Epithelial Sodium Channel Pore Region

STRUCTURE AND ROLE IN GATING*

Shaohu ShengDagger, Jinqing LiDagger, Kathleen A. McNulty, Thomas Kieber-Emmons§, and Thomas R. Kleyman

From the Departments of Medicine, Physiology and § Pathology, School of Medicine, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104

Received for publication, September 5, 2000, and in revised form, September 26, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd2+ was determined. Selected mutants within the amino-terminal portion (alpha Val569-alpha Trp582) of the pore region responded to MTSEA, MTSET, or Cd2+ with stimulation or inhibition of whole cell Na+ current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha -helix. The activation of alpha S580Cbeta gamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alpha Ser583-alpha Ser592) of the pore region, only one mutation (alpha S583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd2+, whereas several other mutant channels were partially blocked by MTSEA or Cd2+ but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha -helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial sodium channels (ENaCs)1 are composed of three homologous subunits, termed alpha -, beta -, and gamma ENaC (1, 2). These subunits assemble to form a hetero-oligomeric, Na+-selective ion channel with a subunit stoichiometry of 2alpha :1beta :1gamma (3, 4), although an alternative subunit stoichiometry has been proposed (5, 6). All three Na+ channel subunits have cytoplasmic amino and carboxyl termini, two transmembrane domains (termed M1 and M2), and a large ectodomain (7-9). Previous studies have shown that selected point mutations within the pore region preceding M2 of each subunit altered functional properties of the channel, including cation selectivity, single channel conductance, and sensitivity to the blocker amiloride (4, 10-14). Specific mutations of residues in a conserved three-residue tract, (G/S)XS (where X is Ser, Gly, or Cys), within the pore region of the three ENaC subunits, rendered channels K+-permeable. Snyder et al. (15) examined the accessibility of a sulfhydryl-reactive methane thiosulfonate (MTS) derivative to substituted cysteine residues within the pore region of human gamma ENaC, and they proposed a structural model of the channel pore similar to that proposed by Kellenberger et al. (11) but distinct from the resolved structure of the KcsA K+ channel pore (16).

We previously reported that selected cysteine substitutions within the carboxyl-terminal domain of the pore region of mouse alpha ENaC (alpha Ser580-alpha Ser592) altered the cation selectivity and amiloride sensitivity of the channel and proposed that this region forms the selectivity filter of the channel (14). In the current study, we systematically examined accessibility of sulfhydryl reagents to alpha beta gamma mENaCs with engineered cysteine within the 24-residue pore region of the alpha -subunit. Channels with selected cysteine mutations within the carboxyl-terminal portion of the pore region responded to the external application of MTS derivatives with an inhibition of amiloride-sensitive Na+ currents. In contrast, we observed a significant increase in amiloride-sensitive Na+ currents following the external application of MTS derivatives or Cd2+ when cysteine residues were introduced at selected sites within the amino-terminal portion of the pore region of alpha mENaC. The pattern of distribution of cysteine mutations that led to MTS-induced activation of Na+ currents suggests that this region has an alpha -helical structure. In addition, the activation of alpha S580Cbeta gamma by an MTS reagent was associated with a dramatic increase in channel open probability. We propose that the ENaC pore region forms part of the outer pore vestibule with an alpha -helix followed by an extended region. ENaC may have limited structural similarities with the KcsA K+ channel (16, 17). In addition, our results suggest that the pore region has a role in ENaC gating.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- All chemicals were from Sigma unless stated otherwise.

Cysteine-scanning Mutagenesis-- Site-directed mutagenesis was performed on mouse alpha ENaC (18) with a sequential polymerase chain reaction method using Pfu DNA polymerase (Stratagene, La Jolla, CA). Amino acids alpha Val569-alpha Ser592 of alpha mENaC were replaced individually with a cysteine residue, and target mutations were conformed by automated DNA sequencing, as described previously (14).

Functional Expression of the Mutant mENaCs in Xenopus Oocytes-- Complementary RNAs (cRNAs) for wild type and mutant alpha -, wild type beta -, and gamma mENaC were synthesized with T3 RNA polymerase (Ambion Inc., Austin, TX). Stage V-VI Xenopus oocyte was injected with 2-4 ng of cRNA for each subunit in 50 nl of H2O. Injected oocytes were maintained at 18 °C in modified Barth's saline (MBS, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM HEPES, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamicin sulfate, pH 7.2).

Two-electrode Voltage Clamp-- Two-electrode voltage clamp was performed 20-72 h after injection at room temperature (22-25 °C) as described previously (14). Data acquisition and analyses were performed using pClamp 6.03 software (Axon Instruments) on a Pentium PC. Oocytes were maintained in a recording chamber with 1 ml of bath solution containing (in mM) 100 sodium gluconate, 2 KCl, 1.8 CaCl2, 5 BaCl2, 10 HEPES, pH 7.2, and continuously perfused at the flow rate of 4-5 ml/min. Pipettes filled with 3 M KCl had resistances of 0.5-5 MOmega . Typically, oocytes were clamped to a series of voltage steps from -140 to +40 mV in 20-mV increments for 450 ms every 2 s, and the whole cell currents were measured at 400 ms. Amiloride-sensitive Na+ currents were defined as the difference of Na+ currents in the absence and presence of 100 µM amiloride in the bath solution.

The susceptibility of mutant channel with engineered sulfhydryl groups to sulfhydryl reagents was examined with the sulfhydryl reagents (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA), [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET), sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES), and Cd2+. MTSEA (2.5 mM), MTSET (1 mM), and MTSES (5 mM) were prepared in bath solution immediately prior to use. Base-line Na+ currents and Na+ currents at 1-3 min following perfusion of the oocytes with a reagent were measured. Oocytes were then washed with bath solution for 3 min, and the currents were monitored for observing the reversibility of the response to the reagent. Bath solution containing 100 µM amiloride was then applied to the oocyte to determine the amiloride-insensitive current. Responses are expressed as ratios of amiloride-sensitive Na+ currents after and before addition of the reagent (I/I0).

