, for the observed differences in Na transport between normal and cystic fibrosis-affected airway epithelia.


INTRODUCTION

Cystic fibrosis (CF) (^1)is a genetic disease in which the ion secretory and absorptive functions of several epithelia, including airway mucosa (nasal and tracheal), pancreas, salivary glands, and sweat glands, are seriously impaired(1, 2, 3, 4) . Among these tissues, airway epithelial dysfunction leads to chronic clinical manifestations that ultimately result in morbidity. In the airways of CF patients, fluid and electrolyte secretion is inhibited because of the defect in the cystic fibrosis transmembrane conductance regulator (CFTR), and absorption due to an increased rate of Na transport is enhanced(5) . These events result in the accumulation of mucus, pulmonary congestion, bronchiectasis, and excessive dryness of the epithelial surface. The relationship between the pulmonary etiology of CF and the primary physiological disturbances in ion transport is not well defined.

Boucher and co-workers(5, 6, 7, 8, 9, 10) initially found that CF nasal epithelia display rates of amiloride-sensitive Na reabsorption 2-3-fold greater than epithelia obtained from normal individuals. Moreover, both the beta-adrenergic agonist isoproterenol and the adenylate cyclase activator forskolin, agents that act via the cAMP second messenger system, stimulate airway Na reabsorption. Aerosolized amiloride, an inhibitor of epithelial Na channels, has been found to inhibit excessive Na reabsorption, to improve mucociliary clearance, and to reduce the decline in pulmonary function(11) . In cell-attached patches of cultured human nasal epithelial cells, Chinet et al.(12) recorded amiloride-sensitive cation channels. These channels had a single channel conductance of 21 pS and a Na/K permeability ratio of 6. However, upon excision, these channels lost their ability to discriminate between Na and K. The same channels were observed in cultured CF nasal tissue(13) . However, in cell-attached patches, the single channel open probability (P(o)) was found to be twice as high compared with that in normal cells, consistent with the earlier results of Duszyk et al.(14) . Grubb et al.(15) found that freshly excised nasal epithelia from CFTR ``knockout'' mice also displayed Na hyperabsorption. These electrophysiologic results contrast with studies on the development of Na transport in the neonatal rat lung, which indicate that increased Na transport results in an increase in absolute levels of Na channel mRNA(16, 17) . Furthermore, Northern analysis of human airway epithelia revealed that all three subunits of a recently cloned epithelial Na channel (ENaC) (18) were present in both normal and CF tissues, but with a lower relative amount in CF compared with normal tissue(19) . Thus, while it is clear that Na reabsorption is increased in CF-affected airway epithelia, the mechanism underlying this enhanced Na transport is not known.

Recent evidence that CFTR can regulate the function of other ion channels (20, 21, 22) prompted us to examine the hypothesis that CFTR may also regulate epithelial Na channels. We co-incorporated immunopurified CFTR and either immunopurified bovine renal Na channel protein or alpha,beta,-rENaC into planar lipid bilayer membranes. The intimate relationship between ENaC (18) and immunopurified bovine renal Na channel (23) has yet to be determined. However, recent experimental evidence suggests that ENaC is one of the components, most likely the core conduction unit, of the immunopurified complex(24) . We examined basal Na channel activity in the absence and presence of CFTR as well as the influence of CFTR on protein kinase A regulation of renal Na channel function. In the presence of CFTR, the single channel activity of both ENaC and the immunopurified renal Na channel was decreased. In addition, the inward rectification of the renal channel induced by PKA-mediated phosphorylation (25) was prevented by co-incorporation of this channel with CFTR. In the absence of CFTR, PKA-mediated phosphorylation increased the activity of the immunopurified renal Na channel, but had no effect whatsoever on ENaC activity either in bilayers or following expression in Xenopus oocytes. (^2)Our results are consistent with the hypothesis that CFTR exerts a tonic inhibition of Na transport through amiloride-sensitive channels, as suggested by the recent observations of Stutts et al.(27) .


EXPERIMENTAL PROCEDURES

Purification of Renal Na Channel and CFTR Protein

Purification of amiloride-sensitive Na channel protein from bovine renal papillary collecting duct cells was performed as described previously(23) . The procedure involved homogenization, differential centrifugation, ion-exchange chromatography, and immunoaffinity purification. Final protein purity was determined by [^3H]methyl bromoamiloride binding as described previously(23) . In all preparations, the specific activity of binding exceeded 1200 pM/mg of protein. CFTR was immunopurified from bovine tracheal epithelium essentially as described (22) . In some cases, CFTR was heterologously expressed in oocytes (see below). Immunopurified renal Na channel protein or CFTR was reconstituted into liposomes as described earlier(22, 23) . Reconstituted proteoliposomes were stored at -80 °C until use.

