Asymmetric Organization of the Pore Region of the Epithelial Sodium Channel*

Jinqing LiDagger §, Shaohu Sheng§, Clint J. Perry, and Thomas R. Kleyman||**

From the Dagger  Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Departments of  Medicine and of || Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, January 7, 2003, and in revised form, January 27, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta -subunit that line the pore, we systematically mutated residues Gln523-Ile536 to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li+ to Na+ current ratios, whereas only cysteine replacement of beta Gly529 resulted in K+-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly529 and beta Ser531 within the previously identified 3-residue tract that restricts K+ permeation. Cysteine substitution preceding (beta Phe524 and beta Gly525), within (beta Gly530) or following (beta Leu533) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln523, beta Phe524, and beta Trp527. MTSET did not inhibit alpha beta G525Cgamma , although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha - and gamma -subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The epithelial sodium channel (ENaC)1 represents the dominant pathway mediating Na+ reabsorption in the distal nephron, distal colon, and airway. ENaC has a critical role in regulating transepithelial Na+ transport and modulating extracellular fluid volume and blood pressure. ENaCs are composed of three homologous subunits, termed alpha , beta , and gamma  (1), with a subunit stoichiometry of 2alpha :1beta :1gamma (2, 3), although an alternative 3alpha :3beta :3gamma subunit stoichiometry has been proposed (4). These subunits have a similar topology, consisting of two transmembrane domains (termed M1 and M2) separated by a large extracellular domain (5-7). A limited region preceding the M2 domain of each subunit may form part of the channel pore. Selected mutations within these pore regions alter ion selectivity, or single channel conductance, as well as amiloride sensitivity (8-13). A critical 3-residue ((G/S)XS) tract within each subunit appears to primarily govern ENaC selectivity (8, 9, 11, 14).

We observed previously that the systematic introduction of cysteine mutations within the alpha -subunit pore region led to changes in the channel selectivity for cations at 6 residues (8). In contrast, Snyder et al. (9) observed that cysteine substitutions at two residues within the pore region of the gamma -subunit (human gamma Ser541 and gamma Ser543) led to changes in cation selectivity, suggesting that the channel selectivity filter is restricted to a limited site within the pore region of the gamma -subunit. To address the role of residues within the beta -subunit in conferring cation selectivity and amiloride sensitivity, we systematically mutated residues within the pore region (beta Gln523-beta Ile536) to cysteine (Fig. 1). Mutant beta mENaCs were co-expressed with wild type alpha mENaC and wild type (or C-terminal truncated) gamma mENaC in Xenopus oocytes, and functional characteristics were examined. As with the alpha -subunit (8), changes in cation selectivity were observed with most mutants within this region. Large (>9-fold) increases in the inhibitory constant of amiloride were observed with mutations at beta Gly525, beta Gly529, and beta Ser531. Responses of mutant channels to sulfhydryl-reactive reagents indicated that the apparent accessibility to (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA) differed depending on whether a residue preceded or followed the selectivity filter. Furthermore, a putative amiloride-binding site, previously identified on the basis of site-directed mutations that dramatically increase the Ki for amiloride (12), was inaccessible to a large sulfhydryl-reactive reagent. Our observations, placed in context with previously published results suggest that ENaC pore regions have an asymmetric organization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All chemicals were purchased from Sigma unless specified otherwise.

Site-directed Mutagenesis-- Mouse ENaC (alpha , beta , and gamma ) cDNAs were previously cloned into pBluescript SK(-) (Stratagene, La Jolla, CA) (15). Amino acids in the region preceding and transitioning into the M2 region of beta mENaC, beta Gln523-beta Ile536 (with the exception of beta Cys534), were individually mutated to cysteine using a polymerase chain reaction method as previously described (8). A truncation of the C terminus of gamma mENaC (R583X) was generated by insertion of a stop codon. This mutation increases function Na+ channel expression in Xenopus oocytes (16). All mutations were confirmed by automated DNA sequencing at sequencing facilities at the University of Pennsylvania or the University of Pittsburgh. T3 RNA polymerase (Ambion Inc., Austin, TX) was used to synthesize cRNAs for mutant and wild type beta mENaCs, wild type alpha mENaC and gamma mENaC from linearized DNAs. cRNAs were stored at -80 °C and diluted in diethylpyrocarbonate-treated water prior to injection.

Channel Expression in Xenopus Oocytes-- Xenopus oocytes (stage V-VI) were pretreated with 2 mg/ml collagenase (type IV) in a Ca2+-free saline solution. ENaC cRNAs (1-3 ng/subunit in 50 nl of H2O) were microinjected into oocytes. The oocytes were incubated at 18 °C in modified Barth's saline (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES-NaOH, pH 7.2, supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamycin sulfate. Whole cell currents were measured 18-96 h after cRNA injections.

Whole Cell Current Measurements-- A two-electrode voltage clamp technique was used to measure the whole cell inward amiloride-sensitive currents in oocytes expressing wild type or mutant alpha beta gamma mENaCs with a DigiData 1200 interface (Axon Instruments, Foster City, CA) and a TEV 200A Voltage Clamp amplifier (Dagan Corp., Minneapolis, MN). Data acquisition and analyses were performed using pClamp 7.0 software with a Pentium II-based PC (Gateway, 2000 Inc., N. Sioux City, SD). Amiloride-sensitive currents were defined as the difference of the current in the absence and the presence of 0.1 mM amiloride. Higher concentrations of amiloride (1 or 2 mM) were used to define amiloride-sensitive currents in oocytes expressing the beta G525C, beta G529C, and beta S531C mutants.

