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J Biol Chem, Vol. 275, Issue 12, 8572-8581, March 24, 2000

Characterization of the Selectivity Filter of the Epithelial Sodium Channel^*

Shaohu Sheng, Jinqing Li, Kathleen A. McNulty, Daniel Avery, and Thomas R. Kleyman^§

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed , , and . Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the -subunit to cysteine and co-expressing these mutants with wild type - and -subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (-Val⁵⁶⁹--Gly⁵⁷⁹) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (-Ser⁵⁸⁰--Ser⁵⁹²) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing G587C or S589C were permeable to K⁺, and mutation of a GSS tract (positions 587-589) to GYG resulted in a moderately K⁺-selective channel. Our results suggest that the C-terminal portion of the pore region within the -subunit contributes to the selectivity filter of ENaC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epithelial sodium channels (ENaCs)¹ mediate sodium transport across apical plasma membranes of epithelial cells that line the distal nephron, the airway and alveoli, and the distal colon. ENaCs are composed of three homologous subunits, termed -, -, and ENaC (1, 2) and have a subunit stoichiometry of 2:1:1 (3, 4) (Fig. 1B), although some reports have suggested an alternative subunit stoichiometry (5, 6). The three subunits have a similar topology, with cytoplasmic amino and carboxyl termini and two transmembrane domains (termed M1 and M2) that are separated by a large ectodomain (Fig. 1A) (7-9). Hydropathy analyses revealed two rather hydrophobic regions (termed H1 and H2) immediately following M1 and preceding M2 (2). The region preceding M2 may enter the membrane, similar to the pore-forming region (P region) of the voltage-gated Na⁺, K⁺, and Ca²⁺ channels (9).

Mutagenesis of selected residues within the H2 region and the segment between H2 and M2 of the -, -, or -subunits of ENaC resulted in changes in cation selectivity, single channel conductance, or sensitivity to amiloride, a putative pore blocker of ENaC (4, 10-13). These findings argued that all three subunits are involved in pore formation and that the region preceding the M2 domain forms a portion of the pore. Snyder et al. (14) recently reported a study describing the results of scanning mutagenesis of the pore region of the -subunit of human ENaC. Both Snyder et al. and Kellenberger et al. (11, 12, 14) have suggested that the pore structure of ENaC is distinct from that of KcsA, a K⁺ channel whose structure was resolved by x-ray crystallography (15).

In this report, we refer to the hydrophobic region preceding the M2 domain as the pore (or P) region of ENaC. We have systematically mutated all 24 residues (Val⁵⁶⁹-Ser⁵⁹²) within the pore region to cysteine in order to identify residues within the mouse -subunit that participate in forming the ENaC pore. The mutated -subunits of mENaC were co-expressed with wild type - and -subunits of mENaCs in Xenopus oocytes in order to identify mutations that affect functional characteristics of the channel pore. Mutations that led to changes in cation selectivity and amiloride sensitivity were largely restricted to the 13 residues within the carboxyl-terminal portion of the pore region of mENaC. Our studies indicate that the C-terminal domain within the putative pore region of the -subunit contributes to the selectivity filter of ENaC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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

Site-directed Mutagenesis-- Three subunits for mouse ENaC (-, -, and mENaC) cDNAs were cloned into pBluescript SK() (Stratagene, La Jolla, CA) from a mouse kidney cDNA library (16). All amino acids in the pore region of mENaC (-Val⁵⁶⁹--Ser⁵⁹²) were replaced individually by cysteine residues through site-directed mutagenesis with the sequential polymerase chain reaction method using Pfu DNA polymerase (Stratagene) (4, 17). The polymerase chain reaction-amplified fragments containing the desired mutations were digested with BsmI and BspEI (New England Biolabs Inc., Beverly, MA) and ligated to wild type cDNA vector that had been digested with the same two restriction enzymes. The subcloned fragment was sequenced in entirety by automated DNA sequencing at the University of Pennsylvania Sequencing Facility to confirm the desired mutation.

Expression in Xenopus Oocytes-- cRNAs for wild type and mutant mENaC, wild type mENaC, and mENaC were synthesized from linearized DNAs with T₃ RNA polymerase (Ambion Inc., Austin, TX) and stored at 80 °C. Stage V-VI Xenopus oocyte pretreated with 2 mg/ml collagenase (type IV) was injected with 2-4 ng/subunit of cRNAs in 50 nl of H₂O (16). After injection, oocytes were incubated at 18 °C in modified Barth's saline (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 gentamycin sulfate, pH 7.2). Two-electrode voltage clamp and patch clamp recordings were performed 20-72 h after injection at room temperature (22-25 °C).