In Vitro Expression of ENaC in Xenopus Oocytes and Oocyte Plasma Membrane Isolation

Membrane vesicles from alpha,beta,-rENaC mRNA-injected and water-injected oocytes were made as described by Perez et al.(28) . Thirty to forty oocytes in each group were washed and homogenized in high K/sucrose medium containing the following protease inhibitors: aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), phenylmethylsulfonyl fluoride (100 µM), and DNase I (2 µg/ml). Oocyte membranes were isolated by discontinuous sucrose gradient density centrifugation and resuspended in 300 mM sucrose, 100 mM KCl, and 5 mM MOPS (pH 6.8). Membrane vesicles were separated into 50-µl fractions and stored at -80 °C until use.

Planar Lipid Bilayer Experiments

Planar lipid bilayers were made from a lipid solution containing a 2:1:2 mixture of diphytanoylphosphatidylethanolamine/diphytanoylphosphatidylserine/oxidized cholesterol (in n-octane; final lipid concentration = 25 mg/ml). Lipids were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). Bilayer formation was ascertained by an increase in membrane capacitance to 300-400 picofarads. Bilayers were bathed in 100 mM NaCl containing 10 mM MOPS/Tris buffer (pH 7.4). All solutions were made with Milli-Q water and were filter-sterilized by passing the solution through 0.22-µm filters (Sterivax-GS filters, Millipore Corp., Bedford, MA). Current measurements were performed using a high gain amplifier circuit (29) as described previously(30) . The electrical continuity was provided by Ag/AgCl electrodes and 3 M KCl, 3% agar bridges that prevented electrode polarization in Cl-free solutions. The reconstituted vesicles or oocyte membranes were applied with a glass rod to one side (trans) of a preformed bilayer with the membrane potential held at -40 mV. Under these experimental conditions, the channels were oriented with the amiloride-sensitive (extracellular) side facing the trans solution and the cytoplasmic side facing the cis solution in over 90% of the incorporations. Voltage was applied to the cis chamber, and the trans chamber was held at virtual ground.

The total number of channels incorporated into any given bilayer membrane was determined by PKA-induced phosphorylation or by imposition of a hydrostatic pressure gradient as described previously in detail (30, 31) . Briefly, PKA + ATP act by increasing the P(o) of immunopurified amiloride-sensitive renal Na channels and CFTR in planar lipid bilayers rather than by recruiting new channels because no channel-containing vesicles are present in the solutions bathing the bilayer. Imposition of a hydrostatic pressure gradient across a channel-containing bilayer also increases P(o), but only for the ENaC channels (31, 32) . Thus, these maneuvers reveal ``silent'' channels already resident in planar lipid bilayers.

Data Analysis

Single channel records were acquired and analyzed using pCLAMP software (Axon Instruments, Inc., Foster City, CA) as described previously(30) . Single channel data were stored digitally and, for analysis, were filtered at 300 Hz with an 8-pole Bessel filter and acquired at 1 ms/point. All data analyses were performed in bilayers containing a single active Na channel. If more than one Na channel was detected, then any data acquired from that experiment was not used in the analysis. We have not found conditions in which only a single CFTR channel could be incorporated into a bilayer. Consequently, in the CFTR experiments, two or three CFTR channels were present.

The existence of conductance sublevels in both ENaC (32) and immunopurified renal amiloride-sensitive Na channels (30, 33) makes the definition of P(o) ambiguous because it depends upon whether the unitary current is assumed to be the main state current (I) or a substate current (i(s)). Mean current (I) is the product of unitary current (I), number of functional channels (N), and their P(o): I = I N P(o). However, P(o) calculated using the same value for unitary conductance within the same set of experiments can represent accurately the changes in activity of the channel. For our purposes (namely, current/voltage (I/V) curves and as a descriptor of channel activity), we employed this approach of calculating P(o), using the most frequently observed main transition state as the unitary current. We have previously shown that using either I or i(s) results in the same P(o) as long as the subconductance levels (m) are equally spaced(24) .