Single Channel Recordings-- Patch clamp was performed in cell-attached mode as described previously (14). Bath and pipette solutions were identical and contained (in mM) 110 LiCl or NaCl, 2 CaCl2, 10 HEPES, pH 7.4, adjusted with LiOH or NaOH, respectively. Na+ or Li+ currents were recorded at -100 mV (membrane potential). To test the effects of MTS on single channel properties of alpha S580Cbeta gamma mENaC, 10 mM MTSET prepared in Na+ bath solution was added into the bath solution giving a 1 mM final concentration of MTSET, and currents were recorded within 1 h. Event files were generated from long current traces (>5 min) using Fetchan 6.05 (Axon Instruments), and open probability was estimated with Histogen 6.05 (Axon Instruments).

Statistical Analyses-- Values are expressed as mean ± S.E. Student's t test was used for significance analysis between wild type and mutant channel using MS Excel 97 (Microsoft, Inc.).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Responses of Mutant mENaCs to External MTS Derivatives-- ENaC pore regions are highly conserved among members of the ENaC/degenerin family (Fig. 1A). Secondary structure predictions of the pore region suggest that the amino-terminal portion may exist as either alpha -helix or beta -sheet, whereas the carboxyl-terminal portion appears to be more irregular in structure. The center portion is predicted to be a turn region (Fig. 1B). To probe the pore region structure, all residues within the alpha mENaC pore region (alpha Val569---Ser592) were systematically mutated to cysteine and coexpressed with WT beta - and gamma mENaC subunits in Xenopus oocytes. We previously observed that all mutants with cysteine substitutions within the pore region of alpha mENaC retained channel activity, although low levels of expressed currents (<200 nA) were observed with two mutants (alpha G587Cbeta gamma and alpha S589Cbeta gamma ) (14).



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Fig. 1.   Sequence alignments and secondary structure prediction of alpha ENaC pore region. A, pore region amino acid sequence alignments of alpha , beta , and gamma mENaC, deg-1 (degenerin from C. elegans), mec-4 (mechanosensitive protein from C. elegans), hBNC1 (human brain sodium channel), and ASIC1 (acid-sensing ion channel 1). Identical amino acids are shaded, and similar residues are boxed. *, analogous site within the C. elegans proteins deg-1 and mec-4 where mutations result in neurodegeneration; black-diamond , proposed amiloride-binding site in alpha -, beta -, and gamma ENaC; , key residues that limit K+ permeation. B, secondary structure prediction of the alpha mENaC pore region was performed with DNASis 2.6 for Windows 95 (Hitachi Software Engineering Co., Ltd., South San Francisco, CA) using Chou-Fasman algorithm. Uppercase indicates probability, and lowercase indicates a possibility that the residue occurs in the indicated conformation.

Wild type alpha beta gamma mENaC responded to 2.5 mM MTSEA with a partial inhibition of whole cell Na+ currents (I/I0 = 0.75 ± 0.05, n = 17; Fig. 2A). This reduction in current is similar to that reported by other investigators (5, 15). The partial inhibition of wild type ENaC by MTSEA is likely due to covalent modification of Cys547 in gamma mENaC that aligns to Ser588 in alpha mENaC, as proposed by Snyder et al. (15). Mutation of gamma Cys547 to serine largely eliminated the MTSEA-induced partial inhibition of Na+ currents, whereas mutation of an adjacent cysteine (gamma C551S) had no effect on the partial inhibition of Na+ currents by MTSEA (data not show). Amiloride-sensitive whole cell Na+ currents in oocytes expressing wild type alpha beta gamma mENaC were not significantly altered following addition of 1 mM MTSET or 5 mM MTSES to the bath solution (Figs. 2A and 4A).



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Fig. 2.   A, accessibility to external 2.5 mM MTSEA, 1 mM MTSET, and 5 mM Cd2+ of wild type and cysteine-substituted mENaCs. Bars (I/I0) indicate ratios of the remaining amiloride-sensitive Na+ current at 2 min after external application of the reagent to the amiloride-sensitive Na+ current before delivery of the reagent into the bath. Data are presented as mean ± S.E., from 4 to 8 oocytes except for wild type response to MTSEA (17 oocytes). Filled bars indicate statistical significance (p < 0.05, mutant versus WT). ND indicates not determined due to low level of expressed currents with alpha G587Cbeta gamma and alpha S589Cbeta gamma . B, helical wheel analysis of alpha mENaC pore residues Thr570-Ser583. Residues where substitution with cysteine resulted in significant changes in amiloride-sensitive Na+ currents following external application of the sulfhydryl reagents are marked with *. Residue Gly579 is not marked based on the small inhibition by Cd2+.

We observed distinct effects of MTS reagents on ENaCs with cysteine substitutions within the amino-terminal (alpha Val569-alpha Trp582) and carboxyl-terminal (alpha Ser583-alpha Ser592) domains of the pore region of the mouse alpha -subunit (Fig. 2A). Within the carboxyl-terminal portion of the pore region of alpha mENaC, only 1 of 8 mutants examined (alpha S583Cbeta gamma ) responded to both MTSEA (I/I0 = 0.04 ± 0.01, n = 4) and MTSET (I/I0 = 0.31 ± 0.09, n = 4) with a large inhibition of the amiloride-sensitive inward Na+ current (Fig. 2A and Fig. 3, B and C). Several mutants (alpha S588C, alpha V590C, and alpha L591C) responded to MTSEA with an inhibition of the amiloride-sensitive inward Na+ current that was significantly greater than WT. These residues are located in close proximity to and on either side of a three-residue tract (alpha Gly587-alpha Ser589) that has a critical role in restricting K+ permeation through the channel (11, 12, 14, 15). One mutant (alpha S588C) that responded to MTSEA with a partial inhibition of whole cell Na+ current is located within this three-residue tract. The MTSEA- and MTSET-induced inhibition of whole cell Na+ currents remained after these reagents were removed from the bath solution (Fig. 3C).