Two-electrode Voltage Clamp-- Two-electrode voltage clamp was performed using a DigiData 1200 interface (Axon Instruments, Foster City, CA) and a TEV 200 Voltage Clamp amplifier (Dagan Corp., Minneapolis, MN). Data acquisition and analyses were performed using pClamp 6.03 software (Axon Instruments) on a 120-MHz Pentium PC (Gateway 2000 Inc., N. Sioux City, SD). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL) with a Micropipette Puller (Sutter Instrument Co., Novato, CA) and had resistance of 0.5-5 megaohms when filled with 3 M KCl and inserted into the bath solution. Oocytes were maintained in a recording chamber with 1 ml of bath solution and continuously perfused with bath solution at a flow rate of 4-5 ml/min. The bath solution used for the measurement of amiloride sensitivity contained 100 mM sodium gluconate, 2 mM KCl, 1.8 mM CaCl₂, 5 mM BaCl₂, 10 mM HEPES, pH 7.2. For most experiments, a series of voltage steps (450 or 900 ms) from 140 to +40 mV in 20-mV increments were performed, and whole cell currents were measured at 400 or 800 ms, respectively, after initiation of the voltage step. Amiloride-sensitive currents were defined as the current difference in the absence and presence of amiloride (100 µM or 1 mM) in the bath solution (16). Amiloride was prepared in bath solution and delivered to the oocyte by perfusion. To determine the inhibition constant (K_i) of amiloride, sodium gluconate bath solutions containing 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, and 10³ M amiloride were sequentially perfused into the recording chamber, and inward currents were recorded before and after each application of amiloride. Amiloride K_i was determined by nonlinear regression analysis (Origin 5, MicroCal Inc., Northampton, MA) using the following equation,

(Eq. 1)

where I and I_o are Na⁺ currents measured in the presence and absence of amiloride, respectively, [A] is the amiloride concentration, and n' is the pseudo-Hill coefficient.

For selectivity experiments, separate bath solutions containing Na⁺, Li⁺, or K⁺ as the predominant cation were used. These solutions all contained the gluconate salt of Na⁺, Li⁺, or K⁺ at a concentration of 100 mM, 1.8 mM CaCl₂, 5 mM BaCl₂, and 10 mM HEPES. The pH was adjusted to 7.2 with NaOH, LiOH, or KOH, respectively. The K⁺, Na⁺, and Li⁺ solutions were sequentially used to bathe the oocytes under continuous perfusion. Oocytes were subsequently bathed in these solutions in the presence of 100 µM amiloride. Macroscopic currents were recorded with each bath solution when a stable membrane potential was observed (typically 2-3 min after initial application of the bath solution). Amiloride-sensitive currents recorded at 100 mV were used to calculate the ratios of K⁺ current (I_K) and Li⁺ current (I_Li) relative to Na⁺ current (I_Na).

Single Channel Recording-- Bath and pipette solutions were identical and contained 110 mM LiCl or NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4, adjusted with LiOH or NaOH, respectively. Oocytes were placed in a hypertonic solution (bath solution supplemented with 200 mM sucrose) for 3-5 min, and vitelline membranes were then removed manually. Oocytes were then transferred to the bath solution and maintained at room temperature (22-25 °C) for at least 30 min before recording. Patch pipettes were pulled in multiple steps and fire-polished with a Micro Forge (Narishige, Japan). Pipettes with tip resistance of 10-30 mega ohms were chosen for patch clamping. Currents were recorded in the cell-attached configuration using PC-ONE Patch Clamp Amplifier (Dagan) and DigiData 1200 interface (Axon) connected to a Pentium II 330-MHz PC (Gateway 2000 Inc.). Single channel recording data were acquired using pClamp 7.0 (Axon) for Microsoft Windows 95 (Microsoft Inc.) at 5 kHz, filtered at 300 Hz by a three-pole low pass Bessel Filter built in the amplifier, and stored on the hard drive disc. To determine single channel conductance, averaged currents recorded at intracellular voltages of 40, 60, 80, and 100 mV were plotted against the applied voltage, and slope conductance was determined by linear curve fitting using MS Excel 97 (Microsoft).

Statistical Analyses-- Data are expressed as mean ± S.E. Student's t test was performed for significance analysis between wild type and mutant channel with MS Excel 97.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Channels with Cysteine Substitutions within the Pore Region of mENaC in Oocytes-- Recent studies suggest that residues immediately preceding the predicted -helical second membrane spanning domain (M2) of the -, - and -subunit of ENaC form part of the channel pore (4, 10-13). The amino acid residues within this region are illustrated in Fig. 1C and are aligned with residues of other members of the ENaC/degenerin family. This region is highly conserved, as expected for a region that might have a critical role in forming the pore of these channels. Each ENaC subunit has two well defined, long hydrophobic domains (2). We used several algorithms to predict the secondary structure of the long second hydrophobic domain containing the putative pore and M2 regions of mENaC. The amino-terminal portion, corresponding to pore region, was predicted to contain both extended and random coiled structures. The carboxyl-terminal portion of this second hydrophobic domain, comprising the -helical second membrane spanning domain (M2), was predicted to begin between residues -Val⁵⁹⁰ and -Ser⁵⁹² (Fig. 1D). We define the pore (or P) region of mENaC to include residues from Val⁵⁶⁹ to Ser⁵⁹².

