©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Triple-barrel Organization of ENaC, a Cloned Epithelial Na Channel (*)

(Received for publication, July 26, 1995; and in revised form, October 10, 1995)

Iskander I. Ismailov (1) Mouhamed S. Awayda (1) Bakhram K. Berdiev (1) James K. Bubien (1) (2) Joseph E. Lucas (1) Catherine M. Fuller (1) Dale J. Benos (1)(§)

From the  (1)Department of Physiology and Biophysics and the (2)Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cloned rat epithelial Na channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the alpha subunit alone and those formed by alpha, beta, and subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either alpha-rENaC alone or alpha- in combination with beta- and -rENaC, produced highly Na-selective (P/P(K) = 10), amiloride-sensitive (K(i) = 170 nM), and mechanosensitive cation channels in planar bilayers. alpha-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13-picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. alpha-, beta-, -rENaC showed primarily the 13-pS level. alpha-rENaC and alpha,beta,-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with >2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of alpha-, beta-, and -rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.


INTRODUCTION

An epithelial amiloride-sensitive Na channel has recently been cloned from rat distal colon (1, 2, 3) and, subsequently, from other tissues(4, 5, 6, 7) . This channel, termed rENaC for rat epithelial Na channel, consists of three homologous subunits alpha, beta, and . While the precise function of any these subunits has not yet been determined, it appears that the conductive portion of the channel resides in the alpha subunit and that beta and are necessary for enhanced ion channel activity in the Xenopus oocyte heterologous expression systems(2, 6, 7) . It is also apparent that the channel's gating properties are influenced by beta and , because truncations in the C-terminal region of these subunits produce a constitutive activation of the channel by increasing single channel open probability (P(O); Refs. 8 and 9). It is these beta subunit mutations that underlie the autosomal dominant genetic hypertensive disorder, Liddle's disease(10, 11, 12, 13, 14) . Moreover, alpha-, beta-, and -ENaC expression has been localized to aldosterone-responsive, Na-reabsorbing epithelial tissues in the rat by in situ hybridization and immunocytochemistry using subunit-specific probes(15, 16) . While amiloride-sensitive Na channels have been shown also to be responsive to antidiuretic hormone in many epithelia(17, 18) , the rat distal colon is refractory to the influence of antidiuretic hormone(19) . Thus, the alpha, beta, and subunits may comprise only a portion of the amiloride-sensitive Na channels expressed by other cell types.

Amiloride-sensitive Na channels studied by the patch clamp technique in native epithelia have displayed a wide variety of single channel properties. For example, single channel conductances ranging from 1 to over 50 pS (^1)have been reported(17) . Patch clamp studies of Xenopus oocyte plasma membranes following coexpression of alpha, beta, and cRNA reveal a channel of 5-8 pS in size(2, 8) , comparable with Na channels present in renal cortical collecting tubules(18) . However, a recent paper by Burch et al.(16) report that the message levels of alpha, beta, and subunits in superficial human proximal airway epithelia are not equal as they are in rat colon, kidney, and salivary gland (15) but exist in a relative ratio of alpha > beta . Interestingly, the single channel conductance of the amiloride-sensitive Na channels found in this epithelium is 19-20 pS(19) . Thus, the possibility exists that different combinations of these subunits could produce channels with different unitary conductances. Another possibility is that the unitary conductances are tissue-specific, depending upon the physical state of the membrane, which in turn is dependent upon ionic conditions and membrane composition.

The purposes of the present work were 2-fold. First, we wished to develop a reconstitution system in which the single channel properties of alpha-, beta-, and -rENaC could be studied, unencumbered by problems inherent in heterologous expression systems. Second, we tested the hypothesis that variations in relative levels of alpha, beta, and have functional consequences on single channel behavior. Our results indicate that either alpha alone, or alpha in combination with one or both of the other subunits of ENaC produce mechanosensitive, amiloride-inhibited, Na-selective ion channels following incorporation into planar lipid bilayers. alpha-rENaC channels showed a complex kinetic pattern: there were two main conductance transitions, one of 13 and the other of 26 pS. In contrast, alpha,beta,-rENaC revealed only the 13-pS state. Different combinations of alpha, beta, or did not produce channels of intermediate conductance. Gating of alpha-rENaC was cooperative, with transitions to a 40-pS level only occurring after the 13-pS level was open. Moreover, upon treatment with the reducing agent dithiothreitol (DTT), both alpha-rENac and alpha,beta,-rENaC fluctuated among three independent 13-pS sublevels. We conclude that these channels are composed of three identical conduction elements and that the differences in the activity of the channels are modulated by the presence of the beta and subunits within the complex.


MATERIALS AND METHODS

In Vitro Translation and Reconstitution of ENaC Subunits

The ENaC plasmids (pSport) were linearized overnight with NotI. The linearized DNA was purified using GeneClean kit (Bio101, La Jolla, CA), followed by in vitro transcription using T7 RNA polymerase according to the manufacturer's instructions (Ribomax kit, Promega Corp., Madison, WI). A 2:1 molar ratio of cap analog m^7 G(5`)ppp(5`)G (NEB, Beverly, MA) to GTP was used in the translation reaction as described previously(20) .