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Fig. 3.   Representative two-electrode voltage clamp recordings showing the responses of mutant mENaCs to external sulfhydryl reagents. A, effect of external 1 mM MTSET on alpha S580Cbeta gamma is shown. Whole cell Na+ current traces were obtained before (MTSET-) and after (MTSET+) application of the reagent, after washout of the reagent, and after perfusion with 0.1 mM amiloride. Current-voltage curves (IV) were obtained by plotting amiloride-sensitive Na+ currents against clamp voltages in the range of -100 to 60 mV. Filled circles represent basal currents, and open circles indicate the currents recorded after MTSET treatment. B, responses of alpha S583Cbeta gamma to MTSEA (left) and MTSET (center) are displayed as IV curves before (filled circles) and after (open circles) external application of the reagents. Currents are amiloride-sensitive Na+ currents. The right panel shows representative voltage ramp recordings from an oocyte expressing alpha S583Cbeta gamma . The oocyte was clamped at 6-s intervals with a linear ramp from -100 mV to 60 mV over 1 s. Top traces are the currents recorded before (0 s) and after (6 and 12 s) perfusion of a bath solution containing 5 mM Cd2+. Current trace in the presence of amiloride is close to zero current level. The zero current level is indicated by a dashed line. C, time courses of MTSET-induced changes in amiloride-sensitive Na+ currents from oocytes expressing WT mENaC (), alpha S576Cbeta gamma (black-triangle), alpha S580Cbeta gamma (), or alpha S583Cbeta gamma (×). Amiloride-sensitive Na+ currents were measured at 5-s intervals at -100 mV and normalized to the current level immediately prior to delivery of a reagent (at time = 1 min). Normalized currents (I/I0) are shown. Solid bar indicates the period (1-4 min) when the oocyte was bathed with 1 mM MTSET. MTSET was washed out over a 3-min period (4-7 min) with bath solution. Dashed bar indicates the period (7-8 min) when 100 µM amiloride was present in the bath solution.

In contrast, channels with cysteine substitutions at multiple sites within the amino-terminal portion of the pore region of alpha mENaC responded to MTSEA (i.e. alpha V572C, alpha S576C, alpha S580C, alpha Q581C, and alpha W582C) or MTSET (i.e. alpha V572C, alpha S576C, alpha N577C, and alpha S580C) with a significant increase in amiloride-sensitive inward Na+ currents (Fig. 2A). Only one mutant (alpha S573Cbeta gamma ) responded to MTSET with an inhibition of whole cell Na+ current (I/I0 = 0.64 ± 0.03, n = 5). The residues within the amino-terminal portion of the pore region where cysteine substitutions responded to MTS reagents with a large change in whole cell Na+ current line one face of an alpha -helix, with the exception of alpha W582C (Fig. 2B), suggesting that this region is alpha -helical in structure.

These increases in whole cell currents occurred rapidly (within a minute) after application of MTS reagents and were not reversible after MTSEA or MTSET were removed from the bath solution (Fig. 3C), indicating that the introduced cysteine residues were irreversibly modified. The inward Na+ currents following stimulation or inhibition by MTSEA or MTSET were completely inhibited by 100 µM amiloride (Fig. 3C). These data suggest that MTS modification of the alpha mENaC pore residues did not interfere with the blocking effect of 100 µM amiloride, although it has been proposed that amiloride blocked ENaC by binding to a site formed by alpha Ser583, beta Gly525, and gamma Gly542 (10). Fig. 3A shows representative recordings of the response of alpha S580Cbeta gamma to 1 mM MTSET.

MTSEA and MTSET introduce positively charged groups to targeted cysteine residues. We tested whether the charges carried by MTS reagents were related to the stimulatory or inhibitory effects on mutant channels by examining the effects of a negatively charged reagent MTSES (5 mM) on alpha S576Cbeta gamma , alpha S580Cbeta gamma , and alpha S583Cbeta gamma . alpha S576Cbeta gamma responded to MTSES with a modest increase in whole cell Na+ current (I/I0 = 1.21 ± 0.04, n = 4), and alpha S580Cbeta gamma responded to MTSES with a large increase in whole cell Na+ current (I/I0 = 3.3 ± 0.1, n = 6, Fig. 4A). In contrast, whole cell currents in oocytes expressing alpha S583Cbeta gamma were unchanged in response to MTSES. When oocytes expressing alpha S583Cbeta gamma were pretreated with MTSES, subsequent treatment with MTSEA still greatly inhibited whole cell Na+ currents (I/I0 = 0.25 ± 0.04, n = 4), suggesting that alpha S583C was still accessible to MTSEA. When either alpha S576Cbeta gamma or alpha S580Cbeta gamma was pretreated with MTSES, the subsequent increase in whole cell Na+ current in response to MTSEA was significantly less than that observed without MTSES pretreatment (Fig. 4B). These data suggest that MTSES reacted efficiently with both alpha S576Cbeta gamma and alpha S580Cbeta gamma but that alpha S583C was largely unmodified by MTSES. Snyder et al. (15) also observed that human ENaC with a cysteine substitution at the site analogous to alpha Ser583 (i.e. gamma G536C) was modified by MTSET but not by MTSES.