Pipettes of borosilicate glass capillaries (Worldwide Precision Instruments Inc., Sarasota, FL) were utilized to pull electrodes with a micropipette puller (Sutter Instrument Co., Novato, CA). The electrodes were filled with 3 M KCl, and the resistances were 0.5-3 megaohms in a 110 mM NaCl bath solution. Oocytes were bathed in a solution containing 110 mM NaCl, 2 mM CaCl2, 2 mM KCl, 10 mM HEPES-NaOH, pH 7.40. For selectivity experiments, separate bath solutions containing either Na+, Li+, or K+ as the predominant cation were used. All measurements were carried out at room temperature (22-25 °C), and the bath solution was continuously perfused at 5 ml/min by gravity. Oocytes were typically incubated in the bath solution for at least 10 min before the current was recorded to allow currents to stabilize. Membrane potentials were clamped from -140 to +60 mV in 20-mV increments with the duration of 900 ms. Currents were measured at 600 ms after initiation of the clamp potential. Intersweep potential was held at the reverse potential. Macroscopic currents were recorded with each bath solution when stable membrane potentials were observed. Amiloride inhibitory constants (Ki) were determined by nonlinear regression analysis with Origin program 5.0 (Microcal Inc., Northampton, MA), using the equation,
I/I<SUB>o</SUB>=<FR><NU>K<SUP>n′</SUP><SUB>i</SUB></NU><DE>K<SUP>n′</SUP><SUB>i</SUB>+[<UP>B</UP>]<SUP>n′</SUP></DE></FR> (Eq. 1)
where Io and I represent inward current carried by Na+ at -100 mV clamping potential in the absence and presence of amiloride (B), respectively, and n' is the Hill coefficient. To determine cation selectivity of wild type and mutant channels, oocytes were sequentially perfused with bath solutions containing K+, Na+, or Li+ as the predominant cation. Oocytes were subsequently bathed in the K+, Na+, or Li+ solutions supplemented with 0.1 or 2 mM amiloride. Amiloride-sensitive currents measured at -100 mV were used to calculate the ratios of the K+ current (IK) and Li+ current (ILi) relative to the Na+ current (INa).

The accessibility of mutant channels with cysteine substitutions within the pore region to sulfhydryl reagents was examined using the sulfhydryl-reactive reagents MTSEA and [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET) (Toronto Research Chemicals, Inc., North York, Canada). Na+ currents were measured before and 3 min following perfusion of oocytes with the methanethiosulfonate (MTS) reagent. Oocytes were washed with an MTS-free NaCl bath solution for >10 min to determine the reversibility of the current response. A bath solution containing 0.1 or 1.0 mM amiloride was then perfused into the oocyte chamber to determine the amiloride-insensitive component of the whole cell current.

Rates of inhibition of Na+ currents by MTSEA were determined by clamping the membrane potential every 5 s at -100 and +60 mV for 900 ms following the arrival of the reagent to the recording chamber. Currents were measured at 600 ms after the initiation of the -100 mV clamp potential. Intersweep potential was held at the reverse potential. The time course of MTSEA inhibition of the currents is shown as relative currents plotted against time. The time constants were obtained by fitting the data with single exponential decay. The rates of inhibition were determined by the equation,
R=(1/T)/C (Eq. 2)
where R, T, and C represent the rate (M-1 s-1), time constant (s), and MTSEA concentration (M), respectively.

Data Analyses-- Data are expressed as mean ± S.E. Student's t test was used for significance analyses with MS Excel 97 software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Channels with Cysteine Mutations of beta mENaC in Xenopus Oocytes-- Several algorithms predicted that the second membrane-spanning domain of alpha mENaC begins between residues alpha Val590 and alpha Ser592 (8). The corresponding residues within beta mENaC are beta Val532 and beta Cys534 (see Fig. 1). In order to examine the role of residues within the pore region of the beta -subunit in conferring cation selectivity, 13 of the 14 residues in the region preceding and transitioning into the second transmembrane domain of beta mENaC, from beta Gln523 through beta Ile536, were individually mutated to cysteine. beta Cys534 is a naturally occurring cysteine. The functional characteristics of these beta mENaC mutations were determined by their co-expression with wild type alpha - and gamma mENaC in Xenopus oocytes (Table I). Average whole cell amiloride-sensitive Na+ currents ranged from -1.6 to -12.5 µA for both wild type and mutant mENaCs, with the exception of alpha beta G529Cgamma and alpha beta S531Cgamma , whose whole cell amiloride-sensitive currents were less than -400 nA at -100 mV (data not shown). We previously observed that channels with cysteine residues at the analogous sites within alpha mENaC expressed low levels of amiloride-sensitive Na+ currents (8, 17). In order to enhance levels of current expression with these mutants, beta G529C and beta S531C were co-expressed with wild type alpha -subunit and a gamma -subunit with a truncation of the C-terminal cytoplasmic domain (gamma R583X; see Table I). Na+ currents measured in oocytes expressing alpha beta gamma R583X were 1.9-fold greater than in oocytes expressing wild type alpha beta gamma ENaC (see Table I), although no change in channel cation selectivity and only a modest (less than 2-fold) change in the Ki for amiloride were observed.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Alignments of residues within the pore regions of alpha -, beta -, and gamma ENaC are illustrated. Residues within alpha mENaC (Gln581-Val594), beta mENaC (Gln523-Ile536), and gamma mENaC (Gln540-Ile553) (GenBankTM accession numbers AF112185, AF112186, and AF112187) are shown. Identical residues are shaded.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Expressed amiloride-sensitive Na+ currents, Li+/Na+ and K+/Na+ current ratios, and inhibitory constant for amiloride (Ki) (mean ± S.E. (n))
Significance was as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus wild type (WT) or alpha ,beta ,gamma R583X. beta G529C and beta S531C were co-expressed with wild type alpha -subunit and gamma R583X.