View larger version (73K): [in this window] [in a new window]   Fig. 1.   Topology, subunit stoichiometry, and pore sequence alignments of ENaC. A, proposed topology for one ENaC subunit. The pore region is shaded. B, subunit stoichiometry of ENaC. The channel is a heterotetramer, formed by two  subunits, one  subunit, and one  subunit, with the two  subunits facing each other. C, amino acid sequence alignments of the pore regions of -, -, and mENaC (GenBankTM accession nos. AF112185, AF112186, and AF112187), human ENaC (-subunit of human ENaC; GenBankTM accession no. U38254), FaNaCh (FMRFamide-activated amiloride-sensitive sodium channel; GenBankTM accession no. X92113), DEG-1 (degenerin from Caenorhabditis elegans; GenBankTM accession no. L34414), MEC-4 (mechanosensitive protein from C. elegans, GenBankTM accession no. U53669), hBNaC1 (human brain sodium channel; GenBankTM accession no. U57352), and ASIC1 (acid-sensing ion channel 1, or proton-gated cation channel 1; GenBankTM accession no. U94403). The multiple sequence alignment was performed with MacVector version 6.5 (MacVector) on a PowerPC (Apple). Identical amino acids are shaded, and similar residues are boxed. The amino acids Val569-Ser592 from mENaC that were mutated in the present study are shown on the top line. D, secondary structural predictions of the regions including and adjacent to the second membrane-spanning domain (M2) of mENaC (residues Pro567-Pro622) were performed by Network Protein Sequence Analysis (available on the World Wide Web). h (boldface type), -helix; e, extended strand; t, -turn; c, random coil; ?, ambiguous site. Prediction methods are DSC (45), GOR1 (46), GOR3 (47), MLRC (48), PHD (49), and Predator (50). The consensus prediction (Sec. Cons.) is shown in the bottom line. The boxed area indicates a region where all methods predict an -helical domain.

All 24 residues (-Val⁵⁶⁹ to -Ser⁵⁹²) within the pore region of mENaC were systematically mutated to cysteine. The functional characteristics of these mutations within mENaC were determined by co-expression with wild type - and mENaC in Xenopus oocytes. Whole cell amiloride-sensitive Na⁺ currents were readily detected following expression of 22 of the 24 mutant channels, in the range of 4 to 40 µA at a test potential of 100 mV. Levels of current expression observed with two mutant channels, G587C and S589C, were less than 200 nanoamperes (nA), differed markedly from that observed with wild type and other -subunit mutants, and did not increase when Ba²⁺ was removed from the bath solution (data not shown).

Mutations within the Carboxyl-terminal Domain of the Pore Region of mENaC Alter Amiloride Sensitivity-- The inhibition constants for amiloride were determined for wild type mENaC and for the mutant mENaCs (Fig. 2A). Modest increases in the K_i for amiloride, in the range of 1.7-3.5-fold, were observed with 8 of 13 mutations within the carboxyl-terminal portion of the pore domain (residues -Ser⁵⁸⁰--Ser⁵⁹²). Given the low magnitude of whole cell currents observed in oocytes expressing the G587C or S589C, K_i values for amiloride were not determined for these mutants. However, the relative inhibition observed with 100 nM and 100 µM amiloride suggested that their amiloride sensitivity was similar to that of wild type mENaC (data not shown). Only one of the cysteine mutations (L575C) within the amino-terminal portion of the pore region (residues -Val⁵⁶⁹--Gly⁵⁷⁹) exhibited a K_i for amiloride (53 ± 5 nM, n = 4) that differed from wild type. Amiloride dose-response curves for wild type mENaC and selected -subunit mutations within the pore region are illustrated in Fig. 2B.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of mutations on amiloride inhibitory constants (Ki values). A, amiloride Ki values for wild type and mutant mENaCs. Macroscopic Na+ currents were measured at the test potential of 100 mV in the absence or presence of increasing concentrations of amiloride in a sodium gluconate bath solution. The amiloride Ki was determined by nonlinear least squares fitting of dose-response data. B, dose-response curves of amiloride blockade for wild type (), W582C (), L584C (), F586C (), and S588C () mENaC. Values and error bars represent the mean ± S.E. for 3-8 oocytes. Lines were created by curve fitting of the data. The p values for comparing mutant and wild type channels are as follows. *, p < 0.05; **, p < 0.01. ND, amiloride Ki was not determined for G587C and S589C.

