Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Michael L. Chalfant1, Jerod S. Denton1, Bakhram K. Berdiev2, Iskander I. Ismailov2, Dale J. Benos2, and Bruce A. Stanton1

1 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755; and 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35233


    ABSTRACT
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (alpha , beta , gamma ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of alpha -, beta -, and gamma -subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the alpha -ENaC subunit was examined in planar lipid bilayers. We report that alpha ,beta ,gamma -ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited alpha ,beta -ENaC, alpha ,gamma -ENaC, and alpha -ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the alpha -ENaC subunit is regulated directly by pHi.

cortical collecting duct; amiloride; patch clamp; hydrogen ion; Xenopus oocyte


    INTRODUCTION
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

SODIUM ABSORPTION across a variety of epithelia, including the renal cortical collecting duct (CCD), frog skin, and urinary bladder, plays a vital role in maintaining Na+ and fluid homeostasis (14). Na+ absorption across these epithelia is a two-step, electrogenic process involving diffusion across the mucosal membrane through amiloride-sensitive, 5-pS, Na+-selective channels and active transport across the basolateral membrane via the Na+-K+-ATPase (14). The rate-limiting step in this process is Na+ entry across the mucosal membrane (14). Na+ channel activity is regulated by a variety of hormones as well as the intra- and extracellular milieu, and thus regulation of Na+ channel activity is an important component of Na+ and fluid homeostasis (14). Protons have long been recognized to influence amiloride-sensitive Na+ absorption in epithelia (12, 13, 16, 21, 25, 27, 28, 40). Acidification of the cytoplasm of frog skin (16, 40) and toad bladder (28) reduces Na+ absorption and the Na+ permeability of the mucosal membrane. Patch-clamp experiments have demonstrated that the activity of Na+ channels in the apical membrane of principal cells in the CCD is inhibited by acidification of intracellular pH (pHi) (30). In contrast, a reduction in pHi has been reported to stimulate Na+ transport in toad bladder (12, 21).

Recent studies indicate that the epithelial Na+ channel (ENaC), which is composed of three subunits, alpha , beta , and gamma , with molecular masses of 70-80 kDa, constitutes the major Na+ entry pathway in the mucosal membrane of epithelial cells that absorb Na+ by an electrogenic mechanism (5, 6, 14, 24). Expression of the alpha -, beta -, and gamma -subunits of ENaC in Xenopus oocytes is sufficient for the expression of Na+ channels with biophysical properties similar to those expressed in epithelia that exhibit pH-sensitive Na+ absorption (6, 14). At least six proteins may associate with and regulate Na+ channels in renal epithelial cells, including a 300-kDa protein that is phosphorylated by protein kinase A (PKA), 95- and 70-kDa proteins, 150- and 55-kDa proteins that are phosphorylated by protein kinase C, and a 40-kDa protein that may be the heterotrimeric G protein subunit Galpha i-3 (1, 3, 35). It is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory or associated protein. Accordingly, because the effect of pH on ENaC subunits has not been reported, we examined the role of pH in regulating various combinations of ENaC subunits expressed in Xenopus oocytes and on alpha -ENaC in planar lipid bilayers. We report that alpha ,beta ,gamma -ENaC currents are regulated by changes in pHi but not by changes in the extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability (Po), reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. A reduction in pHi also inhibited alpha ,beta -ENaC, alpha ,gamma -ENaC, and alpha -ENaC currents. We conclude that pHi is an important regulator of ENaC activity and that the alpha -ENaC subunit is regulated directly by pHi.


    METHODS AND MATERIALS
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Isolation of Xenopus oocytes and injection of cRNA. Well-documented methods for oocyte isolation and cRNA injection were employed (9). Briefly, ovarian lobes were surgically removed from anesthetized frogs (Xenopus laevis) and stored in Ca2+-free OR-2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, with pH 7.5 adjusted with NaOH). Oocytes were isolated and defolliculated using a combination of enzymatic treatment for 2 h (2 mg/ml type 1A collagenase in OR-2; Sigma Chemical, St. Louis, MO) and manual dissection. Defolliculated stage V and VI oocytes were transferred to L-15 medium modified for use with amphibian cells and supplemented with gentamicin sulfate (25 mg/ml). Oocytes recovered overnight at 19°C before injection of cRNA.

Pipettes for microinjecting cRNA were prepared from borosilicate glass using a horizontal puller, followed by beveling of the tip. To examine the effect of pH on rat alpha ,beta ,gamma -ENaC (alpha ,beta ,gamma -rENaC), individual oocytes were injected with a mixture of cRNA for alpha -, beta -, and gamma -rENaC (2.5 ng/subunit in 50 nl of water) or 50 nl of RNase-free water. When the effect of pH on alpha -rENaC was examined, oocytes were injected with 25 ng alpha -rENaC cRNA in 50 nl of water. We also studied oocytes injected with alpha ,beta -rENaC cRNA and alpha ,gamma -rENaC cRNA (2.5 ng/subunit in 50 nl of water). Injected oocytes were kept in 24-well cell culture plates containing modified L-15 medium in groups of 3-6 oocytes/well at 19°C. Electrophysiological analysis was performed 1-4 days after injection.

cRNA preparation. Plasmids containing cDNA encoding the alpha -, beta -, and gamma -subunits of rENaC cloned into the pSport vector were a generous gift of Dr. Bernard C. Rossier (Lausanne, Switzerland) (5, 6). The vector was linearized with Not I and was used as a template for cRNA synthesis using a kit containing T7 RNA polymerase, ribonucleotides, and a 7-methylguanosine cap analog (mMessage mMachine, Ambion, Austin, TX) following the manufacturer's instructions.