Mutations within the Pore Region of beta mENaC Alter the Ki for Amiloride-- Previous studies have shown that the introduction of a cysteine at beta Gly525 dramatically increases the Ki for amiloride (2, 12). We also observed that alpha beta G525Cgamma exhibited a 1400-fold increase in the amiloride Ki, when compared with wild type ENaC (Table I and Fig. 2). Amiloride is a pore blocker, and we examined whether the substitution of cysteine residues at other sites altered the Ki for amiloride. Large increases in the Ki for amiloride inhibition were observed when cysteine residues were placed at two sites (beta G529C and beta S531C; Table I and Fig. 2). These sites are within the critical three-residue tract within ENaC subunits (8-10, 13) that restricts K+ permeation through the channel. Modest increases in the Ki for amiloride inhibition were observed when cysteine residues were placed at other sites, including beta Phe526, beta Val532, and beta Leu533. The Hill coefficients for wild type ENaC and all mutants examined, except for alpha beta G529Cgamma R583X, were between 0.74 and 0.99. The Hill coefficient for alpha beta G529Cgamma R583X was 0.36, suggesting that this mutation led to negative cooperative interactions among sites within the channel that participate in amiloride binding.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Amiloride dose-response curves for selected beta -subunit mutants. Wild type alpha beta gamma channels exhibited a Ki for amiloride of 70 ± 10 nM (n = 7; mean ± S.E.; ). The amiloride Ki for alpha beta G525Cgamma was 97 ± 10 µM (n = 5; ). beta G529C and beta S531C were co-expressed with wild type alpha  and gamma R583X. Ki values for amiloride were increased to 5.1 ± 0.7 and 0.6 ± 0.1 µM, respectively, for alpha beta G529Cgamma R583X (n = 7; black-diamond ) and for alpha beta S531Cgamma R583X (n = 4; black-triangle). Oocytes expressing wild type or mutant ENaCs were bathed in Na+-containing solution, and whole cell currents were recorded at a holding potential of -100 mV during continuous perfusion with increasing concentrations of amiloride. The currents measured in the presence of increasing concentrations of amiloride (IAmil) were normalized to the amiloride-sensitive (1 or 2 mM) component of the whole cell current. Lines were from data fitting with Equation 1.

Mutations within the Pore Region of the beta -Subunit Alter Cation Selectivity Properties of ENaC-- The cation selectivities of wild type and beta -subunit mutant mENaCs were determined by measuring the ratio of inward Li+ or K+ whole cell amiloride-sensitive currents (ILi and IK) relative to the inward amiloride-sensitive Na+ current (INa) at a holding potential of -100 mV. Data are summarized in Table I and Figs. 3 and 4. Oocytes expressing wild type alpha beta gamma mENaC exhibited an ILi/INa of 1.88 ± 0.14 and no detectable amiloride-sensitive inward current when bathed in a solution with K+ as the predominant cation, in agreement with previous studies (8-11). Cysteine substitutions at multiple sites within the beta mENaC pore region altered the ILi/INa of the channel, similar to that observed following cysteine substitutions within this region of the alpha -subunit (8). The introduction of a cysteine residue at 9 of 13 sites with this region of beta -subunit pore altered the ILi/INa of the channel, although the changes in ILi/INa of alpha beta I536Cgamma were modest. Increases in the Li+ to Na+ current ratio of the channel were frequently observed and varied from 2.25 ± 0.14 to 5.56 ± 1.16. Two mutants exhibited a significant reduction in channel ILi/INa. The mutants alpha beta F526Cgamma and alpha beta G529Cgamma R583X exhibited ILi/INa of 1.20 ± 0.03 (p < 0.01, n = 6) and 0.30 ± 0.09 (p < 10-8, n = 5), respectively. The results suggest that multiple residues in the vicinity of the 3-residue tract of beta ENaC (beta Gly529-beta Ser531) that primarily governs ENaC selectivity affect, either directly or indirectly, cation selectivity.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Cation selectivity of wild type (WT) and mutant channels. Oocytes were injected with wild type or mutant ENaCs. Current ratios (ILi/INa) were determined from amiloride-sensitive currents in the presence of a Li+ or Na+ bath solution at a holding potential of -100 mV (A). Nine of 13 beta -subunit mutants exhibited an ILi/INa that differed significantly from wild type ENaC. Current ratios (IK/INa) were determined from amiloride-sensitive currents in the presence of a K+ or Na+ bath solution at a holding potential of -100 mV (B). Only alpha beta G529Cgamma R583X expressed a K+-permeable channel (n = 5). Data from 4-8 oocytes are presented as mean ± S.E. The p values from Student's t tests (wild type versus mutant channels) were as follows: *, <0.05; **, <0.01.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Selected mutations within the 3-residue GGS tract of the selectivity filter alter cation selectivity. Representative whole cell currents recorded in oocytes expressing either wild type ENaC, alpha beta G529Cgamma R583X, or alpha beta S531Cgamma R583X are illustrated in A. Whole cell currents were determined in oocytes bathed in a solution containing Na+, Li+, or K+ at test potentials between -140 mV to +60 mV (20-mV increments). Whole cell currents recorded in oocytes expressing wild type ENaC in the presence of 100 µM amiloride are also shown. Dashed lines indicate zero current. Amiloride-sensitive current-voltage relationships of wild type, alpha beta G529Cgamma R583X, and alpha beta S531Cgamma R583X were obtained in the presence of K+ (), Na+ (black-square), or Li+ (black-triangle) bath solutions (B). Data are presented as mean ± S.E. from 5-8 oocytes.