Mutations within the C-terminal Domain of the Pore Region of mENaC Alter Cation Selectivity-- The cation selectivities for both wild type mENaC and mutant mENaCs were determined by measuring amiloride-sensitive inward currents in oocytes by two-electrode voltage clamp in K⁺, Na⁺, and Li⁺ bath solutions. Wild type mENaC exhibited an amiloride-sensitive Li⁺/Na⁺ current ratio of 1.68 ± 0.06 (n = 16), and an amiloride-sensitive K⁺/Na⁺ current ratio of 0.01 ± 0.01 (n = 16) (Table II and Figs. 3 and 4). No amiloride-sensitive currents were observed in water-injected oocytes (data not shown). The selectivity sequence of Li⁺ > Na⁺ K⁺ observed for mENaC is consistent with that observed with epithelial Na⁺ channels from other species (2, 18-23).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Cation selectivity of wild type mENaC. A, representative whole cell recordings of wild type mENaC in the presence of K+, Na+, and Li+ solutions as described under "Experimental Procedures." The oocyte was injected with 4 ng of -, -, and mENaC cRNAs and was voltage-clamped for 900 ms from 140 to +40 mV in 20-mV increments. Currents represent amiloride-sensitive currents obtained by subtraction of currents in the presence of 100 µM amiloride from the currents in the absence of amiloride. B, current-voltage relationship of wild type mENaC from the same oocyte as in A. Amiloride-sensitive currents in the presence of K+ (), Na+ (), and Li+ () were plotted against test potentials in the range of 140 to +40 mV.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Current ratios of wild type (WT) and mutant mENaCs. A, oocytes injected with wild or mutant cRNAs, and ILi/INa was calculated from amiloride-sensitive currents measured at 100 mV in the presence of a Li+ or Na+ bath solution. B, IK/INa was determined from amiloride-sensitive currents measured at 100 mV in the presence of a K+ or Na+ bath solution. Data are presented as mean ± S.E. from 16 oocytes for wild type and 4-9 oocytes for mutants. The p values from Student's t tests between wild type and mutant channels are as follows. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The ratios of I_Li/I_Na and I_K/I_Na for both wild type and mutant channels measured at a testing potential of 100 mV are illustrated in Fig. 4. Substitution of cysteine residues within the amino-terminal portion of the pore region of mENaC (V569C to G579C) did not produce alterations of Li⁺/Na⁺ and K⁺/Na⁺ current ratios, with the exception of S573C. This mutant showed a modest reduction in the Li⁺/Na⁺ current ratio, when compared with wild type mENaC. These results suggest that these residues are not essential for maintaining the characteristic cation selectivity of ENaC. In contrast, five mutations within the carboxyl-terminal portion of the pore domain (S580C, W582C, L584C, F586C, and S592C) significantly altered the Li⁺/Na⁺ current ratio (Figs. 4 and 5). Two mutants in this region (G587C and S589C) were K⁺-permeable (Fig. 4). These results suggest that multiple residues within the carboxyl-terminal portion of the pore region of ENaC are involved in the ionic permeation mechanism of ENaC.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Point mutations altered Li+/Na+ selectivity. Current-voltage relationships of wild type (WT) mENaC, S580C, W582C, L584C, F586C, and S592C were obtained by plotting amiloride-sensitive currents measured in the presence of K+ (), Na+ (), or Li+ () bath solutions against test potentials in the range of 140 to +40 mV, in 20-mV increments, without curve fitting. Data are presented as mean ± S.E. from 10 oocytes for wild type and 4-9 oocytes for mutants.

Cell-attached patch clamp was performed using Li⁺ as the permeating ion in order to determine whether the changes in Li⁺/Na⁺ selectivity reflected changes in Li⁺ permeation. Of the 12 mutants examined, the single channel Li⁺ conductance was remarkably constant, varying between 7.1 and 9.0 picosiemens (Table I and Fig. 6), with the exception of L591C having Li⁺ conductance of 6.4 ± 0.31 (p < 0.05 versus wild type). Among the mutations associated with changes in Li⁺/Na⁺ selectivity, W582C was the only mutant that exhibited an inward whole cell Na⁺ current that was significantly greater than the inward Li⁺ current (Figs. 4 and 5). The single channel slope conductance of W582C, measured with Na⁺ as the permeating ion, was 7.9 picosiemens, approximately twice that of wild type ENaC (Table I and Fig. 6). The single channel Li⁺/Na⁺ conductance ratio of 0.96 differed from the Li⁺/Na⁺ whole cell current ratio of 0.59 measured from W582C mENaC. Other investigators have used both single channel analyses and whole cell current measurements to examine cation selectivity of selected ENaC mutants and have also noted that these two methods may yield dissimilar results (12).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Single channel recordings of wild type (WT) and W582C mENaC. A and B, single channel current traces recorded in 110 mM Na+ or Li+ solutions (bath and pipette) from oocytes injected with mENaC (A) or W582C mENaC (B) cRNAs. The closed state is indicated as C. Voltages are negative values of applied pipette potentials during recording. Downward deflections indicate inward current. C, current-voltage relationship of Na+ and Li+ currents in the range of 100 to 40 mV through wild type (WT) mENaC () or W582C-mENaC (). Data are presented as mean ± S.E. for 4-7 recordings. Lines were obtained by fitting the data with linear regression.