Statistical Methods

Experiments were analyzed by one-way analyses of variance. The probability (p) that the difference between two population means was significant was determined by computing the t statistic using Student's t test. Data are expressed as the mean value ± S.D. for n experiments.


RESULTS

Effect of CFTR on Single alpha,beta,-ENaC Channels

To test the hypothesis that CFTR influenced epithelial Na channel activity, plasma membrane vesicles prepared from oocytes expressing alpha,beta,-rENaC alone, alpha,beta,-rENaC + CFTR, or CFTR alone were fused to planar lipid bilayer membranes. Fig. 1(top trace) shows a typical current record of alpha,beta,-rENaC activity. The channel had a mean conductance of 13 ± 1 pS, with a time constant of opening of 90 ± 17 ms and a time constant for exit from the closed state of 41 ± 13 ms. This channel was open 50% of the time, with occasional openings of 53 ± 10 ms in duration to a 40-pS level. alpha,beta,-rENaC in bilayers was Na-selective (Naversus K permeability of 10:1, n = 9) and displayed a high sensitivity to amiloride (apparent inhibitory equilibrium constant (K(i)) of 170 ± 35 nM, n = 10). The P(o) of alpha,beta,-rENaC was different when vesicles from oocytes expressing both alpha,beta,-rENaC and CFTR were fused with the bilayer or when oocyte membrane vesicles containing wild-type CFTR were incorporated into bilayers already containing a single alpha,beta,-rENaC. The success rate in achieving simultaneous incorporation of both alpha,beta,-rENaC and CFTR channels from vesicles presumably containing both these channels was 32% (12/38, where 38 represents the total number of successful incorporations of alpha,beta,-rENaC) and 9% for sequential incorporations of CFTR into rENaC-containing bilayers (6/67, where 67 represents the total number of successful incorporations of alpha,beta,-rENaC). However, in only three of these latter six successful experiments did CFTR exert any influence on alpha,beta,-rENaC (see below). The major effect of CFTR was to decrease alpha,beta,-rENaC P(o) by 24%, from 0.51 ± 0.05 to 0.39 ± 0.03 (p < 0.005, n = 15 successful co-incorporations). The Naversus K permeability ratio and amiloride sensitivity were unaffected by CFTR (n = 4 for each) (data not shown). Control experiments using membrane vesicles prepared from water-injected oocytes produced no channel activity at all (600 attempts). In addition, experiments were performed using ENaC- and water-injected oocyte membrane vesicles or liposomes prepared without CFTR. In these cases, the biophysical properties of alpha,beta,-rENaC remained unaltered. Thus, other constituents present in the vesicles were not responsible for the observed effects. The same control experiments were performed using purified renal Na channels (see below).


Figure 1: Single channel records of alpha,beta,-rENaC in planar lipid bilayers and effect of subsequent incorporation of CFTR and PKA-mediated phosphorylation on rENaC activity. Both rENaC and CFTR were expressed in Xenopus oocytes, and the plasma membranes were fused to a bilayer. Bilayers were bathed with symmetrical solutions of 100 mM NaCl and 10 mM MOPS/Tris (pH 7.4). Additions were made sequentially as shown. PKA (1.85 ng/ml), ATP (100 µM), anti-CFTR antibodies (50 ng/ml), and DPC were added to both bathing compartments, while amiloride was added only to the trans bathing solution. The holding potential was +100 mV. Traces are representative of 12 separate experiments.



As described above, in 26 of the 38 experiments in which incorporations of alpha,beta,-rENaC and CFTR coexpressed in oocytes were attempted, only alpha,beta,-rENaC was detected. Because CFTR channel activity could not be recorded in the absence of PKA-mediated phosphorylation and because CFTR by itself oriented in the bilayer in a random fashion (22, 34) , PKA + ATP were first added to the trans solution, followed by addition to the cis bathing solutions. In the 12 successful experiments, PKA + ATP addition to the cis compartment resulted in activation of CFTR, whereas trans addition had no effect. Three CFTR channels are evident in the experiment shown. The addition of anti-CFTR-(505-511) antibodies (34, 35) subsequent to the addition of PKA + ATP inhibited CFTR, leaving alpha,beta,-rENaC unscathed (P(o) = 0.39 ± 0.04; p > 0.1) (Fig. 1, fourth trace, left), thus providing a way to discern ENaC activity. These anti-CFTR-(505-511) antibodies did not have any effect on alpha,beta,-rENaC in the absence of co-incorporated CFTR (n = 5) (data not shown). PKA + ATP had no effect on alpha,beta,-rENaC activity in the absence of CFTR (n = 5).^2 The subsequent addition of 5 µM amiloride or 300 µM DPC completely inhibited alpha,beta,-rENaC and CFTR, respectively (Fig. 1, bottom trace). These results indicate that CFTR, in combination with alpha,beta,-rENaC, preferentially oriented in the bilayer in the same direction as the Na channel, namely, with its presumptive cytoplasmic side facing the cis compartment. Consequently, amiloride or DPC was effective only from the trans compartment, while the anti-CFTR-(505-511) antibodies worked from the cis side. Amiloride and DPC had no effect on CFTR and Na channels, respectively, at these concentrations (n = 6 for each). Therefore, the increase in total membrane current seen upon PKA + ATP addition was due to activation of CFTR alone.