Little or no inward amiloride-sensitive currents carried by K+ were observed with 12 mutations within the pore region of beta mENaC as well as with wild type alpha beta gamma mENaC and with alpha beta gamma R583X (Table I and Fig. 3). As previously observed (11), the introduction of a cysteine residue at beta Gly529 resulted in channels that were permeable to K+ (Table I; Figs. 3 and 4). The IK/INa for beta G529C (8.9 ± 3.6, n = 5) was remarkably different (p < 0.001) from wild type. This channel (alpha beta G529Cgamma R583X) displayed outward rectification and its reverse potential in a K+ bath shifted markedly to the right compared with wild type ENaC (Fig. 4). Kellenberger et al. (11) previously observed that placement of an alanine residue at beta Gly529 resulted in channels that restricted K+ permeation but reversed the channel ILi/INa. In agreement with these data, we observed that alpha beta G529Agamma R583X channels had an IK/INa 0.013 ± 0.013 (n = 7, p > 0.90 versus wild type) and an ILi/INa of 0.60 ± 0.01 (n = 7, p < 0.0001 versus wild type). These results indicate that beta G529 is the only residue in the region of this study crucial for K+ exclusion, supporting the dominant role of the 3-residue (G/S)XS tract in the determination of ENaC cation selectivity.

Selected Mutations Alter the Sensitivity of mENaC to MTS Reagents-- We examined the accessibility of extracellular MTSEA and MTSET to substituted cysteine residues within the pore region of beta mENaC by measuring the effect of these MTS reagents on amiloride-sensitive whole cell Na+ currents. The addition of 0.5 mM MTSEA to the bath solution resulted in an irreversible decrease of the amiloride-sensitive Na+ current by 28 ± 9% (n = 8; Figs. 5 and 6), in agreement with previous observations and reflects, in part, modification of Cys547 in the pore region of gamma mENaC by MTSEA (9, 17). Amiloride (0.1 mM) blocked the residual current. The response of alpha beta gamma R583X to MTSEA was similar to wild type ENaC (data not shown). The effects of external MTSEA on whole cell Na+ currents in oocytes expressing beta -subunit mutants were examined in order to identify residues within pore region accessible to MTSEA. Fractional amiloride-sensitive currents remaining following external MTSEA are shown in Fig. 5A. Cysteine substitutions at only 4 of 13 sites within the pore region of beta mENaC resulted in significantly greater MTSEA-mediated inhibition of amiloride-sensitive Na+ currents than wild type mENaC. These residues were not contiguous but were either flanked by (beta F524C, beta G525C, and beta L533C) or were within (beta G530C) the 3-residue tract (beta Gly529-beta Ser531) that has a critical role in excluding K+ from the channel pore. Both alpha beta G525Cgamma and alpha beta L533Cgamma responded to MTSEA with >80% inhibition of amiloride-sensitive INa.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   Cysteine substitutions of selected residues within the pore region of beta ENaC conferred sensitivity to MTS reagents. Oocytes were injected with wild type or mutant ENaCs. Amiloride-sensitive current in presence of 0.5 mM MTSEA, relative to the amiloride-sensitive current in the absence of MTSEA at a test potential of -100 mV is illustrated in A. Oocytes expressing wild type alpha beta gamma mENaC responded to 0.5 mM MTSEA with a 28 ± 9% (n = 8) inhibition of amiloride-sensitive whole cell Na+ currents. Four (beta F524C, beta G525C, beta G530C, and beta L533C) of 13 mutants exhibited MTSEA-mediated inhibition of amiloride-sensitive whole cell Na+ currents that were significantly greater than the inhibition observed with wild type channels. Amiloride-sensitive currents in presence of 0.5 or 2 mM (beta G529C, beta S531C, beta L535C, and beta I536C) MTSET, relative to amiloride-sensitive currents in the absence of MTSET at a test potential of -100 mV are illustrated in B. Only 3 of 11 mutant channels responded to MTSET with a modest (less than 50%) reduction in amiloride-sensitive whole cell Na+ currents (see below). The IMTSET/I0 values for alpha beta G529Cgamma R583X and alpha beta S531Cgamma R583X were not significant different from the IMTSET/I0 value for alpha beta gamma R583X (1.36 ± 0.10, n = 3, 2 mM MTSET). The p values from Student's t tests (wild type versus mutant channels) were <0.05 (*) and <0.01 (**). Data are presented as mean ± S.E. from 4-8 oocytes (A) or 4-7 oocytes (B).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Representative whole cell recordings (A) and I/V relationships (B) were obtained in the absence or presence of MTSEA in oocytes expressing wild type ENaC, alpha beta G525Cgamma , or alpha beta L533Cgamma . Whole cell currents were determined in a Na+ bath solution at test potentials between -140 mV and +60 mV (20-mV increments) prior to and following the addition of 0.5 mM MTSEA to the bath. Dashed lines indicate zero current. Amiloride-sensitive currents are illustrated in B. Data in B are presented as mean ± S.E. from 4-8 oocytes.