RNA was in vitro translated using a rabbit nuclease-treated cell-free lysate system (Promega) according to the manufacturer's instructions and as described previously(20) . 1.5 units of canine microsomal membranes (Promega) were added to the translation reaction. This resulted in the core glycosylation of the de novo synthesized protein(7) . In vitro translated proteins were purified on a G-75 Sephadex (Pharmacia Biotech Inc.) column as described previously(20) . Fractions enriched in the appropriate ENaC subunits were reconstituted into phospholipid liposomes as described earlier(20) . For liposomes containing alpha,beta,-rENaC, subunits were in vitro translated separately, and each was purified over a gel filtration column. Identical elution fractions were collected and assayed for total S incorporation. The relative ratios of alpha, beta, and subunits reconstituted into vesicles were determined by these S measurements, assuming similar methionine compositions for each subunit. Control liposomes were also prepared from a mock in vitro translation/Sephadex column purification run, following an identical protocol. In this case, ENaC RNA was simply omitted from the reaction mixture. These liposomes were used as control material for the bilayer experiments.

Channel Expression in Xenopus Oocytes and Oocyte Membrane Vesicle Preparation

Membrane vesicles from alpha-rENaC- and alpha,beta,-rENaC mRNA-injected and water-injected oocytes were made essentially as described by Perez et al.(21) . Thirty oocytes in each group were rinsed and homogenized in high [K]/sucrose medium containing multiple protease inhibitors. Membranes were isolated by discontinuous sucrose gradient centrifugation and resuspended in 300 mM sucrose, 100 mM/KCl, and 5 mM MOPS at pH 6.8. This material was aliquoted into 50-µl fractions and stored at -80 °C until use.

Bilayer Experiments and Data Analysis

Lipid bilayers were cast from a phospholipid solution in n-octane containing a 2:1:2 mixture of diphytanoyl-phosphatidylethanolamine/diphytanoyl-phophatidylserine/ oxidized cholesterol (25 mg/ml). Lipids were purchased from Avanti Polar Lipids (Alabaster, AL). Bilayer capacitances ranged from 300 to 400 picofarads. The solution bathing the bilayers consisted of 100 mM NaCl and 10 mM MOPS-Tris buffer (pH 7.4) unless otherwise noted. All solutions were made with Milli-Q water and were filter-sterilized through 0.22-µm Sterivex-GS filters (Millipore, Bedford, MA). The reconstituted vesicles or oocyte membranes were applied with a glass rod to one side (trans) of the preformed bilayer with the membrane voltage held at -40 mV. Under these conditions, channels oriented (>90% of the time, n = 250) asymmetrically, with the amiloride-sensitive (extracellular) side facing the trans solution. Voltage was applied to the cis chamber, and the trans chamber was virtual ground.

Single channel recordings were acquired and analyzed using pCLAMP software as described previously(22, 23) . The recordings were acquired and stored unfiltered. For analysis, they were filtered at 300 Hz with an 8-pole Bessel filter and acquired at 1 ms/point. The 50% threshold-crossing technique was employed to produce events lists. Open and closed dwell time histograms were logarithmically binned and fitted by a sum of exponential functions using maximum likelihood. All data analysis was performed in bilayers containing a single active channel. Application of a hydrostatic pressure difference across a bilayer containing reconstituted ENaC increased open probability to near 1 (20) . This effect was independent of the direction of the hydrostatic pressure gradient, i.e. it was equally effective when the bathing solution level was lowered or raised on either side of the channel-containing bilayer. In multichannel membranes, hydrostatic pressure could thus reveal the total number of channels present in the membrane. Therefore, at the beginning of every experiment, 1 ml of the trans bathing solution was removed to determine the total number of Na channels present. If more than one channel was detected, the membrane was broken and the incorporation procedure repeated.

Patch Clamp Experiments

Cell-attached patch clamp recordings were obtained from defolliculated oocytes previously injected with alpha-rENaC and alpha,beta,-rENaC cRNA (12.5 ng for oocytes expressing alpha-rENaC alone, and 1 ng of each subunit for oocytes expressing all three rENaC subunits) or 50 nl of nuclease-free water at room temperature. The composition of the pipette solution (ND-96) was (in mM): 96 NaCl, 2.4 KCl, 1.8 CaCl(2), 1 MgCl(2), and 5.0 HEPES (pH 7.4). The bathing solution had K substituted for Na to eliminate the resting membrane potential. Blunt-tipped patch micropipettes with a tip resistance of approximately 1-2 M were fabricated using a Narashigi pp73 two-stage micropipette puller. Under these conditions, inward single channel currents (downward current openings) in the cell-attached patches were carried by Na when the pipette was positive relative to the bath. Single channel currents were examined using an EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany). After sealing the pipettes to the cells (seal resistance >10 gigaohms), the cell-attached patches were clamped to a variety of voltages using an S-95 trilevel stimulator (Medical Systems; Greenville, NY). The amplifier gain was 100 mV/pA. This degree of amplification was sufficient to resolve clearly single channel openings >0.3 pA. The resultant ionic currents at each membrane potential were filtered at 300 Hz, digitally recorded at 1 kHz, and filed for analysis by pCLAMP software (Axon Instruments, Sunnyvale, CA). All patch clamp experiments were performed at 21 ± 2 °C.