Two-electrode voltage clamp. For two-microelectrode voltage-clamping experiments, oocytes were bathed in either a HEPES- or sodium acetate-buffered solution. The HEPES-buffered solution contained (in mM) 110 NaCl, 2 KCl, 0.2 CaCl2, 1.0 MgCl2, and 5 HEPES, with pH adjusted to values ranging from 6.4 to 7.4 using either NaOH or HCl, as appropriate. The sodium acetate-buffered solution contained (in mM) 50 NaCl, 60 sodium acetate, 2 KCl, 0.2 CaCl2, and 1.0 MgCl2, with pH titrated to values ranging from 6.4 to 7.4 using NaOH or HCl as appropriate. All experiments were performed at 22-24°C. When filled with 3 M KCl, the microelectrodes had an electrical resistance of 1-3 MOmega . A voltage-clamp amplifier (OC-725, Warner Instrument) was used to measure whole cell current. The bath was grounded via a pair of Ag-AgCl wires immersed in reservoirs filled with 3 M KCl and connected to the bath via glass tubes filled with 4% agar in 3 M KCl. Whole cell currents were passed through a low-pass, four-pole Bessel filter with a cutoff frequency of 100 Hz, digitized at a sampling rate of 2 kHz, and stored on the hard disk of a DOS-based computer for subsequent analysis using pCLAMP software (version 6.03, Axon Instruments, Foster City, CA).

To assess the time course of changes in whole cell current during voltage clamp, a periodic voltage waveform was applied. With a period of 15 s, the clamp potential was hyperpolarized by 10 mV from a holding potential of 0 mV for a duration of 2.5 s. The average current over the final 1 s just before the onset of the test pulse and at the end of the test pulse was used to calculate a chord conductance from Ohm's law. In some experiments, current-voltage (I-V) plots were obtained by applying a series of voltage pulses ranging from -100 mV to +40 mV in increments of 10 mV from a holding potential of 0 mV. Test pulses were applied every 2 s for a duration of 450 ms.

Single-channel current measurements. For patch-clamp experiments, the vitelline membrane was removed as described (26), with minor modifications. Briefly, oocytes were placed in a hypertonic solution containing (in mM) 220 N-methyl-D-glucamine (NMDG) aspartate, 1 MgCl2, 5 EGTA, and 5 HEPES, with pH titrated to 7.4 with NMDG-OH. After several minutes, the vitelline membrane was removed using a pair of fine forceps, and devitellinated oocytes were transferred to the patch-clamp recording chamber (RC-24, Warner Instrument, Hamden, CT) mounted on the stage of an inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan). For cell-attached and inside-out patch-clamp experiments, the pipette solution contained (in mM) 110 NaCl, 1.0 CaCl2, and 5.0 HEPES, with pH titrated to 7.4 with NMDG-OH, and was supplemented with 0.1 mM GdCl3 to block endogenous, stretch-activated cation channels (46). In preliminary experiments, GdCl3 (0.1 mM) had no effect on ENaC currents in oocytes (n = 4). For inside-out patches, the cytoplasmic bath solution contained (in mM) 110 KCl, 2.5 EGTA, and 5.0 HEPES, with pH titrated to values ranging between 8.0 and 6.4 using NMDG-OH or HCl. The solution bathing the cytoplasmic surface of inside-out membrane patches was exchanged rapidly by a perfusion pipette. Patch-clamp pipettes were pulled from borosilicate glass (Corning no. 7052, Garner Glass, Claremont, CA) using a horizontal puller (Sutter Instrument, San Rafael, CA). When filled with the pipette solution, the electrodes had resistances ranging from 1.5 to 5 MOmega . Pipette currents were amplified, filtered with a low-pass, four-pole Bessel filter with a cutoff frequency of 200 Hz, digitized at a sampling rate of 2 kHz, and stored on the hard disk of a DOS-based computer for subsequent analysis using pCLAMP software version 6.03. Single-channel currents were measured as previously described in detail (22). All voltages refer to the cell interior referenced to the patch pipette. After a gigaseal patch was attained and the membrane patch was excised to form the inside-out configuration, recordings were made for the generation of I-V plots and the determination of NPo, the product of the number of observed open channels (N) and Po. NPo was calculated as follows
<IT>NP</IT><SUB>o</SUB> = <LIM><OP>∑</OP><LL><IT>n</IT>=0</LL><UL><IT>N</IT></UL></LIM> <FR><NU><IT>n</IT>(<IT>t</IT><SUB><IT>n</IT></SUB>)</NU><DE><IT>T</IT></DE></FR>
where T was the record time, n was the number of channels open, and tn was the time during which n channels were open. In membrane patches with one channel, Po and mean open and closed times were calculated as described previously (22).

Planar lipid bilayers. The effect of pHi on the single-channel properties of alpha -ENaC was studied in planar lipid bilayers. Stage V-VI Xenopus oocytes were harvested and injected with 5 ng alpha -ENaC cRNA or 50 nl water as described above. Membrane vesicles were prepared 48 h postinjection following the method of Pérez et al. (31). Thirty to forty oocytes in each group were washed and homogenized in a 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 bilayers were made from a phospholipid solution containing a 1:1 mixture of diphytanoyl phosphatidylethanolamine and diphytanoyl phosphatidylserine (in n-octane; final phospholipid concentration 25 mg/ml). Membrane vesicles were applied with a fire-polished glass rod to one side (trans) of a preformed bilayer bathed with a symmetrical solution of 100 mM NaCl and 10 mM MOPS-Tris (pH 7.4). Acquisition and analysis of single-channel recordings were performed as described (20).