Channels with cysteine substitutions at either -Gly⁵⁸⁷ or -Ser⁵⁸⁹ expressed measurable whole cell amiloride-sensitive K⁺ currents (Figs. 4 and 7). The K⁺/Na⁺ current ratios for G587C and S589C were 0.23 ± 0.10 (n = 5) and 1.76 ± 0.22 (n = 6), respectively (Table II). Neither mutant showed a significant change in Li⁺/Na⁺ selectivity from wild type channels. To further explore the role of residues at positions -Gly⁵⁸⁷ and -Ser⁵⁸⁹ in determining cation selectivity, additional mutations were generated, including G587A, G587S, G587T, S589A, S589G, and S589T. As observed with a cysteine mutation at position -Gly⁵⁸⁷, an alanine substitution at this site also resulted in channels that were K⁺-permeable, as determined by measurement of amiloride-sensitive inward K⁺ currents. The K⁺/Na⁺ current ratio for G587A was 0.80 ± 0.16 (n = 7; Table II and Fig. 7). Introduction of an alanine or a glycine residue at position -Ser⁵⁸⁹ also resulted in a mutant channel that was K⁺-permeable. The K⁺/Na⁺ current ratio for S589A was 0.56 ± 0.21 (n = 4; Table II and Fig. 7); the K⁺/Na⁺ current ratio for S589G was 0.32 ± 0.09 (n = 6; Table II and Fig. 8). However, retention of the hydroxyl moiety on the side chain at position -Ser⁵⁸⁹ (S589T), or introduction of a hydroxyl moiety on the side chain at position -Gly⁵⁸⁷ (G587S) allowed channels to discriminate Na⁺ from K⁺. The K⁺/Na⁺ current ratios for G587S and S589T were 0.03 ± 0.03 (n = 4) and 0.04 ± 0.02 (n = 5), respectively, and did not differ significantly from wild type (Table II and Fig. 7). No amiloride-sensitive currents were detected in oocytes expressing G587T and bathed in either K⁺, Na⁺, and Li⁺ solutions. Although G587C and S589C exhibited Li⁺/Na⁺ current ratios similar to that of wild type, other mutations at positions -Gly⁵⁸⁷ and -Ser⁵⁸⁹ did alter the channel's Li⁺/Na⁺ current ratio (Table II). The mutant G587A exhibited a Li⁺/Na⁺ current ratio of 0.97 ± 0.17 (n = 7). The mutants S589A, S589T, and S589G expressed inward Na⁺ currents that were greater than inward Li⁺ currents, with Li⁺/Na⁺ current ratios of 0.88 ± 0.39 (n = 4), 0.86 ± 0.21 (n = 5), and 0.96 ± 0.07 (n = 6), respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Selected point mutations of G587 and S589 alter K+/Na+ selectivity. Current-voltage relationships of the mENaC mutants S589C, S589A, S589T, G587C, G587A, and G587S were obtained by measuring amiloride-sensitive currents in the presence of K+ (), Na+ , or Li+ () bath solution while varying the test potential in the range of 140 mV (or 100 mV for -Ser589 mutants) to +40 mV in 20-mV increments. Data are presented without curve fitting as mean ± S.E. for 4-7 oocytes.

View this table: [in this window] [in a new window]   Table II Selectivity changes from mutations at positions Gly587-Ser589 of mENaC Current ratios were calculated from amiloride-sensitive currents measured at 100 mV in oocytes expressing wild type (WT) or mutant mENaC together with - and mENaC subunits in the presence of 100 mM K+, Na+, or Li+ both solutions. Data are presented as mean ± S.E.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Mutation of GSS (residues -Gly587--Ser589) to GYG renders the channel K+-selective. A-C, representative whole cell currents recorded in oocytes expressing a mutant mENaC containing a GYG tract at residues 587-589 in the presence of 100 mM K+ (A), Na+ (B), or Li+ (C) bath solutions. Current traces in the presence of 100 µM amiloride are shown in panels on the right. D, current-voltage relationship of mutant mENaCs containing the GYG mutation within the -subunit was obtained by plotting amiloride-sensitive currents in the presence of K+ (), Na+ (), or Li+ () against test potentials varying from 140 mV to +40 mV. E, current-voltage relationship of S589G mENaC was obtained as in D. Data are presented as mean ± S.E. from six oocytes.