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Fig. 4.   Effects of external MTSES (5 mM) on wild-type and mutant mENaCs. A, external application of the negatively charged MTSES increased Na+ currents of alpha S576Cbeta gamma and alpha S580Cbeta gamma mENaC without affecting the currents of wild type and alpha S583Cbeta gamma mENaCs. Bars represent amiloride-sensitive Na+ currents (mean ± S.E., from 4 to 8 oocytes) in the presence of 5 mM MTSES normalized to the current level in the absence of MTSES (I/I0). Solid bars indicates values are significantly different from wild type (p < 0.01). B, pretreatment with 5 mM MTSES significantly reduced MTSEA-induced increases in whole cell Na+ currents in oocytes expressing alpha S576Cbeta gamma or alpha S580Cbeta gamma , and the MTSEA induced current inhibition of alpha S583Cbeta gamma . Open bars indicate ratios of currents after and before application of 2.5 mM MTSEA (I/I0). Solid bars represent the ratios of currents after and before application of 2.5 mM MTSEA (I/I0) measured in oocytes pretreated with 5 mM MTSES. * indicates a statistically significant difference (p < 0.001, mutant versus WT). # indicates a statistically significant difference (p < 0.05, MTSES-pretreated oocyte versus none-pretreated oocyte).

Responses of Mutant mENaCs to External Cd2+-- Group IIB divalent cations such as Cd2+ and Zn2+ are able to bind free sulfhydryls with high affinity and therefore have been used as biophysical probes to study the pore structure of ion channels (19-22). Cd2+ was used in this study as its crystal radius (0.92 Å) (21) is nearly same as that of Na+ (0.95 Å) (23). We examined whether extracellular Cd2+ (5 mM) altered whole cell Na+ currents in oocytes expressing either WT or mutant mENaCs. A modest increase in amiloride-sensitive whole cell Na+ current was observed in response to Cd2+ in oocytes expressing WT alpha beta gamma mENaC. A similar response to extracellular Cd2+ was observed with 16 of the 22 mENaC mutants examined. Several mutations within the alpha mENaC pore region responded to Cd2+ with a significant increase (alpha N577C, alpha S580C, and alpha W582C) or a modest decrease (alpha G579C and alpha L584C) in whole cell Na+ current. Similar to MTSEA, Cd2+ abolished whole cell amiloride-sensitive Na+ currents in oocytes expressing alpha S583Cbeta gamma (I/I0 = 0.03 ± 0.01, n = 4, Fig. 2A). The blocking effect of Cd2+ on alpha S583Cbeta gamma was both fast and voltage-dependent, as evidenced by the minimal block of outward currents, compared with the large inhibition of inward currents (Fig. 3B, right panel). This is consistent with the observation of voltage-dependent block of rat alpha S583Cbeta gamma ENaC by external Zn2+ (10).

Role of Pore Region in ENaC Gating-- The introduction of cysteine residues at alpha Val572, alpha Ser576, alpha Asn577, alpha Ser580, alpha Gln581, or alpha Trp582 led to channels that responded to MTSEA, MTSET, MTSES, or Cd2+ with an increase in whole cell Na+ current. These increases in whole cell currents occurred rapidly (within a minute) after external application of the MTS reagent or Cd2+ (Fig. 3C), suggesting that changes in single channel Na+ conductance or open probability occurred. Previous studies demonstrated that the introduction of residues with large side chains at the site analogous to alpha Ser576 of mENaC led to a significant increase of currents in oocytes expressing ASIC2 (or BNC1), an ENaC-related H+-gated ion channel (24). Similar mutations of deg-1 or mec-4 (mechanosensitive proteins in Caenorhabditis elegans) led to neuronal degeneration (25, 26). We examined whether the introduction of a residue with a large side chain at or in proximity to alpha Ser576 increased whole cell Na+ currents. Whole cell Na+ currents in oocytes expressing either alpha V572Fbeta gamma or alpha S576Cbeta gamma were significantly greater than that observed in oocytes expressing WT ENaC, although Na+ currents measured in oocytes expressing alpha V572Cbeta gamma or alpha S576Fbeta gamma were similar in magnitude to WT. In contrast, whole cell currents measured in oocytes expressing alpha S580Fbeta gamma or alpha S580Cbeta gamma were significantly less than that observed in oocytes expressing WT ENaC (Fig. 5).



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Fig. 5.   Expressed amiloride-sensitive Na+ currents (mean ± S.E., n = 6-11 oocytes) of wild type and selected mENaC mutants were obtained from paired measurements. Filled bars indicate statistically significant differences in whole cell amiloride-sensitive Na+ currents (p < 0.05, mutant versus WT). Each oocyte was injected with 1 ng of cRNA for each subunit and clamped in an alternate order 20-30 h after injection.