Statistical analysis. Differences between means were compared by ANOVA and the Bonferroni post hoc comparison test or the paired or unpaired Student's t-test, as appropriate. Statistical analyses were performed with the InStat statistical software package (Graphpad, San Diego, CA). Data are expressed as means ± SE. P < 0.05 is considered significant.


    RESULTS
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Characterization of alpha ,beta ,gamma -rENaC currents. In water-injected oocytes, we did not observe any amiloride-sensitive currents (Fig. 1), consistent with previous observations (6). In contrast, injection of cRNAs encoding the alpha -, beta -, and gamma -subunits of rENaC into oocytes induced the expression of amiloride-sensitive Na+ currents (Fig. 1). The amiloride-sensitive I-V relationship had a positive reversal potential and a slight inward rectification (Fig. 1G). To calculate the ionic selectivity of the amiloride-sensitive currents, ion substitution experiments were conducted in which Na+ was replaced on a equimolar basis with Li+. When the bath solution contained NaCl (110 mM), the reversal potential of the amiloride-sensitive I-V plot was 6.5 ± 0.5 mV (n = 5). Replacing Na+ in the bath solution with Li+ changed the reversal potential of the amiloride-sensitive I-V plot to +19.4 ± 1.2 mV (n = 5; P < 0.0001). From the change in the reversal potential and the Goldman-Hodgkin-Katz equation, we calculate a Li+-to-Na+ permeability ratio of 1.6. Thus, in agreement with previous studies in oocytes, alpha ,beta ,gamma -rENaC is more permeable to Li+ than to Na+ (6, 32, 43).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Representative currents in water-injected oocytes and oocytes injected with cRNA for rat alpha ,beta ,gamma -epithelial Na+ channel (alpha ,beta ,gamma -rENaC; 2.5 ng/subunit). Holding potential was 0 mV. Every 2 s, clamp potential was set to one of a series of test potentials ranging from -100 mV to +40 mV in increments of 10 mV. Duration of test potential was 450 ms. A: whole cell currents (IM) in an oocyte expressing alpha ,beta ,gamma -rENaC. B: whole cell currents from same oocyte as in A exposed to amiloride. C: amiloride-sensitive whole cell currents (INa) obtained by subtracting currents in B from currents in A. D: whole cell currents from an oocyte injected with water. E: whole cell currents from same oocyte as in D exposed to amiloride. F: INa obtained by subtracting currents in E from currents in D. G: current-voltage plot of INa in alpha -beta ,gamma -rENaC-expressing oocytes (open circle ; n = 12) and in water-injected oocytes (bullet ; n = 5). VM, membrane voltage.

As observed by others (10), there is a consistent rundown over time of the amiloride-sensitive currents. The rundown was not abolished by adding glucose (10 mM) or pyruvate (10 mM) to the bathing solution, to support cellular metabolism, or by reducing extracellular Na+ from 110 to 30 mM. Thus, as described below, in all cases we conducted time control studies and examined the reversibility of responses to account for the rundown of ENaC currents.

Acidification inhibits alpha ,beta ,gamma -rENaC currents. To change pHi, oocytes were bathed in sodium acetate solutions titrated to pH values from 7.4 to 6.4. H+-acetate- diffuses across plasma membranes and, once inside the cell, dissociates to H+ and acetate-, thereby reducing pHi (8, 39). The change in pHi is proportional to the pHo (8, 39). For example, reducing pHo from 7.4 to 6.3 in an acetate--buffered bath solution decreased pHi in Xenopus oocytes from 6.8 to 6.1 (8).

Alterations in the pH of the bath solution had no effect on endogenous ion channels in oocytes. A reduction in the pH of the sodium acetate bath solution from 7.4 to 6.4 had no effect on the conductance of oocytes injected with water (0.9 ± 0.2 µS in pH 7.4 vs. 1.0 ± 0.2 µS in pH 6.4; n = 4). In addition, a reduction in the pH of a HEPES bath solution from pH 7.4 to 6.4 also had no effect on conductance in oocytes injected with water (1.4 ± 0.3 µS in pH 7.4 vs. 1.4 ± 0.3 µS in pH 6.4; n = 4). Thus changes in pHi and pHo had no effect on endogenous ion channels in oocytes. In addition, neither sodium acetate nor HEPES had any effect on endogenous ion channels in oocytes.

We next examined the effect of pH on ENaC currents. In time control experiments in which oocytes expressing ENaC were bathed in a sodium acetate solution buffered to pH 7.4, the whole cell conductance decreased slowly with time (Figs. 2A and 3). In contrast, a reduction in pH of the sodium acetate bathing solution from 7.4 to 6.4 rapidly (~15 s) decreased the whole cell, amiloride-sensitive Na+ conductance vs. time controls (Figs. 2B and 3). The inhibition of ENaC conductance was reversible. In a separate set of oocytes, we examined the reversibility of the inhibition of the amiloride-sensitive conductance by acidifying the bath solution from pH 7.4 to 6.4 and then returning the pH to 7.4. In this set of oocytes, when the pH of the bath solution was increased from 6.4 to 7.4, the amiloride-sensitive conductance recovered fully to the value measured originally at pH 7.4 (n = 7; P < 0.05). Thus the effect of acidifying pH on ENaC conductance was fully reversible.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of time and of alterations in pH of bath solution on whole cell chord conductance (Gcell; n = 6/group) in oocytes expressing alpha ,beta ,gamma -rENaC and bathed in sodium acetate solution. Addition of amiloride (10 µM) to bath solution is indicated by letter A in black boxes at top. Bath pH is indicated in upper boxes at top. Gcell was measured between 0 and -10 mV. A: time control illustrating rundown of Gcell at constant pH 7.4. The 2nd application of amiloride elicits a smaller decrease in conductance, indicating that amiloride-sensitive conductance decreases with time. B: acidification of bath solution (from pH 7.4 to 6.4; arrow) rapidly decreased Gcell and reduced amiloride-sensitive conductance compared with control (compare magnitude of decreases in conductance during 2nd application of amiloride in B with magnitude of decreases in conductance during 2nd application of amiloride in A).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of time and alterations in solution pH on amiloride-sensitive whole cell conductance (G; n = 6/group) in oocytes expressing alpha ,beta ,gamma -rENaC. Open bars, time controls; hatched bars, experimental groups. Left: amiloride-sensitive conductance during 1st application of amiloride. Right: amiloride-sensitive conductance during 2nd application of amiloride. Oocytes were bathed in sodium acetate bath solution. * P < 0.05. vs. adjacent open bar.