Our results suggest that a limited domain within the pore region of mENaC, -Gly⁵⁸⁷ through -Ser⁵⁸⁹, has a critical role in restricting K⁺ permeation through the channel, since selected mutations within this region alter K⁺/Na⁺ selectivity. This GSS tract within -subunit is conserved within the -subunit (GGS) and -subunit (SCS) of ENaC. Selected mutations within this limited domain of each of the three subunits alter K⁺/Na⁺ selectivity (11, 12, 14), suggesting that a (G/S)XS tract restricts K⁺ transport through the channel. Interestingly, a G(Y/F)G tract within voltage-gated, Ca²⁺-dependent, and inwardly rectifying K⁺ channels is a critical site for determining K⁺ selectivity (15, 24-27). Therefore, we examined whether substitution of the GSS tract (residues 587-589) within mENaC with GYG (S588Y, S589G) rendered the mutant channel K⁺-selective (Table II and Fig. 8). The K⁺/Na⁺ current ratio of this mutant was 3.92 ± 1.09 (n = 7). This mutant did not discriminate Li⁺ and Na⁺, since the Li⁺/Na⁺ current ratio of this mutant was 1.06 ± 0.30 (n = 7).

Barium chloride at a concentration of 5 mM was included in bath solutions in the present study to minimize the background amiloride-insensitive currents from endogenous K⁺ channels in oocytes. Removal of Ba²⁺ from the bath solution had no effect on the amiloride-sensitive Na⁺ currents measured in oocytes expressing wild type , W582C, G587C, S589C, or the GYG mutant (S588Y-S589G). Increased amiloride-insensitive currents were observed in some oocytes. Another set of experiments in which bath solutions containing chloride (rather than gluconate) as the anion and no Ba²⁺ produced similar results in cation selectivity for wild type , G587S, and S588Y-S589G (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The generation of cysteine mutations systematically throughout the pore region of the -subunit of mENaC (residues -Val⁵⁶⁹ to -Ser⁵⁹²) allowed us to examine the role of residues within this region in conferring cation selectivity. Two distinct domains within this pore region were defined based on analyses of these mutants. Within the N-terminal portion of this region, corresponding to residues -Val⁵⁶⁹ through -Gly⁵⁷⁹, mutations resulted in minimal changes in cation selectivity or amiloride sensitivity. Within this region, a reduced Li⁺/Na⁺ current ratio was observed only with the mutant S573C, and an altered (reduced) K_i for amiloride was observed with the mutant L575C. In contrast, changes in amiloride sensitivity or cation selectivity were observed with all of the mutants within the carboxyl-terminal portion of this domain (-Ser⁵⁸⁰ to -Ser⁵⁹²), with the exception of W585C.

Previous studies of amiloride-induced block of ENaC suggest that this diuretic is a pore blocker and interacts with the external side of the pore (10, 28, 29). In addition, amiloride appears to interact at other sites within the channel protein, including sites within the ectodomain (30-32). As amiloride interacts with the channel pore, it is likely that selected mutations within the outer pore region of ENaC will alter the channel's interaction with this drug. We observed that within the carboxyl-terminal portion of the pore region (residues -Ser⁵⁸⁰ through -Ser⁵⁹²), cysteine substitutions at 9 of 13 residues resulted in mutant channels with increased K_i values for amiloride (Fig. 2). However, these changes in amiloride's K_i were modest, particularly when compared with the nearly 1000-fold increase in K_i observed with a single mutation within the pore region of the - or -subunit of mouse (G525C and G542C) and rat ENaC (G525C and G537C). These glycine residues within rat - and ENaC are located within the pore region, and Schild et al. (10) have proposed that rat -Gly⁵²⁵ and -Gly⁵³⁷ are important sites for amiloride binding. Given their observations, it is somewhat surprising that we found only modest changes in amiloride's K_i in our analyses of mutations throughout the pore region of the -subunit. Murine -Gly⁵²⁵ and -Gly⁵⁴² are within a region that is highly conserved among the three ENaC subunits (Fig. 1C). The residue within mENaC corresponding to -Gly⁵²⁵ and -Gly⁵⁴² is -Ser⁵⁸³, and previous studies have demonstrated that substitution of a cysteine residue at this site results in only a modest increase in amiloride's K_i (4, 10). We confirmed this observation, although we have not examined whether other mutations at this residue (-Ser⁵⁸³) result in larger changes in amiloride's K_i. Our results raise the question of whether the previously identified amiloride binding sites (residues -Gly⁵²⁵ and -Gly⁵³⁷ within rat ENaC) are directly involved in the interaction of amiloride within the channel's pore. An alternative hypothesis is that these mutations alter the structure of the channel's pore and indirectly affect amiloride's interaction with the pore.