RESULTS

In Vitro Translation of rENaC

We have previously demonstrated that in vitro translated alpha-bENaC, the bovine ENaC homolog, migrates as a dimer on SDS-polyacrylamide gel electrophoresis under nonreducing conditions(7) . As seen from the autoradiograph shown in Fig. 1(lane 1), a similar gel pattern was observed for alpha-rENaC in vitro translated in the presence of microsomes and run under denatured but nonreduced conditions. In seven experiments of this type, the presence of a dimer at 172 ± 11 kDa was always observed. However, the relative distribution of alpha-rENaC in the monomeric versus dimeric form differed from one batch of translation reaction to the other. The highest yield of a dimer to a monomer form is shown in the example in Fig. 1. Despite this variability there were never any observed differences in the biophysical properties of the in vitro translated channel incorporated into planar lipid bilayers (see below). Moreover, there was no evidence for the presence of a trimer form of alpha-rENaC (i.e. a product that migrated at 270 kDa was not observed; see lane 1). These observations suggest that the alpha subunit of ENaC may occur in its native state as a covalently linked dimer. As seen from lane 2, this pattern of interaction was not modified by the addition of the beta and subunits. Lane 3 shows that the 180-kDa band is absent following reduction with 20 mM DTT and that the highest molecular mass band observed was one at 92 kDa. This size protein was expected for monomeric and core-glycosylated alpha-rENaC and was similar to previous observations on alpha-bENaC(7) . Co-translation of all three subunits simultaneously did not reveal any forms between 200 and 300 kDa. Lane 4 shows a control, run in the absence of any ENaC message. Thus, the polypeptides seen in lanes 1-3 at molecular masses lower than 92 kDa probably represent partial translation products. The absence of a protein that migrated at a relative molecular mass in the range of the cumulative masses of one alpha, one beta, and one subunit suggests that alpha, beta, and do not covalently interact in a simple 1:1:1 ratio and that electrostatic interactions may be important in stabilizing the native ENaC complex.


Figure 1: SDS-polyacrylamide gel electrophoresis of in vitro translated alpha, beta, and ENaCs. Lane 1 represents in vitro translated alpha-rENaC electrophoresed alone in the absence of DTT or heating (nonreducing conditions). Lane 2 represents in vitro translated alpha-rENaC in combination with equal volumes of unlabeled (cold) beta and rENaC, in the absence of heating or DTT. 0.2% Triton X-100 was used in both cases to solubilize the translated proteins and to allow for incubation with the beta and subunits (lane 2). Note the presence in both lanes 1 and 2 of a band that migrated at a relative molecular mass of 180 kDa, consistent with a dimerization of alpha-rENaC. Lane 3 contains in vitro alpha-rENaC in the presence of 20 mM DTT and after heating for 3 min at 80 °C. Note the disappearance of the dimer form. Molecular mass markers were thyroglobulin and ferritin (330 and 220 kDa, Pharmacia) and phosphorylase B and bovine serum albumin (92 and 70 kDa, Amersham Corp.). Lane 4 shows a control in vitro translation performed in the absence of exogeneously added ENaC transcripts.



Conductance Properties of rENaC

The column-purified in vitro translated polypeptides of alpha-rENaC, or alpha,beta,-rENaC were reconstituted into liposomes and then incorporated into planar lipid bilayer membranes. As shown in Fig. 2, these in vitro translated proteins form functional ion channels (n = 7 for each). This figure depicts channel activity at different applied potentials. There was no effect of voltage on P(o) of either channel. Fig. 2B shows the associated main state single channel current versus voltage curves for each channel. Both alpha-rENaC and alpha,beta,-rENaC channels were linear. In over 1,000 attempted incorporations using control liposomes (see ``Materials and Methods''), no channels of any kind were detected.


Figure 2: A, single channel records of in vitro translated alpha-rENaC and alpha,beta,-rENaC at different applied voltages in planar lipid bilayers. Bilayers were bathed with symmetrical solutions of 100 mM NaCl, 10 mM MOPS-Tris (pH 7.4). Records were filtered at 300 Hz. B, single channel current-voltage curves of alpha-rENaC and alpha,beta,-rENaC in bilayers. Each point represents the single channel current of the main state transition versus applied potential averaged over seven separate experiments (± S.D.).



Examination of the single channel records over several minutes for both alpha-rENaC and alpha,beta,-rENaC indicate continuous activity with no run-down (n = 16; Fig. 3). These channels were kinetically different from alpha-bENaC in bilayers, in that alpha-bENaC channel activity was punctuated by long closed periods lasting up to several minutes(20) . Long (>1-s) closures were never observed for the rENaC channels in over 2 h of recording. alpha-rENaC channels displayed a specific gating pattern; with symmetrical 100 mM NaCl, the largest conductance level was 40 pS, but the channel appeared to fluctuate among 0-, 13-, 26-, and 40-pS levels (see associated all points amplitude histograms, Fig. 3B). These histograms did not fit the binomial algorithm for independent gating, indicating that these conductance levels cannot be produced by three independent channels. Neither the 13- nor 26-pS current transitions were ever observed independently of each other in over 300 separate experiments. In the small number of cases (19/330) in which multiple channels were incorporated into the bilayer, the pattern shown at the top of Fig. 3was simply repeated, i.e. for two channels in the bilayer, 4, 5, and 6 additional states were seen. Moreover, the single channel records indicated that the gating properties of the alpha-rENaC channel were not independent. Openings of the 26-pS level were only observed after the 13-pS state was open, never before. However, albeit infrequently, the 13-pS state would close prior to closure of the 26-pS transition (see Fig. 2A, top).