In experiments with the sodium acetate buffer, both pHi and pHo were reduced (8, 39). Thus it is possible that acidification of the extracellular and/or intracellular solutions inhibited ENaC currents. To determine whether acidification of the extracellular bath solution inhibits ENaC currents, we used HEPES as the buffer with no sodium acetate added (18). Reducing bath solution pH by titrating HEPES-buffered solutions had a nominal effect on pHi. For example, reducing pHo from 7.4 to 6.2 in a HEPES-buffered bath solution decreased oocyte pHi by ~0.1 units (18). Results of experiments reducing pHo on the amiloride-sensitive conductance are shown in Figs. 4 and 5. A reduction of pHo from 7.4 to 6.4 had no effect on the amiloride-sensitive conductance compared with time controls. Thus, because a reduction of pHo had no effect on ENaC currents, it is reasonable to conclude that a reduction in pHi inhibits ENaC currents.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of time and of alterations in pH of bath solution on Gcell (n = 6/group) in oocytes expressing alpha ,beta ,gamma -rENaC and bathed in HEPES-buffered bath solution. Addition of amiloride (10 µM) to bath solution is indicated by letter A in black boxes at top. Bath pH is indicated in upper boxes at top. Gcell was measured between 0 and -10 mV. A: time control illustrating rundown of Gcell at constant pH 7.4. The 2nd application of amiloride elicits a smaller decrease in conductance, indicating that amiloride-sensitive conductance decreased with time. B: acidification of bath solution (pH 7.4 to 6.4; arrow) had no significant effect on Gcell or amiloride-sensitive conductance compared with control (compare magnitude of decrease in conductance during 2nd application of amiloride in B with magnitude of decrease in conductance during 2nd application of amiloride in A).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of time and alterations in bath solution pH on amiloride-sensitive whole cell conductance (n = 6/group) in oocytes expressing alpha ,beta ,gamma -rENaC. Open bars, time controls; hatched bars, experimental groups. Left: amiloride-sensitive conductance during 1st application of amiloride. Right: amiloride-sensitive conductance during 2nd application of amiloride. Oocytes were bathed in HEPES bath solution.

Voltage independence of pH-sensitive alpha ,beta ,gamma -rENaC currents. To determine whether the inhibition of ENaC by a reduction in pHi is influenced by membrane voltage, we examined the effects of voltage on ENaC currents at pH 6.4. At positive voltages, the amiloride-sensitive currents were small, and thus our analysis was restricted to -100 to 0 mV. The percent inhibition of the amiloride-sensitive ENaC current was similar at all voltages and ranged from a low of 81.4 ± 2.7% to a high of 83.6 ± 5.3% (n = 4). Thus the pH-induced inhibition of alpha ,beta ,gamma -rENaC is voltage independent. This result is in agreement with studies in frog skin demonstrating that inhibition of the apical membrane Na+ conductance is independent of voltage over the range from -100 mV to -20 mV (16).

Effect of pHi on single-channel alpha ,beta ,gamma -rENaC currents. In the experiments described above, we did not measure pHi in oocytes, and thus it was not possible to describe the relationship between pHi and ENaC activity. To determine more accurately the relationship between pHi and ENaC activity, we conducted inside-out patch-clamp experiments in which we could control the pH of the solution bathing the cytoplasmic side of the membrane patch. In water-injected oocytes, we never observed ENaC-like channels (i.e., amiloride-sensitive, ~5 pS, Na+-selective channels); however, ENaCs were readily identified in oocytes injected with alpha ,beta ,gamma -ENaC cRNAs. ENaCs were readily identified in cell-attached and inside-out patches due to their low conductance (~5 pS) and slow gating kinetics (e.g., open and closed times >1 s) (14). A representative experiment demonstrating the effect of changing pHi on ENaC activity is shown in Fig. 6. Acidification of the solution bathing the cytoplasmic surface of excised, inside-out membrane patches from pH 7.4 to 6.4 decreased NPo as fast as we could change the bath solution. Moreover, in every membrane patch studied, inhibition of channel activity was reversible when the pH was returned to 7.4 (Fig. 7). Figure 8A summarizes experiments examining the effects of pHi over the range of 6.0-8.0 on channel NPo. A reduction of pHi from 7.4 to 6.9 reduced NPo; however, further reduction in pHi to 6.4 and 6.0 had no additional effect on NPo (Fig. 8A). In contrast, an increase in pHi from pH 7.4 to 8.0 enhanced NPo (Fig. 8A). These results demonstrate that alpha ,beta ,gamma -rENaC is regulated by pHi in the physiological range (i.e., pH 7.0-7.4) (7, 29, 34).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Representative current records illustrating effects of reducing pH of cytoplasmic bath solution [i.e., intracellular pH (pHi)] on alpha ,beta ,gamma -rENaC activity in excised, inside-out membrane patches. Pipette potential was 0 mV. Downward deflections represent inward currents; c, 0 current level. Continuous currents from 1 patch exposed to a series of solutions of different pH. Note that acidification of cytoplasmic solution decreased NPo, the product of the number of observed open channels (N) and single-channel open probability (Po), with no change in unitary current amplitude. In this membrane patch, NPo for entire time of data collection was 0.96 at pHi 7.4 and 0.12 at pHi 6.4.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Reversibility of inhibition of alpha ,beta ,gamma -rENaC activity by pHi in excised inside-out patches. For each patch, NPo was normalized to value of NPo from initial period (pH 7.4). Data are from 4 separate patches, each represented by a different symbol.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 8.   A: effect of pHi on activity (NPo) of alpha ,beta ,gamma -rENaC. NPo values at each pH are normalized to pH 7.4. B: effect of pHi on single-channel conductance (gamma Na); n = 3-8 patches for each pH value. Channels were studied in excised, inside-out membrane patches.