We examined the effects of mutations within the pore region of the mENaC on cation selectivity, as determined by amiloride-sensitive current ratios. Within the C-terminal portion of the pore region, cysteine mutations introduced at multiple residues between -Ser⁵⁸⁰ and -Phe⁵⁸⁶ and a cysteine mutation introduced at -Ser⁵⁹² were associated with significant changes in Li⁺/Na⁺ selectivity (Figs. 4 and 5). Although the G587C and S589C mutants did not alter Li⁺/Na⁺ selectivity, other mutations at these sites including G587A, S589A, S589G, and S589T significantly altered the Li⁺/Na⁺ current ratio (Table II and Fig. 7). Surprisingly, cysteine mutations within the pore region of the -subunit did not appreciably alter single channel Li⁺ conductance (Table I), with the exception of L591C. The changes in Li⁺/Na⁺ selectivity we observed with mutations within this region may reflect changes in single channel Na⁺ conductance, as we observed with W582C, although we cannot rule out the possibility that these mutations affect other parameters, such as open probability.

Channels with substitutions at either position -Gly⁵⁸⁷ or -Ser⁵⁸⁹ expressed low whole cell amiloride-sensitive Na⁺ currents in oocytes (less than 0.5 µA) with the exception of S589T and S589G. In contrast, Kellenberger et al. (11, 12) reported currents in the range of 15-36 µA when rat -Gly⁵⁸⁷ was mutated to Ala or Ser or when rat -Ser⁵⁸⁹ was mutated to Ala, Cys, or Asp and then co-expressed with wild type rat - and ENaC in Xenopus oocytes. However, these amiloride-sensitive whole cell currents were markedly reduced when compared with currents measured in oocytes expressing wild type rat ENaC.

The W582C and F586C mutants exhibited Li⁺/Na⁺ selectivity ratios that were significantly lower than that observed with wild type mENaC. These data suggest that these aromatic residues might have a role in providing a site within the pore that interacts with Li⁺ and Na⁺ in a differential manner. Aromatic amino acid residues have been shown to interact with monovalent cations through cation- interactions (33-35). Our data raise the possibility that tryptophan and phenylalanine residues within a limited domain of the pore region provide binding sites that favor small cations and perhaps discriminate Na⁺ from Li⁺. Alternatively, the aromatic residues might face away from the pore and interact with other residues within the channel to allow the pore to maintain an appropriate diameter to confer cation selectivity. The introduction of mutations at these sites might affect pore diameter and cation selectivity. A proposed role of -Trp⁵⁸² and -Phe⁵⁸⁶ in ion permeation through ENaC is consistent with studies performed on other cation-selective channels with aromatic amino acid residues within their pore regions that are thought to have a role in conferring ion selectivity (36, 37). We did not detect a significant change in cation selectivity following cysteine substitution of another tryptophan (W585C) that is conserved among all three subunits of ENaC as well as Deg-1, Mec-4, and FaNaCh (Fig. 1C). However, Kellenberger et al. (12) observed a modest but significant reduction of I_Li/I_Na with W585C as well as with selected mutations at the corresponding residues within the - and -subunits (-Trp⁵²⁷ and -Trp⁵³⁹). In contrast, Snyder et al. (14) reported no change in Li⁺/Na⁺ and K⁺/Na⁺ selectivity with the equivalent mutation within human ENaC (W538C). Given these data, it is still unclear whether this conserved tryptophan residue has an important functional role within ENaC's selectivity filter.