Figure 3: Single channel records of in vitro-translated alpha-rENaC and alpha,beta,-rENaC in planar lipid bilayers. A, records are shown for +100 mV holding potential and are representative of nine separate experiments. Record was filtered at 300 Hz using an 8-pole Bessel filter prior to the acquisition and were sampled at 1000 Hz using a Digidata 1200 interface. Bathing solutions contained symmetrical 100 mM NaCl plus 10 mM MOPS-Tris (pH 7.4). Dotted lines indicate zero current. B, all point amplitude histograms. Histograms were generated by pCLAMP software from a record of 5 min in length. C, single channel events dwell time histograms. Time constants were calculated from single exponential fits for each state. This experiment is representative of nine and seven separate trials for alpha-rENaC and alpha,beta,-rENaC, respectively. Bin width was 1 ms.



When alpha,beta,-rENaC, in a 1:1:1 (w/w/w) combination, was incorporated into bilayers, only a 13-pS conductance level was observed ( Fig. 2and Fig. 3, bottom). However, brief openings of <250 ms in a duration to 40 pS were occasionally seen. Again, these openings occurred on the top of 13-pS conductance level that was, in essence, constitutively open. Event dwell time histograms were constructed for all the conductance levels of alpha-rENaC and alpha,beta,-rENaC channels by setting a threshold at 50% of the open level of each substate (Fig. 3C). The dwell histograms in each sublevel for each channel were all fitted by a single exponential function. The closed state time constants were 70 ± 9 and 40 ± 5 ms for alpha-rENaC and alpha,beta,-rENaC, respectively. The open state time constants for alpha-rENaC and alpha,beta,-rENaC, respectively, were (in ms) 77 ± 10 and 72 ± 3 (13-pS state), 35 ± 8 and 51 ± 3 (26-pS state), and 91 ± 11 and 52 ± 3 (40-pS state). From this analysis, the time constant for exit from the closed state is nearly twice as long for alpha-rENaC than for alpha,beta,-rENaC.

Because alpha,beta,-rENaC expressed in Xenopus oocytes displays a Na-selective, 5-pS single channel conductance with relatively long lived open and closed conductance states as measured by patch clamp(2, 8) , we wanted to assess directly whether bilayer reconstitution protocol utilizing in vitro translated polypeptides could affect conductance and/or open and closed times. Therefore, we compared rENaC single channel properties determined from patch clamp measurements of rENaC-expressing oocytes with those made in bilayers using in vitro translated proteins or subsequent to fusion of rENaC-expressing oocyte plasma membranes. Oocytes were injected either with alpha-rENaC or alpha,beta,-rENaC cRNA and then either they were patch-clamped or their plasma membranes were used for fusion to planar bilayers. Fig. 4shows the results of these maneuvers. Patch clamp recordings of oocytes expressing alpha-rENaC revealed channels with a large conductance of approximately 18 pS. Interposed among the large transitions were two additional conductance levels of 6 and 12 pS each. In recordings made from three separate oocytes, these alpha-rENaC channels behaved in this manner. Except for the absolute values of the conductance states, this kinetic behavior was very similar to that observed for alpha-rENaC in the bilayer (Fig. 2). However, in contrast to the bilayer, channel activity occurred in bursts rather than continuously. Patch clamp recordings made from oocytes expressing alpha,beta,-rENaC typically showed long lived 6-pS channels, similar to what was previously reported(2) . Native oocyte membranes from Xenopus oocyte expressing either alpha-rENaC or alpha,beta,-rENaC fused to bilayer membranes revealed channels with similar kinetic behavior to those measured by patch clamp, but only with larger conductance states. In all other respects, however, these channels behaved identically to those observed for in vitro translated protein incorporated into bilayers. We conclude, therefore, that the bilayer is an appropriate system in which to study rENaC.


Figure 4: Single channel records of alpha-rENaC and alpha,beta,-rENaC expressed in Xenopus oocytes. A, patch clamp recordings. Traces are representative of data obtained from three separate oocytes for each ENaC. Oocytes were bathed in high K medium in order to depolarize the resting membrane potential to 0 mV (see (2) ) B, oocyte membrane vesicles incorporated into planar lipid bilayers. For the bilayer experiments, a holding potential of 100 mV was employed in order to match the estimated holding potential of the cell-attached patches of oocyte membranes. Traces are representative of 12 separate experiments each for alpha-rENaC and alpha,beta,-rENaC.



Effects of Hydrostatic Pressure on rENaC Channel Activity

Based upon our experience with alpha-bENaC(20) , we next tested the hypothesis that both alpha-rENaC and alpha,beta,-rENaC could be activated by an imposition of a hydrostatic pressure gradient across the bilayer membrane. As shown in Fig. 5, channel activities of both alpha-rENaC and alpha,beta,-rENaC were significantly enhanced by membrane stretch. A hydrostatic pressure gradient (DeltaP) was established by removal of 1 ml of bathing solution from one compartment of the bilayer chamber, which was equivalent to 0.26 mm Hg. For alpha,beta,-rENaC, single channel open probability (P(O)) increased from 0.15 ± 0.04 to 0.65 ± 0.08 (n = 7), and for alpha-rENaC a DeltaP of 0.26 mm Hg increased P(O) from 0.60 ± 0.08 to 0.91 ± 0.08 (n = 8). There was no apparent change in single channel conductances following stretch, but the relative frequencies of channel conductance levels were altered. In the case of alpha-rENaC, the 40-pS level predominated after stretch, while for alpha,beta,-rENaC, it appeared that three 13-pS conductance levels were present. All point amplitude histogram analysis of these records after stretch revealed that these three conductances could be fit by a binominal distribution, indicating that stretch induced independent gating of the component sublevels.