The amiloride-sensitive current (INa) in oocytes expressing ENaC is proportional to the number of functional channels in the membrane (N), the Po, and the single-channel conductance (gamma ). Regulation of ENaC currents by pHi could be referable to changes in N, Po, and/or gamma . To determine whether pH affected INa by altering gamma , we examined the effect of alterations in pHi between 6.0 and 8.0 on gamma . As shown in Fig. 8B, changes in pHi between 6.0 and 8.0 had no effect on gamma , which was 5.2 ± 0.1 pS at pH 7.4. Thus regulation of ENaC currents by pHi involved changes in N and/or Po but not changes in gamma . These observations are similar to the behavior of Na+ channels in rat CCD in response to the acidification of the cytoplasmic bath solution from 7.4 to 6.9 (30) and are in agreement with studies examining the effect of pHi on alpha -rENaC in planar lipid bilayers presented below (Effect of pHi on alpha -rENaC in planar lipid bilayers).

Most membrane patches contained more than one ENaC, thereby precluding a kinetic analysis of all membrane patches studied. However, in five patches a single channel was present in the membrane. A kinetic analysis of those channels revealed that acidification of the cytoplasmic solution from pH 7.4 to 6.9 decreased the Po from 0.46 ± 0.18 to 0.22 ± 0.15 (P < 0.05). Moreover, acidification reduced the channel mean open time from 2.9 ± 1.3 to 1.2 ± 0.5 s (P < 0.05) and increased the channel mean closed time from 3.9 ± 1.7 to 8.6 ± 2.6 s (P < 0.05). These observations are similar to the behavior of Na+ channels in rat CCD in response to the acidification of the cytoplasmic bath solution from 7.4 to 6.9 (30) and are in agreement with studies examining the effect of pHi on alpha -rENaC in planar lipid bilayers presented below (Effect of pHi on alpha -rENaC in planar lipid bilayers).

Effect of pH on different combinations of alpha -, beta -, and gamma -rENaC subunits. To identify the subunit(s) of rENaC that are regulated by pH, we examined the effects of reducing pH on various combinations of ENaC subunits expressed in Xenopus oocytes. As illustrated in Fig. 9, a reduction in the pH of the sodium acetate-buffered bath solution from 7.4 to 6.4 reduced ENaC conductance in oocytes expressing alpha ,beta ,gamma -ENaC, alpha ,beta -ENaC, alpha ,gamma -ENaC, and alpha -ENaC. We did not examine the effect of pH on beta ,gamma -rENaC because these subunits do not form amiloride-sensitive Na+ channels in oocytes (6). These studies demonstrate that the alpha -rENaC subunit is regulated by pH and that beta - and gamma -rENaC are not required for pH regulation of the channel.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of reducing extracellular pH from 7.4 to 6.4 (sodium acetate bath solution) on various combinations of alpha -, beta -, and gamma -rENaC or on alpha -rENaC alone. Data are expressed as %change in Na+ conductance when bath solution pH was reduced from 7.4 to 6.4 (n = 6-11/group). %Change in conductance in oocytes expressing alpha ,beta ,gamma -rENaC (81%) was not statistically different from change in conductance reported for alpha ,beta ,gamma -rENaC in Fig. 3. * %Change is significant at P < 0.05. ** %Change is significant at P < 0.05 and is different from %change of other combinations tested.

Effect of pHi on alpha -rENaC in planar lipid bilayers. To determine more accurately the relationship between pHi and alpha -rENaC activity, we examined the regulation of alpha -rENaC by pHi in channels reconstituted in planar lipid bilayers (Fig. 10). A sequential reduction in pHi (i.e., cis bath solution) from 7.4 to 6.9 and to 6.5 progressively decreased the Po from 0.62 ± 0.06 to 0.29 ± 0.05 to 0.08 ± 0.04 (P < 0.05), respectively, without changing the single-channel conductance (n = 3-9/pH). In contrast, a similar reduction in pHo (i.e., trans bath solution) had no effect on Po (Fig. 10). Thus the alpha -rENaC subunit expressed in planar lipid bilayers, like alpha -rENaC expressed in oocytes, is inhibited by a reduction in pHi but not by a reduction in pHo.1


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 10.   A reduction in pHi reduces Po of alpha -rENaC subunit expressed in planar lipid bilayers. A: representative current records illustrating effect of reducing pHi on Po. pH of cis solution (corresponding to intracellular solution; Ref. 19) was reduced sequentially from 7.4 to 6.9 to 6.5. Upward deflections represent channel openings. Dotted lines indicate channel closed state. B: summary of all experiments examining effect of changes in pH of cis solution and trans solution (corresponding to extracellular solution; Ref. 19) on channel Po. Data were normalized to Po at pH 7.4.