A similar sidedness to PKA action was noted in the series of experiments performed in which CFTR was incorporated into a bilayer already containing an active alpha,beta,-rENaC. The total membrane current was increased in three experiments (out of a total of 67) when PKA + ATP were added to the cis side of the bilayer chamber. In these same three experiments, the P(o) of alpha,beta,-rENaC was decreased after the presumed insertion of CFTR, but before PKA + ATP addition. In 63 of the remaining 64 experiments in which the activity of alpha,beta,-rENaC was not altered upon the presumed insertion of CFTR, the cis addition of PKA and ATP did not result in any change of membrane current, suggesting that CFTR was not present. However, in 2 of these 64 experiments, the subsequent addition of PKA and ATP to the trans side revealed increased CFTR channel activity, with no concomitant alteration of alpha,beta,-rENaC P(o). Also, an increase in CFTR channel activity upon PKA + ATP addition to the cis compartment was noted in a single experiment. In this experiment, the activity of alpha,beta,-rENaC was not initially altered upon the insertion of CFTR, nor was it altered after CFTR activation with PKA + ATP. One explanation for these observations is that while both alpha,beta,-rENaC and CFTR are present in the membrane, they are not in close enough proximity to interact. These results are consistent with our previous observations of a directed orientation of ENaC channels incorporated into bilayers held at -40 mV (32) and a random orientation of CFTR channels incorporated into bilayers by themselves (34) . These results also suggest that a parallel orientation of CFTR and ENaC may be critical for their interaction.

To ensure that the anti-CFTR-(505-511) antibodies did not alter the activity of alpha,beta,-rENaC in the presence of wild-type CFTR, a series of anion replacement experiments was performed with CFTR-impermeable MOPS anion substituted for Cl (Fig. 2). Incorporations of alpha,beta,-rENaC and CFTR were performed using vesicles isolated from oocytes expressing either channel. In 27 experiments, no CFTR channel activity was observed when PKA and ATP were added to either or both bathing solutions. This result was expected because CFTR, although activated by phosphorylation, could not conduct MOPS. However, at the end of each experiment, dry CsCl was added to both compartments to a final concentration of 100 mM (note: in five separate experiments, we found that ENaC in bilayers was essentially impermeable to Cs). In 7 of the 27 experiments, CFTR channel activity became evident after the addition of CsCl. In five of these seven experiments, the initial P(o) of alpha,beta,-rENaC was lower subsequent to the putative incorporation of CFTR. This decrease in P(o) averaged 23.1 ± 2.0%. There was no change in the P(o) of alpha,beta,-rENaC in two of these experiments. This lack of effect of CFTR on alpha,beta,-rENaC presumably occurred because CFTR was not oriented properly in the bilayer, as evidenced by the inability of DPC to inhibit CFTR when applied to the same compartment as amiloride. The addition of PKA and ATP did not affect alpha,beta,-rENaC, similar to what was observed in Cl-containing solutions. All of these results on ENaC are summarized in the current/voltage curves shown in Fig. 3. Fig. 3demonstrates that CFTR decreased current through ENaC at all potentials independent of whether or not CFTR was conductive.


Figure 2: Effect of CFTR and PKA-mediated phosphorylation on single channel activity of alpha,beta,-rENaC in planar lipid bilayers. Bilayers were bathed with symmetrical 100 mM MOPS and 10 mM MOPS/Tris (pH 7.4) solutions. All other conditions were the same as described for Fig. 1. The membrane was held at +100 mV. Results are representative of five experiments.