Figure 5: Single channel records of in vitro translated alpha-rENaC and alpha,beta,-rENaC in planar lipid bilayers in the absence and presence of a hydrostatic pressure gradient. These records are typical of seven individual experiments. Conditions are the same as indicated in the legend to Fig. 2. A 0.26 mm Hg hydrostatic pressure gradient was produced by the addition of 1 ml of bathing solution to the cis compartment.



Ion Selectivity of rENaC in Planar Bilayers

When alpha-rENaC or alpha,beta,-rENaC channels were bathed with asymmetric solutions of NaCl (a 10-fold gradient) under nonstretched conditions, a reversal potential of 57 ± 3 mV was measured, and a permeability ratio for Naversus Cl of 10:1 for each channel type was calculated. These values did not change upon stretch (n = 9 for each channel type). These results are similar to those previously reported for alpha-bENaC(20) . Likewise, P/P for each channel was determined from reversal potential measurements made under biionic conditions in the absence of and in the presence of a 0.26 mm Hg hydrostatic pressure gradient across the membrane (Fig. 6). Under nonstretched conditions, both channels were 10-fold more permeable to Na than to K. However, upon stretch, each channel lost some of its ability to discriminate between Na and K, decreasing to 3:1 and 4:1 for alpha-rENaC and alpha,beta,-rENaC, respectively.


Figure 6: Mean current-voltage relationships of alpha-rENaC (A) and alpha,beta,-rENaC (B) in planar lipid bilayers under biionic conditions in the absence and presence of a hydrostatic pressure gradient. For alpha-rENaC under nonstretched conditions, P/P was 10:1 (n = 13). Stretch decreased P/P to 3:1 (n = 11). For alpha,beta,-rENaC, P/P was 10:1 and 4:1 under control (n = 17) and stretched (n = 13) conditions, respectively. Bilayers were bathed with 100 mM solutions of NaCl (trans) and KCl (cis), each containing 10 mM MOPS-Tris (pH 7.4). Each point represents the mean ± S.D.



Gating Properties of rENaC

The records depicted in Fig. 2and Fig. 3indicate that the apparent single channel kinetic behavior of alpha,beta,-rENaC was different from that of alpha-rENaC. alpha-rENaC has one small 13-pS conductance level that is almost continuously open and a larger 40-pS level, while alpha,beta,-rENaC primarily displays the 13 pS level with only brief openings to the 40-pS level. Based on the observations that both alpha-rENaC and alpha,beta,-rENaC display kinetic properties indicative of subconductive type behavior and that beta and themselves do not form channels, it is likely that the conduction pathway(s) of rENaC is formed by the alpha subunit(s). We next examined whether these channels were comprised of more than one conduction element. The results presented in Fig. 1indicate that disulfide bond formation occurs between two of the subunits. Thus, we postulated that disrupting disulfide bonds would reveal more fundamental kinetic behavior. Therefore, we tested the effects of 25 µM of the reducing agent DTT on alpha-rENaC and alpha,beta,-rENaC in bilayers. Fig. 7summarizes the results of a typical experiment. First, either ENaC was sensitive to DTT only from the trans (i.e. outside) bathing solution. Second, concentrations of DTT below 25 µM had no effect on single channel properties, and concentrations larger than 50 µM irreversibly damaged coordinated channel activity (data not shown). As can be seen in the figure, treatment of either alpha-rENaC or alpha,beta,-rENaC with DTT resulted in the appearance of three indistinguishable 13-pS conductance states. As beta and , independently or together, do not induce channel activity, at least as expressed in oocytes(2, 7) , it is plausible that both alpha-rENaC and alpha,beta,-rENaC consist of a minimum of three protochannels formed by alpha subunits that gate in a concerted fashion. In the presence of DTT, this synchronous gating may be disrupted, thus permitting independent operation of these three conductive elements.


Figure 7: Effect of DTT on alpha-rENaC and alpha,beta,-rENaC in planar lipid bilayers. DTT was added at a final concentration of 25 µM to the trans bathing solution. All other conditions were the same as indicated in the legend to Fig. 2. This experiment was repeated 15 times each for alpha-rENaC and alpha,beta,-rENaC, with identical results.



To further test the hypothesis that ENaC consists of three individual protochannels formed by alpha subunits, we cross-linked all of the subunits present in the functional complex together. The sulfhydryl reactive reagent 5,5-dithiobis(2-nitrobenzoate) (DTNB) was used as the cross-linking reagent. Again, DTNB was only effective from the trans side of the bilayer. Fig. 5shows that DTNB treatment of either alpha-rENaC or alpha,beta,-rENaC produced channels that fluctuated between a 0- and 40-pS level. Thus, the kinetic behavior of the channels indicated that the three putative individual alpha subunit proto-channels may operate in concert. However, the complete opening of this channel complex when in its native form occurred in two steps, the second twice the size of the first (Fig. 3A). Thus, we hypothesized that one of the alpha subunit protochannels was anchored to the complex by a noncovalent interaction. To test this idea, we exposed rENaC to elevated salt concentrations in the hope of minimizing electrostatic interactions between subunits (the bulk of the amino acids comprising each ENaC subunit lies in a large extracellular loop (24, 25, 26) ). Thus, the prediction was that both alpha-rENaC and alpha,beta,-rENaC should gate in a very similar manner, with one of the protochannels behaving as an independent lower conductance channel and the two disulfide-linked protochannels operating in effect as a single higher conductance unit. This experiment has been performed a total of six times each for alpha-rENaC and alpha,beta,-rENaC, and the results are summarized in Fig. 8. Raising the NaCl concentration of the bilayer bathing solution from 0.1 to 1.5 M resulted in the appearance of three conductance levels for both alpha-rENaC and alpha,beta,-rENaC. The conductance levels of rENaC were increased to 19, 38, and 57 pS by the elevated [Na]. Moreover, the current transitions appeared to fluctuate randomly from the zero conductance level to each of the three higher levels, unlike the kinetic behavior of the channels at 0.1 M salt. In addition, both alpha-rENaC and alpha,beta,-rENaC responded identically to elevated salt and to DTNB treatment, suggesting that the core conduction elements of both channels are identical.