Residues within the pre-M2 region comprise the external portion of the ENaC pore, and several investigators have proposed that the external pore gradually narrows as it enters the membrane-spanning region to form the selectivity filter that restricts K+ (9, 11, 12). If this model is correct, the relative accessibility of beta Gly525 and beta Leu533 to MTSEA should differ, and this difference should be reflected in the rates of channel inhibition in response to MTSEA. Fig. 7 illustrates the rate of current loss over the initial 180 s following the addition of MTSEA to the bath solution. The rate of current reduction in response to MTSEA for alpha beta G525Cgamma channels (36.7 ± 1.1 M-1 s-1, n = 6) was approximately twice the rate observed for alpha beta GL533Cgamma channels (17.4 ± 1.4 M-1 s-1, n = 5, p < 0.001) (Fig. 7 and Table II). These results suggest that beta G525C is more accessible to external MTSEA than beta L533C, supporting a gradually narrowing pore geometry.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Time course for MTSEA inhibition of ENaC currents. Relative currents (mean ± S.E.) measured at -100 mV from oocytes (five or six oocytes for each group) expressing alpha beta G525Cgamma (open square), alpha beta G525Cgamma C547S (solid square), alpha beta L533Cgamma (open triangle), and alpha beta L533Cgamma C547S (solid triangle) following MTSEA application were plotted against time. Lines are from curve fitting of the data to Equation 2.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Rate of MTSEA-mediated inhibition of mutant mENaCs (mean ± S.E.)

As MTSEA inhibited wild type channels, it is possible that the introduction of cysteine residues at beta Gly525 or beta Leu533 enhanced the accessibility of gamma Cys547 to MTSEA (see above) (9, 17). We examined the effect of MTSEA on alpha beta G525Cgamma C547S and alpha beta L533Cgamma C547S channels. MTSEA inhibited both alpha beta G525Cgamma C547S and alpha beta G525Cgamma to a similar extent, and the rates of inhibition of these channels by MTSEA were similar (see Fig. 7 and Table II), suggesting that current inhibition of alpha beta G525Cgamma by MTSEA is due to covalent modification of the introduced sulfhydryl group at beta Gly525. In contrast, the extent of inhibition of alpha beta L533Cgamma by MTSEA was considerably greater than that of alpha beta L533Cgamma C547S (Fig. 7). Furthermore, the rate of inhibition of alpha beta L533Cgamma by MTSEA (17.4 ± 1.4 M-1 s-1, n = 5) was significantly greater than the rate of inhibition of alpha beta L533Cgamma C547S (11.3 ± 1.4 M-1 s-1, n = 5, p < 0.05), suggesting that the beta L533C mutation enhanced the accessibility of MTSEA to gamma Cys547. Since MTSEA-mediated inhibition of alpha beta L533Cgamma C547S was greater than both alpha beta gamma C547S and wild type mENaC, it is likely that the inhibition of alpha beta L533Cgamma mENaC by MTSEA was due to both a direct modification of beta L533C and the enhanced modification of gamma Cys547.

MTSET substitutes a large cationic group (trimethylaminoethyl) onto the sulfhydryl group of cysteine residues, compared with the smaller aminoethyl modification with MTSEA. We examined the accessibility of selected substituted cysteine residues within the pore region of the beta -subunit to MTSET. The amiloride-sensitive Na+ currents carried by wild type ENaC were not affected by MTSET, as previously observed (9, 17). Surprisingly, only modest reductions in amiloride-sensitive INa were observed with 3 of 11 mutants studied (Figs. 5 and 8). Residues with substituted cysteines that responded to MTSET preceded the 3-residue tract (Gly529-Ser531) that has a critical role in excluding K+. Furthermore, the mutant alpha beta G525Cgamma did not respond to MTSET (n = 6). This result was unexpected, since Snyder et al. (9) observed that the equivalent mutation in human ENaC (alpha beta G527Cgamma ) was inhibited by MTSET (67% reduction in current). These differences may reflect the use of channels derived from different species (mouse versus human). In contrast, the introduction of a cysteine residue at the analogous site within the alpha -subunit of mouse and human ENaC (alpha S583Cbeta gamma ) or the gamma -subunit of human ENaC (alpha beta gamma G536C) responded to external MTSET with a large (>= 70%) reduction in amiloride-sensitive INa (9, 17).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 8.   Representative whole cell recordings (A) and I/V relationships (B) were obtained in the absence or presence of MTSET in oocytes expressing wild type (WT) ENaC, alpha beta Q523Cgamma , and alpha beta W527Cgamma . Whole cell currents were determined in a Na+ bath solution at test potentials between -140 and +60 mV (20-mV increments) prior to and following the addition of 0.5 mM MTSET to the bath. Dashed lines indicate zero current. Amiloride-sensitive currents are illustrated in B. Data in B are presented as mean ± S.E. from 4-7 oocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent studies suggest that a limited region preceding the M2 domain of each subunit forms part of the ENaC pore. Selected mutations within these regions alter ion selectivity, single channel conductance, amiloride sensitivity, and channel gating (8-14, 18, 19). Changes in IK/INa, indicating that channels exhibited a measurable inward K+ current, were only observed with mutations within a 3-residue tract (Gly587-Ser588-Ser589) of the alpha -subunit. Selected mutations of the first (Gly587) or third (Ser589) residues resulted in channels that were K+-permeable. Snyder et al. (9) observed that cysteine substitutions of the third residue within a homologous 3-residue tract (Ser541-Cys542-Ser543) within the pore region of the human gamma -subunit led to changes in cation selectivity; only mutations in gamma Ser543 resulted in K+-permeable channels. A 3-residue tract within the beta -subunit (Gly529-Gly530-Ser531) is present in a position homologous to the GSS and SCS tracts within the alpha - and gamma -subunits, respectively, that restricts K+ permeation through the channel (see Fig. 1). Only substitution of cysteine (not alanine) in the first residue (beta Gly529) of this tract resulted in expression of K+-permeable channels, in agreement with previous observations (11). Our results, together with previous studies that have examined the effects of systematic amino acid substitutions within the pore regions of the alpha - and gamma -subunit, suggest that the selectivity filter of the channel resides primarily within a conserved 3-residue tract.