    DISCUSSION
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

The major new finding of this study is that alpha ,beta ,gamma -ENaC currents are regulated directly by changes in pHi in the physiological range but not by changes in pHo. Although it is possible that other proteins in addition to alpha ,beta ,gamma -ENaC may regulate Na+ channels in epithelial cells, these subunits are not required for pHi regulation of ENaC.2 Acidification reduced and alkalinization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced Po, reduced single-channel open time, and increased single-channel closed time, without altering single-channel conductance. A reduction in pHi also inhibited alpha ,beta -ENaC, alpha ,gamma -ENaC, and alpha -ENaC currents. Thus our data demonstrate that alpha -ENaC alone is sufficient for pHi regulation of rENaC.

Other members of the ENaC/FaNaC/DRASIC/degenerin superfamily are also regulated by pH and are, like ENaC, H+-gated, Na+-selective channels. The recently cloned acid-sensing ion channel (ASIC), MDEG1, and DRASIC are activated directly by a reduction in the pH of the extracellular solution but not the intracellular solution (23, 41, 42). Accordingly, H+ not only affects ENaC-mediated Na+ absorption but also influences mechanotransduction, neurotransmission, and nociception by regulating Na+ uptake via MDEG1, FaNaC, and ASIC, respectively (23, 41, 42). It is interesting to note that ENaC may mediate salt taste perception in taste cells on the surface of the tongue and that sour taste, perhaps via H+, alters the perception of salt (14). Thus H+, in addition to regulating ENaC-mediated transepithelial Na+ transport, may also play a role in modulating the perception of salt by ENaC.

The decrease in ENaC activity with acidification of the cytoplasmic solution is consistent with observations that a reduction in pHi inhibits Na+ absorption across frog skin (16, 40) and toad bladder (28). In addition, a reduction in pHi also reduces the Na+ permeability of the mucosal membrane of frog skin (16, 40), toad bladder (28), and principal cells in rabbit CCD (38). In contrast, a reduction in pH has been reported to stimulate Na+ transport in toad bladder (12, 21). Moreover, neither acidosis nor alkalosis affects Na+ absorption across rabbit CCD perfused in vitro (4, 38) or rat distal tubule perfused in vivo (37). These apparent differences in the reported effects of pH may be related to the fact that a number of parameters in addition to the Na+ permeability of the mucosal membrane influence transepithelial Na+ absorption, including the electrochemical gradient for Na+ across the mucosal membrane and the rate of Na+ efflux across the basolateral membrane via the Na+-K+-ATPase, all of which may be regulated by pHi (38, 45).

Changes in pHi between 8.0 and 7.0, but not pHo, influenced ENaC gating. Several different molecular mechanisms have been proposed to account for pH-dependent regulation of ion channels. First, protons may bind in the channel pore and block the channel, as proposed for the voltage-dependent proton block of the frog nerve Na+ channel (44). However, because inhibition of ENaC by H+ is voltage independent, it is unlikely that H+ inhibits the channel by this mechanism. Second, protons may bind near the pore and influence ion conduction through an electrostatic interaction. Channel subconductance states caused by this type of regulation have been observed for the Torpedo Cl- channel (15), the olfactory cyclic nucleotide-gated cation channel (33), and the ATP-sensitive cardiac inwardly rectifying K+ channel (11). This type of inhibition is also unlikely for ENaC, because a reduction in pHi neither affected the channel conductance nor promoted the appearance of subconductance states (Fig. 6). Third, changes in pH may regulate ENaC currents in part by altering interfacial potentials (17). Finally, protonation of key amino acid(s) may alter channel conformation and shift the equilibrium between the open and closed state such that the closed state is favored. Although our analysis of single-channel open and closed times favors this mechanism, additional studies are required to determine how protons regulate ENaC.

Alterations in pHi but not pHo regulate ENaC, and thus it is likely that amino acids in the amino and carboxy termini of alpha -rENaC, which are thought to be cytoplasmic, are involved in pH regulation (36). A highly conserved histidine (His-94) in the amino terminus of alpha -rENaC is a likely candidate. In preliminary studies, we found that mutation of His-94 eliminated ENaC currents in oocytes, thereby precluding an examination of the role of His-94 in the regulation by protons. There are numerous other highly conserved candidate amino acids in the amino and carboxy termini (5, 6, 14, 24). Additional studies are required to address the role of these amino acids in pH modulation of ENaC activity.

In summary, we report that alpha ,beta ,gamma -ENaC is regulated directly by changes in pHi in the physiological range but not by changes in pHo. Although expression of all three subunits is required for maximum ENaC currents, expression of alpha -rENaC alone is sufficient for pHi regulation of rENaC. Although several proteins may associate with and regulate the alpha ,beta ,gamma -ENaC complex, it does not appear that these regulatory subunits are required or necessary for pHi regulation.