Figure 8: Composite figure showing the effects of high salt concentration and DTNB on alpha-rENaC and alpha,beta,-rENaC in planar lipid bilayers. Each perturbation was performed a minimum of six times for each channel type. Other conditions were as indicated in legend to Fig. 2.



To better understand the biophysical consequences of DTT and high salt treatment of rENaC, amplitude histogram analyses of current records made under these experimental conditions were performed. All points and events amplitude histograms are presented in Fig. 9. The overall distribution of the all points amplitude histograms was binomial, thus suggesting equal probability of the channel residing in any conductance level (cf., Fig. 3B). The events amplitude histogram revealed a disruption of concerted gating following DTT or high salt treatment in that transitions to all conductance levels occurred independently. Such an outcome of events amplitude histogram may be due to counting channel openings ``from'' a fixed zero current level ``to'' a conductance sublevel. In order to overcome this limitation we have constructed amplitude histograms with a gradually sliding zero level, the level that channel resides in becomes an ``apparent zero-current level'' for the next transition. This maneuver permits the construction of a histogram of absolute values of amplitudes of transitions. The resulting histograms (Fig. 10) show that the predominant transition in the case of DTT-treated rENaC was 13 pS, while high salt treatment produced, in equal probability, transitions of 19 and 38 pS. Taking into account that the increased conductance of the channels in this latter case was due to elevated [Na], these results support the hypothesis that rENaC consists of a minimum of three conductive elements, two of which may be linked by disulfide bonds and the third noncovalently anchored to the covalently linked complex. Histograms for DTT and high salt-treated alpha-rENaC and alpha,beta,-rENaC were almost identical ( Fig. 9and Fig. 10), indicating that the conductive pore of these channels was formed by alpha-rENaC.


Figure 9: All points and events amplitude histograms of DTT and high salt-treated alpha-rENaC and alpha,beta,-rENaC. Experimental conditions were the same as described in the legends to Fig. 4and Fig. 5, respectively. Events lists were produced by pCLAMP software using 50% amplitude threshold technique with a minimum event duration. All points amplitude histograms are shown in gray, while the events amplitude histograms are shown in black.




Figure 10: Events amplitude histograms of DTT and high salt-treated alpha-rENaC and alpha,beta,-rENaC, using a ``sliding'' zero-current level. Experimental conditions were the same as described in the legends to Fig. 4and Fig. 5, respectively. Events lists were produced by pCLAMP software using 50% amplitude threshold (with 3-ms duration) technique. Transitions from 0-pS level, from 13-pS level, from 26-pS level, and from 40-pS level were sorted manually in a pStat events list spreadsheet session and processed by the pStat routine to produce events amplitude histograms.



Effect of Amiloride on rENaC

The effect of amiloride on alpha-rENaC and alpha,beta,-rENaC is summarized in the dose-response curves presented in Fig. 11. Amiloride inhibited both of these channels with very similar efficacy. The apparent amiloride-inhibitory constant (K(i)) was 170 ± 25 nM (n = 12) for both channels. Treatment of alpha-rENaC or alpha,beta,-rENaC with either DTT or DTNB had no significant effect on K(i). Likewise, bathing either alpha-rENaC or alpha,beta,-rENaC with symmetrical 1.5 M NaCl solutions had only a minor effect on K(i), shifting it to 250 ± 30 nM (n = 3) and 230 ± 20 nM (n = 3) for each channel, respectively, consistent with amiloride acting as a competitive inhibitor of these channels(18, 27) . The best fit of these amiloride dose-response curves was achieved with a Michaelis-Menten formalism using a Hill coefficient of 3. All other channel properties, i.e. ion selectivity, amiloride sensitivity, and substate behavior were preserved in the bilayer.


Figure 11: Amiloride dose-response curves of in vitro translated alpha-rENaC and alpha,beta,-rENaC in planar lipid bilayers under different experimental conditions. Points in plots are mean ± S.D. for at least six experiments under each condition. Amiloride was sequentially added in increasing concentrations to the trans bathing solution.