The introduction of cysteine residues at specific sites within the pore region of the alpha - and beta -subunit, other than within the conserved 3-residue tract, led to changes in ILi/INa (8) (Fig. 3A), suggesting that residues adjacent to the conserved 3-residue tract that restricts K+ permeation influence cation selectivity. It remains unclear at this time whether ENaC has an extended selectivity filter structure or whether amino acids adjacent to the conserved 3-residue tract modify ILi/INa by indirectly altering the structure of the selectivity filter. Although Snyder et al. did not observe changes in Li+ to Na+ permeability ratios following cysteine substitutions within the pore region of the human gamma -subunit, other than within the conserved 3-residue tract (9), we cannot exclude the possibility that cysteine substitutions within this region of gamma mENaC alter ILi/INa.

Previous studies examining the effects of amino acid substitutions or deletions on the efficacy of channel inhibition by amiloride indicated that selected mutations of residues at a homologous site within the beta - and gamma -subunits of ENaC (beta Gly525 and gamma Gly542 in mENaC) led to dramatic increases (100 to >1000-fold) in the Ki for amiloride (2, 12). In contrast, cysteine substitutions throughout the pore regions of the alpha -subunit resulted in modest increases in amiloride Ki at 9 residues, including the residue (alpha Ser583) that is a site homologous to beta Gly525 and gamma Gly542 (8, 12). Mutations at position alpha Ser588 within the 3-residue tract of the alpha -subunit that restricts K+ permeation exhibited amiloride Ki values that were 20-50-fold higher that wild type (11, 20). We also observed large (~9-73-fold) increases in the Ki for amiloride following cysteine substitutions of selected residues (alpha beta S531Cgamma and alpha beta G529Cgamma , respectively) within the 3-residue tract of the beta -subunit that restricts K+ permeation through the channel. Kellenberger et al. observed that mutations at position beta Gly529 resulted in increases in the Ki for amiloride of 40-130-fold (11). Whereas Schild et al. (12) suggested that amiloride binds to alpha Ser583, beta Gly525, and gamma Gly542 (in mENaC), it is quite possible that amiloride also interacts with residues within the narrow selectivity filter.

We examined the accessibility of substituted cysteine residues within the pore region of the beta -subunit to methanethiosulfate analogs. Channels with cysteine substitutions at sites (beta F524C, beta G525C, beta L533C) on either side of the 3-residue tract that restricts K+ permeation through the channel were blocked by MTSEA (Fig. 5), indicating that these residues were accessible to MTSEA. Our results suggest that beta G525C was more readily accessible to modification by MTSEA than beta L533C (see Fig. 7 and Table II). MTSEA is a membrane-permeant reagent (21), and it is possible that MTSEA gained access to beta L533C via the internal aspect of the channel pore. Furthermore, the inhibition of alpha beta L533Cgamma by MTSEA was due, in part, to modification of gamma Cys547.

The response of these mutant channels to the larger MTSET was modest at best. Furthermore, MTSET did not alter currents in oocytes expressing alpha beta G525Cgamma . This was a surprising result, since several groups have proposed that amiloride interacts directly with this residue and with residues at the homologous sites within the alpha - and gamma -subunits (i.e. alpha Ser583 and gamma Gly542) and that beta Gly525 must be external to beta Gly529 (9-11). These investigators proposed a "funnel-like" structure for the ENaC pore region that gradually narrows as it enters the membrane-spanning region to form the selectivity filter that restricts K+ and then transitions to an alpha -helical second membrane-spanning domain (see Refs. 9-11). This proposed structure differs from the structure of the pore region of the KcsA K+ channel, whose outer pore is formed by an alpha -helix that enters the membrane followed by an extended region containing the selectivity filter that is directed toward the extracellular space (22). Our previous study examining of the pore (pre-M2) region of alpha ENaC predicted that this region is formed by an alpha -helical domain that transitions into an extended selectivity filter in a manner analogous to the K+ channel pore (17). The residues that form the putative amiloride-binding site (alpha Ser583, beta Gly525, and gamma Gly542) are at the site where the pore alpha -helix transitions to an extended selectivity filter (17). Our earlier work indicated that the second membrane-spanning domains of ENaC have a secondary selectivity site, and we suggested that M2 may have an "inverted teepee" structure similar to that of KcsA (18). The apparent lack of accessibility of alpha beta G525Cgamma to MTSET is consistent with a pore region structure similar to that of the KcsA K+ channel. However, both Zn2+ (12) and MTSEA (Fig. 5A) were able to block alpha beta G525Cgamma , indicating the introduced sulfhydryl group was accessible to smaller reagents. In contrast, our observation that alpha beta G525Cgamma is more accessible than alpha beta L533Cgamma to MTSEA was consistent with a "funnel-like" structure of the ENaC pore region. If the ENaC pore has a "funnel-like" structure, the apparent lack of accessibility of beta G525C to MTSET suggests that this residue resides deeper within the pore region than the analogous residues in the alpha - and gamma -subunits. Alternative structural approaches are needed to resolve the pore region structure.