    ACKNOWLEDGEMENTS

B. A. Stanton is grateful for the support and encouragement of Dr. Ralph E. Stanton, who published his first paper on pH regulation of cell function in 1923 (37a). We thank Flora Ciampolillo for valuable technical assistance.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34533, DK-51067, and DK-07301 (to B. A. Stanton) and DK-37206 (to D. J. Benos) and a grant from the National Kidney Foundation (to M. L. Chalfant).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1 The responses to changes in pHi are somewhat different in alpha ,beta ,gamma -ENaC expressed in oocytes (Fig. 8) vs. alpha -ENaC expressed in planar lipid bilayers (Fig. 10). A reduction in pHi below 7 has no additional effect on alpha ,beta ,gamma -ENaC (Fig. 8), whereas a reduction in pHi below 7 decreases alpha -ENaC activity (Fig. 10). This difference suggests, but does not prove, that the beta - and gamma -ENaC subunits may influence the pHi sensitivity of the alpha -ENaC subunit. It is also possible that the response to changes in pHi may depend on the experimental preparation (i.e., oocytes vs. bilayers). It is important to note, however, that this subtle difference in the response to pHi does not change our major conclusion that changes in pHi within the physiological range (i.e., 7.0 to 7.4), but not pHo, modulate ENaC activity.

2 It is possible that the regulatory proteins associated with ENaC in epithelial cells are also expressed in Xenopus oocytes and present in lipid bilayers. Thus we cannot categorically exclude the possibility that pH regulates ENaC indirectly. However, indirect regulation by pHi is unlikely for two reasons. First, PKA does not activate ENaC in lipid bilayers or oocytes (2), most likely because the regulatory subunit that is phosphorylated by PKA is not present in bilayers or expressed in oocytes. Second, studies by Benos and colleagues (3) have shown that recombinant ENaC synthesized by rabbit reticulocytes and ENaC isolated from Xenopus oocytes have similar biophysical properties and are regulated in a similar fashion in lipid bilayers. Thus, because channels produced in vitro by rabbit reticulocytes are unlikely to be associated with regulatory proteins, the most parsimonious conclusion is that pHi directly regulates ENaC.

Address for reprint requests: B. A. Stanton, Dept. of Physiology, Dartmouth Medical School, 615 Remsen Bldg., Hanover, NH 03755-3836.

Received 12 August 1998; accepted in final form 6 November 1998.


    REFERENCES
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

1.   Ausiello, D. A., J. L. Stow, H. F. Cantiello, J. B. De Almeida, and D. J. Benos. Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Galpha i-3 protein. J. Biol. Chem. 267: 4759-4765, 1992[Abstract/Free Full Text].

2.   Awayda, M. S., I. I. Ismailov, B. K. Berdiev, C. M. Fuller, and D. J. Benos. Protein kinase regulation of a cloned epithelial Na+ channel. J. Gen. Physiol. 108: 49-65, 1996[Abstract].

3.   Benos, D. J., M. S. Awayda, I. I. Ismailov, and J. P. Johnson. Structure and function of amiloride-sensitive Na+ channels. J. Membr. Biol. 143: 1-18, 1995[Medline].

4.   Boudry, J. F., L. C. Stoner, and M. B. Burg. Effect of acid lumen pH on potassium transport in renal cortical collecting tubules. Am. J. Physiol. 230: 239-244, 1976[Medline].

5.   Canessa, C. M., J. Horisberger, and B. D. Rossier. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[Medline].

6.   Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J.-D. Horisberger, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

7.   Chaillet, J. R., A. G. Lopes, and W. F. Boron. Basolateral Na-H exchange in the rabbit cortical collecting tubule. J. Gen. Physiol. 86: 795-812, 1985[Abstract].

8.   Choe, H., H. Zhou, L. G. Palmer, and H. Sackin. A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating. Am. J. Physiol. 273 (Renal Physiol. 42): F516-F529, 1997[Medline].

9.   Dascal, N. The use of Xenopus oocytes for the study of ion channels. CRC Crit. Rev. Biochem. Mol. Biol. 22: 317-387, 1987.

10.   DuVall, M. D., S. Zhu, C. M. Fuller, and S. Matalon. Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC. Am. J. Physiol. 274 (Cell Physiol. 43): C1417-C1423, 1998[Abstract/Free Full Text].

11.   Fan, Z., T. Furukawa, T. Sawanobori, J. C. Makielski, and M. Hiraoka. Cytoplasmic acidosis induces multiple conductance states in ATP- sensitive potassium channels of cardiac myocytes. J. Membr. Biol. 136: 169-179, 1993[Medline].

12.   Garty, H., C. Asher, and O. Yeger. Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions. J. Membr. Biol. 95: 151-162, 1987[Medline].

13.   Garty, H., E. D. Civan, and M. M. Civan. Effects of internal and external pH on amiloride-blockable Na+ transport across toad urinary bladder vesicles. J. Membr. Biol. 87: 67-75, 1985[Medline].

14.   Garty, H., and L. G. Palmer. Epithelial sodium channels: function, structure, and regulation. Physiol. Rev. 77: 359-396, 1997[Abstract/Free Full Text].

15.   Hanke, W., and C. Miller. Single chloride channels from Torpedo electroplax. Activation by protons. J. Gen. Physiol. 82: 25-45, 1983[Abstract].

16.   Harvey, B. J., S. R. Thomas, and J. Ehrenfeld. Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium. J. Gen. Physiol. 92: 767-791, 1988[Abstract].

17.   Hille, B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992, p. 427-429.

18.   Humphreys, B. D., L. Jiang, M. N. Chernova, and S. L. Alper. Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes. Am. J. Physiol. 267 (Cell Physiol. 36): C1295-C1307, 1994[Abstract/Free Full Text].

19.   Ismailov, I. I., M. S. Awayda, B. K. Berdiev, J. K. Bubien, J. E. Lucas, C. M. Fuller, and D. J. Benos. Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. J. Biol. Chem. 271: 807-816, 1996[Abstract/Free Full Text].

20.   Ismailov, I. I., V. G. Shlyonsky, O. Alvarez, and D. J. Benos. Cation permeability of a cloned rat epithelial amiloride-sensitive Na+ channel. J. Physiol. (Lond.) 504: 287-300, 1997[Abstract].