Effect of Different Combinations of alpha,beta,-rENaC Subunits on Channel Activity in Bilayers

Our observations, made both in patch clamp studies of rENaC-expressing oocytes and in planar bilayers, indicate that alpha-rENaC and alpha,beta,-rENaC display characteristic single channel properties; namely, alpha-rENaC consisted of a small (13 pS in bilayers, 6 pS in patch) and a large (2 times the conductance of the small) conductance state, while alpha,beta,-rENaC primarily exhibited only the small conductance level. Because amiloride-sensitive Na channels recorded in both native epithelial and cultured cells exhibit a wide range of single channel conductances (17, 18) , and because different relative levels of alpha, beta, -rENaC mRNA have been detected within a given tissue(15, 16, 28) , we utilized the in vitro translation-bilayer reconstitution system for rENaC subunits to test the hypothesis that varying ratios of rENaC subunit components may modify the resultant single channel kinetic properties and/or conductance. Table 1presents the results of these experiments. First, beta or alone or in combination, do not produce channels of any kind, confirming the observations initially made in oocytes(2, 6, 7) . Likewise, alphabeta or alpha yielded channels identical to alpha-rENaC described above. An excess of beta and relative to alpha in the reconstitution mixture produced channels indistinguishable from those of alpha,beta, in a 1:1:1 ratio. If alpha exceeded beta and by more than 7-fold, only alpha-type channels were seen. Although these experiments do not address the actual stoichiometry of the subunit composition of functional rENaC, they do, nonetheless, suggest that the variable conductances observed in different cells and tissues cannot be attributed solely to different ratios of ENaC subunits comprising the functional channel complex.




DISCUSSION

In this work, we report the successful incorporation of alpha-rENaC and alpha,beta,-rENaC into planar lipid bilayers. ENaC protein was obtained either from a rabbit reticulocyte lysate in vitro translation system or following expression in Xenopus oocytes and isolation of oocyte plasma membranes. The results obtained using either of these preparations were identical. Our experiments also indicate that the alpha-rENaC subunit alone or in combination with other alpha subunits acts as the conductive element of the channel complex. However, a high degree of concerted gating occurs between these putative conduction elements and those covalently linked by disulfide bonds. The kinetic behavior of ENaC suggests that a functional channel unit is comprised of a minimum of three conductive elements formed by alpha subunits. ENaCs are highly Na-selective, are inhibited with high affinity by the diuretic amiloride, and are mechanosensitive.

Comparison of rENaC in Bilayers and by Patch Clamp-A number of biophysical experiments were performed on alpha-rENaC and alpha,beta,-rENaC to compare their properties when expressed in Xenopus oocytes and when purified and reconstituted into planar lipid bilayers. For alpha-rENaC, the overall kinetic behavior of the channel was similar in patch clamp and bilayer experiments, namely a small conductance level on top of which another conductance level (twice the size of the small one) would open. The absolute value of the small conductance level differed (13 pS in the bilayer and 6 pS in the patch). For alpha-rENaC and alpha,beta,-rENaC, a 6-pS (patch) or 13-pS (bilayers) level was routinely measured. That these amiloride-sensitive channels expressed in oocytes are ENaCs is supported by the fact that they were never observed in water-injected oocytes. We conclude, therefore, that the microenvironment in which ENaC resides determines in large measure its conductance and mean open and closed times (31, 32, 33) . Aside from these changes, the channels displayed comparable amiloride sensitivities, ion selectivities, and gating patterns.

The existence of subconductive levels within a single ion channel has been reported for many ion channels including the acetylcholine receptor(34) , the glycine, GABA, and glutamate receptors(35, 36, 37) , the dihydropyridine-sensitive Ca channel(38) , inwardly rectifying K channels(39, 40) , the ryanodine receptor cation channel(41) , and gramicidin(33) . It is not clear why subconductive behavior has not been observed in patch records of alpha,beta,-rENaC channels. One possible explanation is that these channels have not yet been analyzed at high time resolution. Another reason may be that upon drawing the oocyte membrane into the tip of a patch electrode, sufficient tension may have already been applied to produce what appear to be three independent, small conductance channels (cf., Fig. 3and Fig. 4; (2) and (8) ). The fine details of channel conductances appear to be influenced by the methods of observation.

Kinetic Behavior of ENaC in Bilayers

Visual inspection of alpha-rENaC transitions in bilayers reveals that the channel fluctuates either between 0 and 13 pS or between 13 and 40 pS but never (in at least 2 h of recording) between 0 and 26 pS. Residence of the channel in its 26-pS level was rare and short lived and occurred only when the channel transited from its 40-pS level (see current trace and associated amplitude and dwell time histograms, Fig. 2, A, B, and C). Moreover, transition to the highest conductance level (40 pS) occurred in a 26-pS step and only when the channel occupied its 13-pS level. This pattern of channel behavior was different when the bilayer was bathed with high salt-containing solutions (1.5 M, Fig. 6). Under these conditions, the channel flickered between 0 and 13 pS, and 0 and 26 pS stochastically and episodically reached its 40-pS level. Likewise, when alpha-rENaC was treated with DTT, transitions occurred independently between all three equally spaced conductance levels. High salt or DTT treatment of alpha,beta,-rENaC produced an identical pattern of channel activity as for alpha-rENaC ( Fig. 5and Fig. 6).

Both high salt and DTT disrupt protein-protein interactions. Thus, the change in biophysical properties associated with these treatments implies that a multimeric form of alpha-rENaC underlies channel behavior. Because these three levels represent subconductance states of a single channel entity(23) , this kinetic behavior strongly suggests that ENaC is composed of a minimum of three conductive elements and that a pore is formed within each one of these elements. The observations that the same kind of channel activity following DTT treatment is seen for ENaC composed of only alpha or of alpha,beta, and that beta and cannot form ion channels by themselves (Table 1) suggest that the conduction element is the alpha subunit of ENaC. Whether a monomer or dimer (or higher form) of alpha-ENaC acts as the unit conduction element cannot be deduced from these experiments.