The specific sites within the 3-residue tract where mutations allowed for K+ permeation are not identical among the three ENaC subunits. Selected mutations of the first residue (mouse beta Gly529) in this tract within the beta -subunit (Fig. 3) (11), the third residue in this tract (equivalent to mouse gamma Ser548) within the gamma -subunit (9), and primarily at the third residue (alpha Ser589) (8, 10, 11) but also the first residue (alpha Gly587) (8) within the alpha -subunit resulted in K+-permeable channels. Furthermore, whereas mutations in beta Gly525 and gamma Gly542 led to large changes in the Ki for amiloride (2, 12) (Fig. 2), mutations in alpha Ser583 led to only a modest increase in the Ki for amiloride (8). These results suggest that the ENaC pore region has an asymmetric organization.

We propose a model of this region where the beta ENaC pore region is shifted down toward the cytoplasmic face of the plasma membrane, relative to these regions within the alpha - and gamma -subunits (see Fig. 9). Several lines of evidence support an asymmetric structure of ENaC pore region. Although beta G525C was not modified by MTSET (see Fig. 5), channels with cysteine substitutions at the corresponding sites in the alpha - and gamma -subunits were blocked by MTSET (8, 9). Furthermore, only mutations in the first residue within the beta -subunit 3-residue selectivity filter resulted in K+-permeable channels, whereas mutations in the third residue of the selectivity filter within the alpha - and gamma -subunit allowed for K+ permeation. Sequence comparisons revealed that the ENaC pore region residues are highly conserved, but are not identical (Fig. 1). At several sites, glycine residues are present in both the beta - and gamma -subunits, including beta Gly522/gamma Gly539 and beta Gly525/gamma Gly542, whereas a serine residue is at the corresponding site in the alpha -subunit. Pore (pre M2) regions of alpha -, beta -, and gamma -subunits have 2, 5, and 3 glycine residues, respectively. Given the unique structural role of glycine residues in proteins, it is possible that the number of glycine residues in the pore regions of different subunits contributes to their secondary structure and degree of flexibility. Differences have been reported in the single channel properties of alpha beta , alpha gamma , and alpha beta gamma ENaCs (23) and may reflect different pore structures that are contributed by different subunits. The accessibility pattern of introduced cysteine residues within the pore regions of ENaC subunits also supports an asymmetric arrangement of the pore region residues. For example, MTSET significantly increased whole cell currents in oocytes expressing channels with a cysteine substitution at alpha Ser576 or at the corresponding site in the beta -subunit, by presumably locking the channel in open state (17, 19). In contrast, MTSET did not alter whole cell currents in channels with a cysteine substitution at the corresponding site in the gamma -subunit (9). Comparisons of the effects of MTSET on mutant ENaCs with cysteine substitutions in the pore regions of the alpha - and gamma -subunits revealed significant differences in the responses at multiple sites (9, 17). Asymmetries in the pore regions of voltage-gated Na+ and Ca2+ channels have also been observed (24-28), in contrast to the reported symmetric organization of the pore region of K+ channels (22).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 9.   Model of the pore regions of alpha -, beta -, and gamma mENaC illustrating an asymmetric organization. The N-terminal portions of the alpha , beta , and gamma mENaC pore regions are shown as cylinders representing possible helical structures, and the C-terminal portions are shown as thick curves, indicating nonhelical structures. Side chains of selected residues are shown as broken or solid wavy lines. The three dashed horizontal lines indicate relative levels corresponding to sites involved in channel gating (Gating), amiloride-binding (Amiloride), and selectivity filter (Filter) from previous and current studies. The accessibility of introduced cysteines near these sites to external MTS reagents is shown to the right of each level line. yes indicates that an MTS reagent significantly altered channel activity (current); no indicates that an MTS reagent failed to produce significant change in channel activity (current). nd, not determined.

In summary, our results confirm the crucial role of the previously identified 3-residue (beta Gly529-beta Gly530-beta Ser531) tract of beta ENaC in cation selectivity and suggest that residues surrounding this tract also contribute to Li+/Na+ selectivity. Based on the observation that beta G529C and beta S531C dramatically decreased amiloride sensitivity, we speculate that the ENaC selectivity filter, in addition to the previously described amiloride binding site (alpha Ser583-beta Gly525-gamma Gly542), may interact with amiloride. Differences in the accessibility of introduced cysteine residues within the pore regions of the alpha -, beta -, and gamma -subunits to external sulfhydryl reagents were observed (see above and Refs. 9 and 17). We propose that ENaC pore region residues are arranged asymmetrically, similar to voltage-gated Na+ and Ca2+ channels but distinct from K+ channels.