Figure 3: Mean current versus applied voltage relationships of alpha,beta,-rENaC in presence or absence of wild-type CFTR before and after PKA + ATP activation in planar lipid bilayers. Experimental conditions are indicated by the symbol legend on the figure. Each point represents the mean ± S.D. for at least three separate experiments under each condition. Ionic conditions were identical to those described for Fig. 1.



Given the usual variability in the gating of ion channels over long periods of time, we have performed a rigorous analysis of alpha,beta,-rENaC activity in the presence and absence of CFTR (before and after the addition of PKA + ATP (Fig. 4, A and B, respectively). P(o) was calculated for each minute of recording (total of 10 min for each experimental condition) starting at the third minute after the addition of reagents. Clearly, the P(o) of alpha,beta,-rENaC was significantly higher in the absence of CFTR than in its presence. p values for these unpaired measurements were less than 0.01 and 0.05 over the time course of these experiments.


Figure 4: Bar graphs of single alpha,beta,-rENaC P(o)versus time in absence (white bars) and presence (hatched bars) of CFTR. This analysis was performed in the absence of (A) or following (PKA + ATP)-mediated phosphorylation (B). The vertical bars represent the means ± S.D. of 17 separate experiments.



Effect of CFTR on Single Immunopurified Renal Na Channels

The influence of CFTR on an amiloride-sensitive Na channel that was sensitive to PKA-mediated phosphorylation was next examined. For these experiments, immunopurified bovine renal papillary collecting duct amiloride-sensitive Na channel (NaCh) (22, 23) and immunopurified bovine CFTR were used. Typically, immunopurified renal Na channels reconstituted into planar lipid bilayers displayed a very low P(o) (0.02 ± 0.01)(23, 25, 30, 33) . Under these circumstances, it was pointless to expect further inhibition of this mostly closed channel by the presence of CFTR. To overcome this problem, we reduced [Na] to 10 mM (leaving [Na] at 100 mM) in order to increase single channel P(o) by a factor of 5(33) . All other biophysical characteristics of this channel were unaltered by the imposition of this Na gradient. Fig. 5depicts representative current traces at ±80 mV under these conditions. The same experimental sequence as with rENaC was employed: incorporation of NaCh with or without CFTR, PKA + ATP addition, and lastly, addition of channel inhibitors. Control traces (Fig. 5, top trace) displayed discrete channel openings with a P(o) of 0.11 ± 0.02 at both ±80 mV when the membrane was bathed with a [Na] of 100 mM and a [Na] of 10 mM (n = 155). Both mean and unitary current/voltage (I/V) relationships of the channel were linear and intersected the voltage axis at +52 ± 3 mV (Fig. 6), suggesting that the channel had a cation/anion permeability ratio of 9:1. As described earlier(30, 33) , this channel displayed a main open state unitary conductance of 40 pS, with two equally spaced subconductive states of 13 pS.


Figure 5: Channel activity of immunopurified renal Na channel before and after co-incorporation of wild-type CFTR in planar bilayer. The bathing solution in the trans compartment contained 100 mM NaCl and 10 mM MOPS/Tris (pH 7.4), and that in the cis compartment contained 10 mM NaCl, 90 mMN-methyl-D-glucamine chloride, and 10 mM MOPS/Tris (pH 7.4). The holding voltage was ±80 mV. This experiment is typical of 21 such experiments.




Figure 6: Mean current versus applied voltage relationships of immunopurified renal Na channel in presence or absence of wild-type CFTR before and after PKA + ATP activation in planar lipid bilayers. Conditions are stipulated by the symbol legend on the graph. Each symbol, and the error bars indicate the mean ± S.D. for at least six separate experiments for each condition. Ionic conditions were the same as described for Fig. 5.



The presumed incorporation of CFTR did not change NaCh P(o), although it affected the NaCh gating pattern in a way that the channel resided predominantly in its main 40-pS conduction state. In addition, the Na/K permeability ratio of the phosphorylated renal Na channel was unaltered by CFTR, namely, 20:1 (n = 7). Given a negligible change in P(o), this effect on gating by itself could not account for the extent of the increase in Na transport observed in CF airway epithelia. Moreover, it was not clear if this effect was due to the presence of CFTR in the bilayer membrane. Therefore, we used PKA and ATP to clarify this issue. The success of co-incorporation, as with alpha,beta,-rENaC, was good (21/59, where 59 represents the total number of successful incorporations of NaCh) when proteoliposomes containing both NaCh and CFTR were fused with the bilayers. The success rate of co-incorporation was significantly lower when attempts to insert CFTR were made subsequent to the successful incorporation of NaCh (4/96, where 96 represents the total number of successful incorporations of NaCh).