We performed single channel analyses of alpha S580Cbeta gamma before and after treatment with MTSET, to test whether the modification of the introduced cysteine residue altered channel gating (Fig. 6). The single channel slope conductance for Na+ of alpha S580Cbeta gamma was 3.7 ± 0.3 pS (n = 11), slightly less than the conductance of wild type mENaC (4.3 pS) (14). This is consistent with our previous observation that the single channel slope conductance for Li+ of alpha S580Cbeta gamma was nearly identical to WT ENaC and that the Li+/Na+ current ratio for alpha S580Cbeta gamma was 1.36-fold greater than that of WT (14). The open probability of alpha S580Cbeta gamma was 0.07 ± 0.02 (n = 5), determined at potentials between -100 and -60 mV. The reduced single channel Na+ conductance and open probability were consistent with the reduced whole cell Na+ currents observed in oocytes expressing alpha S580Cbeta gamma , when compared with oocytes expressing WT ENaC (Fig. 5). Following treatment with MTSET, alpha S580Cbeta gamma exhibited a dramatic change in gating characteristics. When patches were made shortly (within minutes) following MTSET treatment, channels were primarily open but exhibited frequent transitions to the closed state (Fig. 6B). However, when patches were made minutes later following MTSET treatment, channels remained open, and very few brief closures were observed (Fig. 6, C and D). The open probability was not determined due to too few transitions between open and closed states despite long (>10 min) recordings; however, open probability was clearly >0.9 (Fig. 6, C and D). The single channel conductance of MTSET-modified alpha S580Cbeta gamma was 2.3 ± 0.1 pS (n = 4), lower than that of unmodified channels (equaling a 38% decrease). In many recordings we only observed noise, comparable to open channel noise, with no clear transitions suggesting that the channel remained open over a recording period of 5-10 min (data not shown). These data indicate that MTSET converted alpha S580Cbeta gamma to a lower conductance channel, but one that was nearly continuously open. These MTSET-induced changes in conductance and open probability are the likely mechanisms of the MTSET-induced increase in whole cell Na+ current observed with this mutant channel (Figs. 2A and 3A).



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Fig. 6.   External MTSET markedly increased the open probability of alpha S580Cbeta gamma mENaC. Single channel recordings were performed in the cell-attached configuration at -80 or -100 mV (negative value of pipette potential) with 110 mM NaCl solution (A-D) or 110 mM LiCl solution (E and F) in both the pipette and bath. Scales are displayed next to the recordings on the right and closed states are marked with short bars. Traces in A and E are single channel recording traces of the mutant channel prior to MTSET treatment. The channel is characterized with short open time and long close time that result in a low open probability. Trace in B shows a representative recording obtained shortly (within minutes) after MTSET treatment. It displays increased open probability due to increased open time and reduced close time as well as decreased single channel conductance. The estimated Na+ slope conductance was 2.3 ± 0.1 pS (n = 4) from MTSET-treated cells and 3.7 ± 0.3 pS (n = 11) from untreated cells). Recordings in C and D were obtained more than 5 min after MTSET treatment. The channel stays open most of the time with very short close states. F shows a Li+ current trace following MTSET treatment. The channel was primarily open but exhibited a few transitions to the closed state that can be observed when the trace is displayed in expanded time scale (low trace). The unitary Li+ current was decreased (0.3 pA) when compared with untreated channels (0.7 pA) at -80 mV.

Similar single channel analyses were performed with Li+ as the conducting ion in the pipette, as well as in the bath solution. We observed that MTSET treatment of oocytes expressing alpha S580Cbeta gamma reduced the unitary Li+ current from 0.7 to 0.3 pA at -80 mV and locked the channel in an open state as only brief closures were observed (Fig. 6F). The Li+ current reduction in response to MTSET was larger than Na+ current reduction following MTSET treatment. These data obtained from single channel analyses with Li+ as the conducting ion were consistent with the effect of MTSET on amiloride-sensitive whole cell Li+ currents measured in oocytes expressing alpha S580Cbeta gamma . Unlike the effects of MTSET on whole cell alpha S580Cbeta gamma Na+ currents, Li+ currents were not significantly increased by MTSET (I/I0 = 0.93 ± 0.06, n = 4). It is likely that the MTSET-induced reduction in Li+ unitary current balances the MTSET-induced increase in alpha S580Cbeta gamma open probability.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The substituted cysteine accessibility method has been used to probe pore structure of various ion channels (27). In this study, we used this approach to examine the pore structure of alpha ENaC. Several distinct effects of MTS reagents and Cd2+ on alpha beta gamma mENaCs were observed with cysteine substitutions within the pore region of the alpha -subunit. MTSEA, MTSET, and Cd2+ inhibited whole cell Na+ currents in oocytes expressing alpha S583Cbeta gamma , as previously reported (4, 10). Surprisingly, this is the only mutant that displayed significant block by these three reagents. The lack of an inhibitory effect of MTS reagents on engineered cysteines near alpha Ser583 indicates that this residue is located within a restricted site. Interestingly, another MTS reagent (MTSES) with a negative charge did not inhibit alpha S583Cbeta gamma . Furthermore, MTSES pretreatment of oocytes expressing this mutant channel failed to prevent the subsequent inhibition by MTSEA (Fig. 4B), indicating that MTSES did not efficiently modify alpha S583Cbeta gamma . Schild et al. (10) proposed that alpha Ser583 is located in the electrical field of the ENaC pore, as Zn2+-induced inhibition of alpha S583Cbeta gamma was voltage-dependent. We also observed that Cd2+-induced block of alpha S583Cbeta gamma mENaC was voltage-dependent (Fig. 3B). These results suggest that a negative potential within the vicinity of alpha Ser583 hinders the access of MTSES. This notion is reminiscent of the proposal that the pore helix of KcsA K+ channels generates a negative potential at its carboxyl terminus that contributes to the stabilization of cations in the pore cavity (28).

At sites amino-terminal to alpha Ser583, only alpha S573Cbeta gamma and alpha G579Cbeta gamma responded to MTSET or Cd2+ with a partial inhibition of the whole cell Na+ current. Several mutant channels with cysteine mutations within the amino-terminal portion of the pore region (i.e. amino-terminal to alpha Ser583) responded to sulfhydryl reagents with significant increases in whole cell current. These residues (alpha Val572, alpha Ser576, alpha Asn577, alpha Ser580, alpha Gln581, and alpha Trp582) line one face of an alpha -helix, with the exception of alpha W582C (Fig. 2B), consistent with an alpha -helical structure as suggested by secondary structure predictions (Fig. 1B). These stimulatory effects do not appear to rely on the positive charges carried by these reagents, as the negatively charged MTSES also stimulated alpha S576Cbeta gamma and alpha S580Cbeta gamma (Fig. 4A). Snyder et al. (15) also observed MTSET-induced activation of Na+ currents with cysteine mutations in this region of human gamma ENaC, although only three mutants responded with a gain of function in response to MTSET, and the location of these residues was not suggestive of an alpha -helical structure. These differences in responses of alpha - and gamma -subunit mutants to MTS reagents may reflect, in part, the presence of two alpha -subunits and only one gamma -subunit within each channel protein. Another possibility is that the three ENaC subunits may not be arranged symmetrically along pore axis. This latter notion is supported by the observations that mutations at homologous sites in different subunits led to different changes in channel selectivity, amiloride sensitivity, and divalent cation sensitivity (10, 12, 15). Although KcsA core pore is formed by four identical subunits in a symmetrical manner (16), studies on voltage-gated Na+ channels suggested that the four-pore segments from Domains I-IV are arranged asymmetrically (19, 20).