    ACKNOWLEDGEMENT

We thank Kathleen McNulty for generating the gamma R583X construct.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK54354.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.

§ Recipient of a postdoctoral fellowship award from the Cystic Fibrosis Foundation.

** To whom all correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. E-mail: kleyman@pitt.edu.

Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M300149200

    ABBREVIATIONS

The abbreviations used are: ENaC, epithelial sodium channel; mENaC, mouse ENaC; M2, second transmembrane domain; cRNA, complementary RNA; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl) methanethiosulfonate hydrobromide; MTSET, [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide; KcsA, K+ channel from Streptomyces lividans.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Canessa, C. M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J. D., and Rossier, B. C. (1994) Nature 367, 463-467[CrossRef][Medline] [Order article via Infotrieve]
2. Kosari, F., Sheng, S., Li, J., Mak, D. O., Foskett, J. K., and Kleyman, T. R. (1998) J. Biol. Chem. 273, 13469-13474[Abstract/Free Full Text]
3. Firsov, D., Gautschi, I., Merillat, A. M., Rossier, B. C., and Schild, L. (1998) EMBO J. 17, 344-352[Abstract/Free Full Text]
4. Snyder, P. M., Cheng, C., Prince, L. S., Rogers, J. C., and Welsh, M. J. (1998) J. Biol. Chem. 273, 681-684[Abstract/Free Full Text]
5. Canessa, C. M., Merillat, A. M., and Rossier, B. C. (1994) Am. J. Physiol. 267, C1682-C1690[Medline] [Order article via Infotrieve]
6. Renard, S., Lingueglia, E., Voilley, N., Lazdunski, M., and Barbry, P. (1994) J. Biol. Chem. 269, 12981-12986[Abstract/Free Full Text]
7. Snyder, P. M., McDonald, F. J., Stokes, J. B., and Welsh, M. J. (1994) J. Biol. Chem. 269, 24379-24383[Abstract/Free Full Text]
8. Sheng, S., Li, J., McNulty, K. A., Avery, D., and Kleyman, T. R. (2000) J. Biol. Chem. 275, 8572-8581[Abstract/Free Full Text]
9. Snyder, P. M., Olson, D. R., and Bucher, D. B. (1999) J. Biol. Chem. 274, 28484-28490[Abstract/Free Full Text]
10. Kellenberger, S., Gautschi, I., and Schild, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4170-4175[Abstract/Free Full Text]
11. Kellenberger, S., Hoffmann-Pochon, N., Gautschi, I., Schneeberger, E., and Schild, L. (1999) J. Gen. Physiol. 114, 13-30[Abstract/Free Full Text]
12. Schild, L., Schneeberger, E., Gautschi, I., and Firsov, D. (1997) J. Gen. Physiol. 109, 15-26[Abstract/Free Full Text]
13. Kellenberger, S., and Schild, L. (2002) Physiol. Rev. 82, 735-767[Abstract/Free Full Text]
14. Kellenberger, S., Auberson, M., Gautschi, I., Schneeberger, E., and Schild, L. (2001) J. Gen. Physiol. 118, 679-692[Abstract/Free Full Text]
15. Ahn, Y. J., Brooker, D. R., Kosari, F., Harte, B. J., Li, J., Mackler, S. A., and Kleyman, T. R. (1999) Am. J. Physiol. 277, F121-F129[Medline] [Order article via Infotrieve]
16. Schild, L., Canessa, C. M., Shimkets, R. A., Gautschi, I., Lifton, R. P., and Rossier, B. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5699-5703[Abstract]
17. Sheng, S., Li, J., McNulty, K. A., Kieber-Emmons, T., and Kleyman, T. R. (2001) J. Biol. Chem. 276, 1326-1334[Abstract/Free Full Text]
18. Sheng, S., McNulty, K. A., Harvey, J. M., and Kleyman, T. R. (2001) J. Biol. Chem. 276, 44091-44098[Abstract/Free Full Text]
19. Snyder, P. M., Bucher, D. B., and Olson, D. R. (2000) J. Gen. Physiol. 116, 781-790[Abstract/Free Full Text]
20. Waldmann, R., Champigny, G., and Lazdunski, M. (1995) J. Biol. Chem. 270, 11735-11737[Abstract/Free Full Text]
21. Holmgren, M., Liu, Y., Xu, Y., and Yellen, G. (1996) Neuropharmacology 35, 797-804[CrossRef][Medline] [Order article via Infotrieve]
22. Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77[Abstract/Free Full Text]
23. Fyfe, G. K., and Canessa, C. M. (1998) J. Gen. Physiol. 112, 423-432[Abstract/Free Full Text]
24. Perez-Garcia, M. T., Chiamvimonvat, N., Marban, E., and Tomaselli, G. F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 300-304[Abstract/Free Full Text]
25. Benitah, J. P., Tomaselli, G. F., and Marban, E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7392-7396[Abstract/Free Full Text]
26. Wu, X. S., Edwards, H. D., and Sather, W. A. (2000) J. Biol. Chem. 275, 31778-31785[Abstract/Free Full Text]
27. Chiamvimonvat, N., Perez-Garcia, M. T., Ranjan, R., Marban, E., and Tomaselli, G. F. (1996) Neuron 16, 1037-1047[CrossRef][Medline] [Order article via Infotrieve]
28. Koch, S. E., Bodi, I., Schwartz, A., and Varadi, G. (2000) J. Biol. Chem. 275, 34493-34500[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.