With regard to the orientation of these two channels in the membrane, the situation was complex. In 58 out of the 59 experiments in which proteoliposomes presumably containing both NaCh and CFTR were fused with the bilayer, the subsequent addition of PKA + ATP to the cis compartment activated NaCh. CFTR was simultaneously activated, however, only in 13 of these experiments. Moreover, in seven experiments, CFTR channels were seen following the trans addition of PKA + ATP. In a single experiment, the cis addition of PKA + ATP activated CFTR, whereas NaCh was activated by PKA + ATP added to the trans side.

The cis addition of PKA + ATP activated NaCh in 92 out of the 96 experiments in which CFTR was incorporated into a bilayer already containing NaCh, while in the remaining four, NaCh was activated by the trans addition of PKA + ATP. In only 2 of these 92 experiments did the cis addition of PKA + ATP simultaneously activate CFTR. The subsequent addition of the phosphorylating mixture to the trans compartment revealed CFTR channels only twice more. Therefore, in total, the shift in gating of NaCh to the main conductance mode occurred in 15 experiments and only when NaCh and CFTR were oriented in the membrane in the same fashion (i.e. when PKA + ATP activated both channels simultaneously when added to the same side of the bilayer). The identity of these channels was established using their respective specific inhibitors (anti-CFTR-(505-511) for CFTR and amiloride for NaCh). The effects of amiloride were reversible, thus permitting further identification of the CFTR channel. These pharmacological tests independently confirmed the sidedness of the incorporation.

In the presence of anti-CFTR-(505-511), the characteristic pattern of (PKA + ATP)-mediated stimulation of NaCh activity was similar to that reported earlier for NaCh in the absence of CFTR(33) , i.e. a shift to long-lived, 13-pS conductance sublevels (Fig. 5, bottom two traces). However, certain biophysical properties of these renal Na channels in the presence of CFTR were altered compared with those measured in its absence. First, as indicated earlier, the presence of wild-type CFTR promoted a shift of NaCh gating into its largest (40 pS) conduction state. Second, the P(o) of the renal Na channel, PKA-phosphorylated in the presence of CFTR, was significantly lower (P(o) = 0.44 ± 0.05) than observed under the same experimental conditions in the absence of CFTR (P(o) = 0.62 ± 0.07; p < 0.005). Third, the amiloride sensitivity of this purified renal Na channel decreased following phosphorylation, from an apparent K(i) of 0.7 ± 0.1 µM in controls (n = 19) to 2 ± 0.3 µM in the presence of CFTR (n = 15), rather than to 20 µM previously observed in the absence of CFTR(33) . Fourth, in the presence of CFTR, PKA-mediated phosphorylation did not produce an inward rectification of NaCh gating, unlike what occurred in the absence of CFTR(25, 33) . The elimination of the phosphorylation-induced voltage-dependent gating of NaCh in the presence of CFTR can be more clearly seen in the mean current (i.e. the current averaged over the entire period of observation at each applied voltage) versus applied voltage (I/V) curves shown in Fig. 6. It is apparent from this graph that CFTR effectively prohibited the PKA-mediated induction of inwardly rectified gating of NaCh, significantly lowering its P(o) at negative applied potential and thus lowering inward current. Again, the use of control liposomes devoid of CFTR left unaltered the properties of the renal Na channel with regard to PKA-mediated phosphorylation (n = 100).