ENaCs with cysteine substitutions at sites carboxyl-terminal to alpha Ser583 either did not responded to MTS reagents and Cd2+ or, alternatively, were partially inhibited (i.e. alpha L584Cbeta gamma , alpha S588Cbeta gamma , alpha V590Cbeta gamma , and alpha L591Cbeta gamma ). MTSET did not inhibit channels with introduced cysteines carboxyl-terminal to alpha Ser583, a region encompassing the proposed selectivity filter. Given several reports indicating alpha Gly587 and alpha Ser589 have an important role in conferring cation selectivity and restricting K+ permeation (11, 12, 14), residues alpha Ser587-alpha Ser589 and adjacent residues are likely facing the conducting pore. A simple explanation for a lack of an inhibitory effect of MTSET on channels with cysteine substitutions in this region is that these sites are not accessible to the reagent.

Structure of the ENaC Pore Region-- The effects of MTS reagents and Cd2+ were most prominent when cysteine residues were placed at sites amino-terminal to alpha Ser583. Only modest changes in whole cell currents were observed in response to MTSEA or Cd2+ when cysteine residues were placed carboxyl-terminal to alpha Ser583. As these accessible residues (defined by a large response to MTS reagents or Cd2+) preceded alpha Leu584, our results support a model of the pore region of the channel that has been proposed by both Kellenberger et al. (11) and Snyder et al. (15). These groups suggested that pore is formed by residues that enter the membrane spanning region from an extracellular site and transitions to an alpha -helical second membrane spanning domain. As the pore enters the membrane, it gradually narrows to form a site that restricts K+, analogous to a funnel that narrows to its spout. This model is based, in part, on the observation that the substitution of cysteine residues in the beta - or gamma -subunits at a position analogous to alpha Ser583 (beta G525C and gamma G542C) resulted in a large increase in the Ki values of amiloride. Schild and co-workers (10, 11) proposed that amiloride interacts directly with these residues and that alpha Ser583 must be external to alpha Ser589. A model of the outer vestibule of the pore, incorporating an alpha -helical structure that transitions to a narrow selectivity filter, is illustrated in Fig. 7B. This model is consistent with that proposed by Palmer (29) 10 years ago.



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Fig. 7.   Structural models of the alpha ENaC pore region. A, structure of KcsA pore region (right) and a model for alpha mENaC pore region (left) are presented in stick model with ribbon rendering (9 shin green lines) using the modeling software HyperChem 5.1 (Hypercube Inc., Gainesville, FL). For KcsA K+ channel, structure of residues P41-Y60 was generated from coordinates obtained from the Protein Data Bank (code: 1BL8) and the numbering of the residues is shown in C of this figure. The structural model for ENaC pore region was generated by mutating KcsA pore region residues to Val572-Ser592 of alpha mENaC according to the alignments shown in C. Residue alpha Phe586 was omitted and energy minimization was not performed. Element colors are as followings: cyan for carbon, blue for nitrogen, and red for oxygen. Residues corresponding to Val572, Ser573, Ser576, Asp577, Ser580, Gln581, Trp582, and Ser583 of alpha mENaC are displayed in violet color. Substituted cysteines at these sites were accessible to sulfhydryl reagents from extracellular side judged by significant changes in amiloride-sensitive Na+ currents in response to these reagents. KcsA residues homologous to the residues in voltage-gated and inward rectifier K+ channels that are accessible to external sulfhydryl reagents are also shown in violet color. B, an alternative model for the alpha mENaC pore region was generated by rotating residues Ser12-Ser20 (corresponding to Ser583-Ser592 of alpha mENaC) from the model A (left panel) by 180° along the X axis. Externally accessible residues in alpha mENaC are also highlighted in violet color as in A. This model includes all 24 residues (Val569-Ser592) of alpha mENaC pore region. The key residues retaining K+/Na+ selectivity (Gly19 and Ser21, corresponding to Gly587 and Ser589 of alpha mENaC) are located in the narrowest region of the pore. C, sequence alignments and comparison of accessibility to external sulfhydryl reagents between K+ channels and ENaC. The alignments of the pore region residues of KcsA, Shaker K+ channel, inward rectifier K+ channel Kir 2.1 and alpha mENaC were performed by aligning the Gly587-Ser588-Ser589 of alpha mENaC to the K+ channel GYG track. A gap was introduced in K+ channels to enhance overall alignments. The plus sign (+) indicates sites where cysteine substitutions within the pore region result in channels that were inhibited (or activated) by MTS reagents, Ag+, or Cd2+ and the minus sign (-) shows residues not accessible to the reagents. The plus/minus sign (±) indicates sites where cysteine substitutions result in channels that were modestly inhibited by MTSEA or Cd2+. The X indicates the sites not tested for sulfhydryl reagent accessibility.