21.   Leaf, A., A. Keller, and E. F. Dempsey. Stimulation of sodium transport in toad bladder by acidification of mucosal medium. Am. J. Physiol. 207: 547-552, 1964.

22.   Light, D. B., F. V. McCann, T. M. Keller, and B. A. Stanton. Amiloride-sensitive cation channel in rat inner medullary collecting duct cells. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F278-F286, 1988[Abstract/Free Full Text].

23.   Lingueglia, E., J. R. De Weille, F. Bassilana, C. Heurteaux, H. Sakai, R. Waldmann, and M. Lazdunski. A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J. Biol. Chem. 272: 29778-29783, 1997[Abstract/Free Full Text].

24.   Lingueglia, E., N. Voilley, R. Waldmann, M. Lazdunski, and P. Barbry. Expression cloning of an epithelial amiloride-sensitive Na+ channel: a new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett. 318: 95-99, 1993[Medline].

25.   Lyall, V., G. M. Feldman, and T. U. Biber. Regulation of apical Na+ conductive transport in epithelia by pH. Biochim. Biophys. Acta 1241: 31-44, 1995[Medline].

26.   Methfessel, C., V. Witzemann, T. Takahashi, M. Mishina, S. Numa, and B. Sakmann. Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflügers Arch. 407: 577-588, 1986[Medline].

27.   Palmer, L. G. Voltage-dependent block by amiloride and other monovalent cations of apical Na channels in the toad urinary bladder. J. Membr. Biol. 80: 153-165, 1984[Medline].

28.   Palmer, L. G. Modulation of apical Na permeability of the toad urinary bladder by intracellular Na, Ca, and H. J. Membr. Biol. 83: 57-69, 1985[Medline].

29.   Palmer, L. G., L. Antonian, and G. Frindt. Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone. J. Gen. Physiol. 102: 43-57, 1993[Abstract].

30.   Palmer, L. G., and G. Frindt. Effects of cell Ca and pH on Na channels from rat cortical collecting tubule. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F333-F339, 1987[Abstract/Free Full Text].

31.   Pérez, G., A. Lagrutta, J. P. Adelman, and L. Toro. Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers. Biophys. J. 66: 1022-1027, 1994[Abstract].

32.   Puoti, A., A. May, C. M. Canessa, J.-D. Horisberger, L. Schild, and B. C. Rossier. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells. Am. J. Physiol. 269 (Cell Physiol. 38): C188-C197, 1995[Abstract/Free Full Text].

33.   Root, M. J., and R. MacKinnon. Two identical noninteracting sites in an ion channel revealed by proton transfer. Science 265: 1852-1856, 1994[Medline].

34.   Silver, R. B., G. Frindt, E. E. Windhager, and L. G. Palmer. Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F557-F564, 1993[Abstract/Free Full Text].

35.   Smith, P. R., and D. J. Benos. Epithelial Na+ channels. Annu. Rev. Physiol. 53: 509-530, 1991[Medline].

36.   Snyder, P. M., F. J. McDonald, J. B. Stokes, and M. J. Welsh. Membrane topology of the amiloride-sensitive epithelial sodium channel. J. Biol. Chem. 269: 24379-24383, 1994[Abstract/Free Full Text].

37.   Stanton, B. A., and G. Giebisch. Effects of pH on potassium transport by renal distal tubule. Am. J. Physiol. 242 (Renal Fluid Electrolyte Physiol. 11): F544-F551, 1982[Abstract/Free Full Text].

37a.   Stanton, R. E. The selective reabsorption of potassium by animal cells. III. The effect of hydrogen ion concentration upon the retention of potassium. J. Gen. Physiol. 5: 461-467, 1923[Free Full Text].

38.   Tabei, K., S. Muto, H. Furuya, Y. Sakairi, Y. Ando, and Y. Asano. Potassium secretion is inhibited by metabolic acidosis in rabbit cortical collecting ducts in vitro. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F490-F495, 1995[Abstract/Free Full Text].

39.   Tsai, T. D., M. E. Shuck, D. P. Thompson, M. J. Bienkowski, and K. S. Lee. Intracellular H+ inhibits a cloned rat kidney outer medulla K+ channel expressed in Xenopus oocytes. Am. J. Physiol. 268 (Cell Physiol. 37): C1173-C1178, 1995[Abstract/Free Full Text].

40.   Ussing, H. H., and K. Zerahn. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Scand. 23: 110-127, 1951.

41.   Waldmann, R., F. Bassilana, J. de Weille, G. Champigny, C. Heurteaux, and M. Lazdunski. Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J. Biol. Chem. 272: 20975-20978, 1997[Abstract/Free Full Text].

42.   Waldmann, R., G. Champigny, F. Bassilana, C. Heurteaux, and M. Lazdunski. A proton-gated cation channel involved in acid-sensing. Nature 386: 173-177, 1997[Medline].

43.   Waldmann, R., G. Champigny, F. Bassilana, N. Voilley, and M. Lazdunski. Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. J. Biol. Chem. 270: 27411-27414, 1995[Abstract/Free Full Text].

44.   Woodhull, A. M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61: 687-708, 1973[Abstract/Free Full Text].

45.   Wright, F. S., and G. Giebisch. Regulation of potassium excretion. In: The Kidney: Physiology and Pathophysiology, edited by D. W. Seldin, and G. Giebisch. New York: Raven, 1992, p. 2209-2247.

46.   Yang, X. C., and F. Sachs. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243: 1068-1070, 1989[Medline].


Am J Physiol Cell Physiol 276(2):C477-C486
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society