Effect of G551D CFTR on Single Immunopurified Renal Na Channels

The next set of experiments was performed to test the hypothesis that a mutant form of CFTR, namely, G551D CFTR, would influence the P(o) and PKA sensitivity of renal NaCh. We used the same experimental design as described for NaCh, except that G551D CFTR immunopurified from L cells overexpressing this mutant (34) was used in place of CFTR. In these experiments, no change in the biophysical properties of NaCh was observed (n = 51). One complication in these experiments is that because this form of CFTR is nonconductive(36) , we were unable to identify its presence and/or orientation in the planar lipid bilayer. We have previously shown that in 24 out of 26 separate experiments(34) , G551D CFTR co-reconstituted with a preparation containing an outwardly rectified chloride channel (ORCC), but devoid of CFTR, produced a change in the rectification properties of ORCC in the negative potential quadrant. Therefore, we exploited this effect to resolve the issue of whether G551D CFTR was present in the bilayer membrane (Fig. 7). Proteoliposomes containing both ORCC and G551D CFTR were fused with bilayers already containing a NaCh. To investigate the properties of NaCh, we eliminated the electrical activity of ORCC by switching to NaMOPS solutions. Again, as was done in the previous experiments with alpha,beta,-rENaC, the presence of ORCC was assessed at the end of every experiment by adding dry CsCl to both compartments to achieve a final concentration of 100 mM. In four experiments of this design (i.e. the use of CsCl in the absence of DIDS) out of a total of 137 experiments performed, an activated ORCC with altered rectification properties was observed at the end of the experiment with PKA, ATP, and CsCl, added to both sides of the bilayer after inhibition of an activated NaCh with amiloride. We verified that, in these four experiments, ORCC (and therefore CFTR; see (34) ) was oriented in the same direction as NaCh because 100 µM DIDS inhibited ORCC from the same side as amiloride inhibited NaCh. Nonetheless, none of these 137 experiments demonstrated biophysical properties of NaCh (e.g. conductance, P(o)) different from the control. Moreover, PKA and ATP always activated NaCh, inducing an inward rectification of its gating (Fig. 8). Therefore, we conclude that G551D CFTR, unlike wild-type CFTR, does not interact with renal Na channels to alter their kinetic and PKA regulatory properties.


Figure 7: Effect of G551D CFTR on single immunopurified renal Na channel before and after PKA + ATP phosphorylation in planar lipid bilayer. Bilayers were bathed with symmetrical 100 mM NaMOPS and 10 mM MOPS/Tris (pH 7.4) solutions. All other conditions were the same as indicated for Fig. 1. G551D CFTR and an outwardly rectified chloride channel were contained in the same proteoliposome preparation(29) . The holding potential was ±80 mV. This experiment is representative of four such experiments.




Figure 8: Mean current versus voltage relationships of immunopurified renal Na channel in presence or absence of G551D CFTR before and after PKA + ATP activation in planar lipid bilayers. Each symbol represents the mean ± S.D. (n = 4). Ionic conditions were the same as indicated for Fig. 7.




DISCUSSION

The results presented in this paper demonstrate a direct interaction between CFTR and amiloride-sensitive epithelial Na channels. Our findings are consistent with those observed in native epithelia, namely, that in the absence of cell membrane CFTR (i.e. cystic fibrosis), basal Na transport rate is enhanced(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . Furthermore, our experiments demonstrate that CFTR can directly modulate single Na channel P(o), again consistent with previous patch-clamp data(12, 13) . Na channels of normal upper airway epithelia are not responsive to elevations in intracellular cAMP (7) . When CFTR is absent, these airway Na channels can be activated by cAMP(7) . The results with PKA-sensitive renal NaCh suggest that its interaction with CFTR may be more complex than that of alpha,beta,-rENaC. The effect of CFTR on NaCh channels occurred primarily at the level of the gating of these channels following PKA-induced phosphorylation, resulting in an inhibition of Na current in the physiologically relevant direction (inward). If ENaC subunits are part of the native Na channel complex of upper airway epithelia, cAMP sensitivity must be conferred by additional protein components because ENaC itself cannot be activated by PKA + ATP either in the presence or absence of CFTR (Fig. 1).^2 Moreover, in native epithelia, CFTR appears to uncouple the airway Na channel's sensitivity to cAMP, opposite to CFTR's effect on outwardly rectified chloride channels (34) . The fact that CFTR does not totally uncouple PKA sensitivity of the renal Na channel studied here is consistent with the minimal renal function impairment in patients with CF (37, 38) and suggests major differences in PKA regulatory pathways between renal and airway Na channels. Moreover, distal lung Na channels studied in mammalian alveolar type II cells are activated by PKA-mediated phosphorylation(39) . We speculate that the hormonal regulation of amiloride-sensitive Na channels will thus be regional and specifically designed to accommodate the physiological needs of the specific tissue in which the channels are located.