Schild et al. (10) previously demonstrated that ENaCs with acidic residues at position alpha Ser580 (or at the analogous positions beta Gly522 and gamma Gly534) were inhibited by extracellular Ca2+ in a voltage-dependent manner, suggesting that these residues (i.e. amino-terminal to alpha Ser583, beta Gly525, and gamma Gly537, respectively) were accessible to extracellular Ca2+. These data support the proposed pore structure of Kellenberger et al. (11) and Snyder et al. (15), whereby the interaction of Ca2+ with alpha S580D would block the pore of the channel (see Fig. 7B). However, we observed that oocytes expressing alpha S580Cbeta gamma mENaC responded to MTSEA, MTSET, and Cd2+ with a large increase in amiloride-sensitive currents. If the alpha S580C side chain extends into the pore lumen, its modification by MTS reagents or Cd2+ would be expected to inhibit the channel and not activate it. Furthermore, channels with substituted cysteine residues at selected sites near alpha Ser580 (i.e. alpha Val572, alpha Ser576, alpha Asn577, alpha Gln581, or alpha Trp582) also responded to MTSEA, MTSET, or Cd2+ with an activation of amiloride-sensitive currents. If this proposed pore structure is correct, it is reasonable to predict that channels with cysteine residues substituted at multiple sites amino-terminal to the selectivity filter would respond to MTSEA, MTSET, or Cd2+ with a large inhibition of whole cell currents. However, of the mutants we have examined, only alpha S583Cbeta gamma responded to these reagents with a large inhibition of Na+ current. Our results suggest that periodic residues within the amino-terminal portion of the alpha mENaC pore region are accessible to sulfhydryl reagents externally applied but do not directly face the conducting pore.

The outer pore of the KcsA K+ channel is formed by an alpha -helix that enters the membrane followed by an extended region directed toward the extracellular space (16) (Fig. 7A). Is it necessary to propose a pore structure for ENaC that basically differs from other highly selective cation channels? Previous studies have suggested that the pore regions of the voltage-gated Na+ and Ca2+ channels are similar in structure to voltage-gated K+ channels, although they clearly differ in mechanisms for achieving cation selectivity (30). Our data suggest that pore region residues amino-terminal to alpha Gln581 form an alpha -helix, and our previous results (14) suggested that residues extending from alpha Ser580-alpha Ser589 form an extended selectivity filter. This secondary structure is similar to that of the KcsA K+ channel. Furthermore, within the pore regions of K+ channels (Shaker and inward rectifier Kir2.1) and alpha mENaC, the pattern of accessibility to substituted cysteine residues to cysteine-reactive reagents is strikingly similar (Fig. 7C) (31-34). Our previous observation that mutation of the GSS tract within the selectivity filter of the alpha -subunit of mENaC (alpha Gly587-alpha Ser589) to the K+ channel selectivity filter signature sequence GYG rendered the mutant channel K+-selective (14) is also consistent with the notion that K+ channels and ENaC may have similar pore structures.

Fig. 7A illustrates a model of the ENaC alpha -subunit pore region using the structure of the KscA K+ channel, aligning the GYG tract within KscA with the GSS tract within alpha ENaC. Residues alpha Val572-alpha Ser580 form an alpha -helix; alpha Leu584-alpha Ser589 form an extended selectivity filter; and alpha Gln581-alpha Ser583 are located at the turn region where the alpha -helix transitions into the selectivity filter. Residues where substituted cysteines responded to MTS reagents or Cd2+ line one face of the helix, with the exception of alpha Trp582 (Fig. 2B). Our model places alpha Ser592 at a location external to the GSS track, consistent with the previous observation that the mutant alpha S592Ibeta gamma rat ENaC was blocked by external Ca2+ in a voltage-dependent manner (13). This model (Fig. 7A) is not consistent with amiloride interacting directly with beta G525C or gamma G542C, residues proposed to interact directly with amiloride (10). These residues would be within the internal portion of the selectivity filter. However, these mutations (i.e. beta G525C or gamma G542C) might change the structure of the pore and indirectly alter the Ki values of amiloride, as suggested by Schild et al. (10).

The introduction of cysteine residues carboxyl-terminal to the GYG tract of K+ channels conferred sensitivity to MTS reagents (Fig. 7, A and C). If ENaC and K+ channels share a similar pore structure, the introduction of cysteine residues carboxyl-terminal to alpha Ser589 would be expected to confer sensitivity to MTS reagents. However, we only observed a modest block of Na+ currents in response to MTSEA when cysteine residues were present at alpha Val590 or alpha Leu591, and no changes in whole cell Na+ currents were observed in response to MTSET or Cd2+. These results suggest that K+ channels and ENaC differ in their pore structure, although the nature of these differences has yet to be defined. Based on our data and previous studies, we propose that the amino-terminal portion of the alpha ENaC pore region forms an alpha -helix, and the carboxyl-terminal portion forms a selectivity filter, like that of K+ channels. Furthermore, we propose that the transition from selectivity filter to alpha -helical second membrane spanning domain within ENaC has a structure that differs from K+ channels.

Role of Pore Region in ENaC Gating-- Our observation that mutant mENaCs with cysteine substitutions within the amino-terminal portion of the pore region (preceding alpha Ser583) responded to MTSEA, MTSET, or Cd2+ with an increase in whole cell Na+ current led us to examine whether this region has a role in ENaC gating. In agreement with our observation that MTS reagents activated whole cell Na+ currents in oocytes expressing alpha S580Cbeta gamma , MTSET induced a large increase in open probability of alpha S580Cbeta gamma , indicating that this residue is within a domain that controls ENaC gating. Our data suggest alpha Ser576 and alpha Ser580 are two crucial residues in this gating domain, as all three MTS reagents dramatically stimulated whole cell Na+ currents in oocytes expressing these mutant channels. This proposed role of alpha Ser576 in channel gating is in agreement with previous observations suggesting a similar role of the residue at the analogous position in degenerins and in H+-gated Na+ channels. The introduction of bulky residues at a site analogous to alpha S576C within the C. elegans proteins deg-1 and mec-4 lead to neurodegeneration that is proposed to occur as a result of an unregulated activation of a putative mechanosensitive cation channel (35, 36). Similarly, selected mutations at an analogous site within this pore region of related H+-gated Na+ channels, termed BNC (or ASIC2), resulted in sustained channel activation that was independent of acidification (24). Based on these observations, we propose that the ENaC pore region participates in channel gating.