As a first approximation, a simple kinetic model of ENaC can be described as follows:

or

where C represents the closed state and O(1), O(2), and O(3) the 13-, 26-, and 40-pS open states, respectively. As indicated above, there were only a few transitions to 26 pS that were observed, and these only occurred from the 40-pS conductance level and had a time constant of 35 ± 8 ms (Fig. 2, B and C). Three possible explanations can account for these data: 1) if the 26-pS transition is comprised of two concertedly linked 13-pS openings, it may be that there is simply a short-lived half-closed state associated with the closing and opening of this 26-pS level; 2) if opening of the 13-pS level is required for the subsequent opening of the 26-pS level, it may be that a brief transient closing of the 13-pS level triggers the closing of the 26-pS level; and 3) if a 13-pS level transiently dissociates from the complex in the lipid bilayer, a 26-pS level may be observed. The first possibility would predict zero residence in the 26-pS level, assuming that closure of the double protochannel was reversible. This second explanation does not account for the transition from 13 through 26 to 40 pS. If the third possibility were true, a transition from 0 to 26 pS would be expected, but the data do not support this. Thus, we simplified the scheme to contain two predominant transitions: one of 13 pS and the other 26 pS in size. When both are open, the conductance of the channel is 40 pS.

There is certain probability P that the channel will reside in any of the given states, and because at any given time the channel must be in one of them, the sum of these probabilities must equal one.

Also, for a system in equilibrium the percentage of channels in any given state must remain constant. Therefore, the rate of transition out of one state must equal the rate of transition into it. The constants k, m, k(1), and m(1) are measures of transition rates between these states. Therefore, the net transition rate out of a state is the product of the rate constant and the probability of the channel being in that state.

If the values for each of the rate constants are determined, it will be possible to calculate the values for the probabilities P(C), P

where T equals one divided by the sum of all of the rate constants leading away from the state(42) . Thus,

Substituting with the experimental data (Fig. 2C), we can calculate the following: k = 14.5 s; k(1) + m = 13.5 s; and m(1) = 11.5 s.

This gives a unique solution for the rate constants k and m(1). However, to complete the model, we need values for k(1) and m. The appropriate equation can be obtained by calculating the probability of the channel proceeding to the 40-pS state from the 13-pS state (i.e. P

Thus, we calculate the values of the rate constants k(1) = 4.95 s and m = 9.0 s. Using these calculated values, we can now solve equations 3, 4, and 6 to calculate the probability of the channel being in any of the possible three states: P(C) = 0.14; P

From the all points amplitude histograms (Fig. 3B), we can compute the probability of finding a channel in any given state by calculating the area under each individual curve and dividing by the total area under the histogram. This analysis yields the following results: P(C) = 0.14 ± 0.05; P

A comparison of the values for the probabilities calculated from the histogram analysis and those derived from the kinetic simulation are in good agreement. This simulation thus formalizes the kinetic behavior of a triple-barrel model for ENaC. This triple-barrel model of ENaC is similar to that proposed for inwardly rectifying K channels(39, 40) . Interestingly, while there is little homology between ENaC and IRK1 or ROMK1 at the nucleotide and amino acid levels(43, 44) , the membrane topology of both classes of ion channel are similar in that they each have only two putative membrane-spanning domains(24, 25, 26) .

Are Biophysically Distinct Amiloride-sensitive Na channels Referable to Different Combinations of alpha, beta, and Subunits?

Steroid hormones increase transepithelial Na transport in target epithelia such as colon, lung, and renal cortical collecting tubule(45) . These tissues contain message for all three subunits of ENaC(2, 5, 6, 15, 16, 46) . Steroid hormones have also been found to increase the abundance of alpha-rENaC mRNA relative to beta and in lung(28, 30) . In rats fed a low Na diet to elevate circulating aldosterone levels, -rENaC mRNA (in contrast to alpha and beta) was found to be raised specifically(29) .

Although expression of alpha-rENaC in oocytes produced an amiloride-sensitive current(1) , the absolute magnitude of this current was greatly augmented by co-expression with beta- and -rENaC(2) . The roles that each subunit plays in channel formation are unknown as is the stoichiometry of alpha,beta, comprising the functional rENaC. Nonetheless, our results indicate that co-reconstituting different relative quantities of alpha,beta,-rENaC does not produce amiloride-sensitive Na channels with altered kinetic signatures. The large diversity in kinetic and conductance properties of these native channels in cells as measured via patch clamp (17, 18) is thus likely to result, at least in part, from tissue-specific factors such as auxillary or regulatory proteins. In fact, biochemical purification studies of amiloride-sensitive Na channels have revealed a different pattern of polypeptide composition of the channel complex, depending upon the source material(47, 48, 49) . However, a thorough analysis of variations in alpha, beta, and subunit ratios on single channel properties will only be achieved once the functional significance of any channel-associated proteins are elucidated.


FOOTNOTES

*
This work was supported by NIH Grant DK37206. 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.

§
To whom correspondence should be addressed: University of Alabama at Birmingham, Dept. of Physiology & Biophysics, 1918 University Blvd., 706 BHSB, Birmingham, AL 35294-0005.

(^1)
The abbreviations used are: pS, picosiemens; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; DTNB, 5,5-dithiobis(2-nitrobenzoate).


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Bernard Rossier for the generous gift of ENaC cDNA and Dr. Roger White for helpful discussions. We also thank Charlae Starr and Ann Harter for excellent secretarial assistance.


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