Stutts et al.(27) recently reported that transfection of alpha,beta,-rENaC into Madin-Darby canine kidney cells resulted in the appearance of an inward Na current following induction by an overnight incubation with dexamethasone and butyrate. Moreover, this induced Na current was amiloride-sensitive and could be further augmented by forskolin. Cotransfection of these cells with CFTR significantly lowered basal amiloride-sensitive Na current and obliterated the forskolin response. Likewise, in similar experiments using 3T3 fibroblasts, permeable cAMP analogs stimulated whole cell Na currents in cells expressing rENaC only, but inhibited them in cells coexpressing rENaC and CFTR. Thus, these authors concluded that CFTR acts ``as a cAMP-dependent negative regulator of Na channels.'' While the results presented in the present paper are consistent with their conclusion, they are not as dramatic as those reported by Stutts et al.(27) . We suggest that the interaction between CFTR and amiloride-sensitive Na channels is complex and may involve many other factors in addition to direct protein-protein associations. For example, in many Na-transporting epithelial tissues, particularly renal distal tubules in which both Na channels and CFTR are simultaneously present(40) , amiloride-sensitive Na transport can still be activated by cAMP(41) . Our observations are also consistent with the hypothesis that ENaC channels represent the core conduction element of NaCh, and its responsiveness to regulatory inputs is due to associated polypeptides(24, 26, 42, 43) .

There is no apparent theoretical explanation for how a CFTR channel in a relatively large two-dimensional artificial membrane can influence the function and regulation of a single Na channel. It is likely that energetically favorable protein-protein interactions occur, especially between molecules that are predisposed for such interaction. Similar interactions between CFTR and an outwardly rectified anion channel in bilayers have been demonstrated(22) , thus establishing precedence for the phenomenon. Moreover, in four experiments, the conduction mutant G551D CFTR was not able to substitute for CFTR in producing diminution of the P(o) of ENaC and prohibiting the PKA-induced inward rectification of the renal Na channel. We cannot rule out the possibility that in these four experiments, G551D CFTR and NaCh did not establish protein-protein interaction in the bilayer (e.g. because of the presence of ORCC). However, we did not observe any effect of G551D CFTR in any of the other 51 experiments performed without ORCC. In fact, in only one experiment did we observe both wild-type CFTR and the renal Na channel present in the same membrane and oriented in the same direction, but apparently non-interacting. Taken together, these observations suggest that, while wild-type CFTR does not have to be functional in order to exert influence over Na channels, the first nucleotide-binding domain region of CFTR is critical for the interaction to occur because that is the locus of the G551D mutation. Moreover, in experiments in which G551D CFTR was inserted into NaCh-containing bilayers, PKA and ATP were present on both sides of the membrane without eliciting any inhibitory effect of G551D CFTR on NaCh function. This should be contrasted with the situation in which the addition of ``extracellular'' ATP activated ORCC when G551D CFTR was present(34) .

In summary, we have shown that functional CFTR can directly influence amiloride-sensitive Na channel activity in planar bilayers. CFTR not only decreases single ENaC P(o), but also decreases the extent of elevation of renal Na channel P(o) following PKA-induced phosphorylation. Moreover, CFTR can prohibit the induction of voltage-dependent gating of the immunopurified renal Na channel by (PKA + ATP)-mediated phosphorylation. Thus, at physiologically relevant negative voltages, the mean current through these Na channels would be reduced in the presence of CFTR, thus accounting for the observations made in native epithelia (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK 37206 and DK48764. 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 Cystic Fibrosis Foundation Grant CFF R464.

Supported by Cystic Fibrosis Foundation Postdoctoral Fellowship CFF 981.

**
To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., 706 BHSB, Birmingham, AL 35294-0005. Tel.: 205-934-6220; Fax: 205-934-2377; :Benos{at}PhyBio.BHS.UAB.Edu.

(^1)
The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; pS, picosiemens; ENaC, epithelial Na channel; rENaC, rat ENaC; NaCh, Na channel; PKA, protein kinase A; MOPS, 4-morpholinepropanesulfonic acid; DPC, diphenylamine-2-carboxylic acid; ORCC, outwardly rectified chloride channel; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid.

(^2)
M. S. Awayda, I. I. Ismailov, B. K. Berdiev, C. M. Fuller, and D. J. Benos, submitted for publication.


ACKNOWLEDGEMENTS

We thank Charlae Starr and Ann Harter for excellent assistance in preparing the manuscript and Dr. J. K. Bubien for helpful discussions. We also thank Dr. Bernard Rossier for the kind gift of the rENaC clones and Dr. Gail Johnson for the gift of the purified catalytic subunit of PKA.


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