Aside from increasing open probability, external application of MTSET reduced both Na+ and Li+ unitary currents of alpha S580Cbeta gamma . The observed changes in whole cell amiloride-sensitive Na+ currents of alpha S580Cbeta gamma in response to MTSET reflects opposing effects on open probability (increased) and unitary current (decreased). The mechanism of MTSET-induced reduction in unitary current is unknown. This may reflect changes in the conformation of the open channel due to covalent binding of MTSET to alpha S580C; alternatively, the positively charged MTSET may partially block the pore.

Waldmann et al. (13) reported that alpha S589I and alpha S589F (analogous to alpha Ser588 of mENaC) reduced rat ENaC open probability from 0.89 to 0.09 and 0.04, respectively, and increased Na+ unitary conductance from 4.6 to 10 pS without changing Li+ conductance. This study provided evidence that alpha Ser588, located within the selectivity filter of ENaC, has a role in ENaC gating. We have also observed that alpha S588Cbeta gamma mENaC has very low open probability resulting from short open and long close times (data not show). Fyfe et al. (37) studied the functional properties of ENaCs with chimeric gamma -beta subunits. Their results also suggested that this region regulates ENaC gating. These observations that mutations within both amino-terminal and carboxyl-terminal portions of the pore region resulted in changes in channel gating suggest a collaborative role of these two regions in ENaC gating through intradomain interactions. We propose a working model for ENaC gating. In this model, the pore helix (amino-terminal portion of pore region) undergoes rotational movement. This movement could be in response to conformational changes at other sites within ENaC, such as the ectodomain, the M2 domains, or cytoplasmic domains (i.e. the amino terminus (38)). The rotation of the pore helix leads to changes in the diameter of the selectivity filter, which in turn allows ion translocation through the pore. A recent study of KscA gating observed movement of reporter cysteines introduced at the carboxyl-terminal end of the pore helix in association with the channel gating (45). If the ENaC pore structure shares the fundamental design of the KcsA K+ channel pore, alpha Ser576 and alpha Ser580 are expected to be close to the carboxyl-terminal end of the pore helix of ENaC. Substitution of large side chains at these two sites might hinder rotational movement of the pore helix, essentially locking the channel in an open state. Our model suggests that there are extensive interactions between the amino-terminal and carboxyl-terminal portions of the pore region, and we propose that this occurs, in part, through hydrogen bonding. ENaC pore regions have a large number of serine residues (7 in alpha mENaC, 2 in beta mENaC, and 4 in gamma mENaC). Given the proposed subunit stoichiometry of 2alpha , 1beta , and 1gamma , a total of 20 serine residues are present within the pore regions of the channel. These serine residues, as well as other polar residues and backbone carbonyl oxygens, are capable of forming a network of hydrogen bonds. A sliding model of gating is also plausible. A relative sliding movement between alpha Ser576-Ser580 and alpha Ser588-Ser592 could lead to channel transitions between open and closed states.

Our model predicts a connection between ENaC gating and permeation and favors a dynamic selectivity filter. Gating and permeation have been generally considered as independent processes of ionic channels since 1952 (39). However, recent studies have challenged this concept (40, 41). The notion that pore regions function as ion channel gates has been proposed for cyclic nucleotide-gated channel (42). Recent studies support a dynamic selectivity filter challenging another long term notion of a rigid selectivity filter (43).

Alternatively, if the ENaC pore is similar to that proposed by Kellenberger et al. (11) and Snyder et al. (15) (Fig. 7B), gating might also involve rotational movement of the alpha -helical region preceding the selectivity filter. This gating mechanism was proposed by Adams et al. (24) in their studies of activation of the H+-gated Na+ channel BNC (or ASIC2).

We observed that several alpha ENaC mutants in the pore helix, including alpha V572Fbeta gamma and alpha S576Cbeta gamma , expressed whole cell currents in oocytes that were significantly greater than that observed with WT ENaC, consistent with the notion that mutations within this region affect channel gating. We anticipate that selected mutations within this domain of human ENaC are likely to be found in the clinical setting of salt-sensitive hypertension due to enhanced ENaC-mediated Na+ transport in the distal nephron. In this context, Melander et al. (44) reported a mutation within human ENaC (gamma N530K), a position analogous to mouse alpha Asn577, in a patient with hypertension and diabetes, although a causal relationship was not established. In summary, our data suggest that the amino-terminal pore region of alpha ENaC is alpha -helical in structure and that this region is involved in channel gating.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK54354 and DK50268 and by the Department of Veterans Affairs.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. Section 1734 solely to indicate this fact.

Dagger Recipients of postdoctoral fellowship awards from the Cystic Fibrosis Foundation.

Present address and to whom correspondence should be addressed: Renal Division, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213. Tel.: 412-647-3121; Fax: 412-647-6222; E-mail: kleyman@pitt.edu.

Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M008117200


    ABBREVIATIONS

The abbreviations used are: ENaC, epithelial sodium channel; WT, wild type; MTS, methane thiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSET, [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; KcsA, potassium channel from Streptomyces lividans; ASIC, acid-sensing ion channel; BNC, brain sodium channel; pS, picosiemen..


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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