The 7-residue tract (-Ser⁵⁸⁰--Phe⁵⁸⁶) where cysteine substitutions lead to changes in Li⁺/Na⁺ selectivity of ENaC is adjacent to a GSS tract (residues -Gly⁵⁸⁷--Ser⁵⁸⁹) that is conserved within the ENaC/degenerin superfamily (Fig. 1C). Cysteine substitutions of either -Gly⁵⁸⁷ or -Ser⁵⁸⁹ significantly increased the K⁺ permeability of the channel. Our results demonstrating that mutations at position -Ser⁵⁸⁹ (S589A and S589C) increase K⁺ permeability are largely in agreement with results reported by Kellenberger et al. (11). However, the significant change in K⁺/Na⁺ selectivity we observed with selected mutations at position -Gly⁵⁸⁷ (G587C or G587A) differs from results reported by Kellenberger et al., where both G587S and G587A showed no K⁺ permeability (12). Our observation that two distinct mutations at this site (G587C or G587A) resulted in measurable amiloride-sensitive whole cell K⁺ currents provides strong evidence that this residue has an important role in restricting K⁺ permeation through the pore. Furthermore, our observation that the G587S mutation resulted in no significant change in cation selectivity is in agreement with the results of Kellenberger et al. (12). Interestingly, Kellenberger et al. observed a significant increase in I_K/I_Na with mutations at rat G529 (G529C and G529S), a position analogous to mouse and rat -Gly⁵⁸⁷ (12). An equivalent mutation in human ENaC (S540C) displayed a 5-fold increase in the Li⁺/Na⁺ current ratio without an effect on the K⁺/Na⁺ current ratio (14). These data, taken together, suggest that the limited (G/S)XS tract within the three ENaC subunits restricts K⁺. This tract is remarkably reminiscent of the G(Y/F)G tract within the pore region of K⁺-selective cation channels that has been identified as a key site for conferring cation selectivity based on mutagenesis and x-ray crystallographic studies (15, 24-27). Substitution of GSS (residues -Gly⁵⁸⁷--Ser⁵⁸⁹) in mENaC with the residues GYG altered the cation selectivity of mENaC such that the channel was primarily K⁺-selective, with a K⁺/Na⁺ current ratio of 3.9 (Table II and Fig. 8). This is the most K⁺-selective mutant that we observed in our analyses of the 30 mutants that were generated within the pore region of the -subunit. These data suggest that the ENaC pore may share some degree of structural similarity with the pore of the KscA K⁺ channel.

Substitution of amino acid residues with hydroxyl groups (serine or threonine) at position -Gly⁵⁸⁷ or -Ser⁵⁸⁹ (G587S and S589T) resulted in channels that retained wild type K⁺/Na⁺ selectivity. We observed that substitution of other residues at position -Gly⁵⁸⁷ or -Ser⁵⁸⁹ (G587C, G587A, S589C, S589A, S589G) led to significant increases in K⁺/Na⁺ selectivity. The weaver mouse has a serine for glycine substitution within the GYG tract (residues 156-158) of the pore region of a G protein-gated inwardly rectifying K⁺ channel (G156S) (38). Interestingly, channels with the weaver mutation were permeable to both Na⁺ and K⁺, suggesting that the presence of a serine residue at this site may be important for allowing Na⁺ to pass through the channel (39-41). Taken together, these data suggest that serine or threonine residues within the GSS tract of the -subunit of ENaC have a role in conferring Na⁺ selectivity. However, one caveat is that we have been unable to detect measurable amiloride-sensitive whole cation (Na⁺, Li⁺, or K⁺) currents when G587T was expressed in oocytes.

Proposed Models for ENaC Selectivity Filter: ENaC Selectivity Is Achieved through Cation Interactions at Multiple Sites within the Selectivity Filter-- The changes in cation selectivity we observed with mutations within the carboxyl-terminal portion of the pore domain suggest that residues within this region, whose secondary structure is predicted to contain an extended region, line the selectivity filter of the channel. We propose a selectivity model whereby ENaC selectivity is achieved through multiple sites within the outer pore region (Fig. 9). Our results suggest that there are a minimum of two critical sites within the pore region of mENaC that are primarily responsible for K⁺/Na⁺ and Li⁺/Na⁺ selectivity. A permeant ion must pass both sites, or "barriers," to traverse the membrane. One selectivity site is formed by the (G/S)XS tract (residues -Gly⁵⁸⁷--Ser⁵⁸⁹ within mENaC). This site is responsible for excluding K⁺ and presumably divalent cations according to the size of the dehydrated ion, allowing both Li⁺ and Na⁺ to traverse the site. We propose that K⁺ is excluded from this site on the basis of size, since its dehydrated radius (1.33 Å) is too large to pass through this site. Changes in the diameter of the pore might be expected to alter interactions with Na⁺ (or Li⁺) and lead to changes in the Li⁺/Na⁺ selectivity ratio, as we observed with the G587A, S589A, S589G, and S589T mutants. We propose that cations pass a second site that consists of a tract including residue -Trp⁵⁸² (Fig. 9). Since several mutations within the (G/S)XS tract are K⁺-permeable, this second site does not appear to strictly exclude K⁺. Tryptophan residues are proposed to stabilize dehydrated cations via cation- interactions (33, 34), and it is conceivable that the Trp⁵⁸² side chain extends into the pore's lumen and participates in solvation of cations. Alternatively, backbone carbonyl residues extend into the pore lumen, and the tryptophan side chain interacts with other residues within the channel to maintain appropriate channel diameter.

View larger version (31K): [in this window] [in a new window]   Fig. 9.   ENaC selectivity models. A, a model of the ENaC selectivity filter with two crucial sites formed by 1) the GXS tract (residues -Gly587 --Ser589, indicated as S589) and 2) -Trp582 (W582). The GXS site is external to the -Trp582 site. B, a similar model to A; however, the GXS is internal to -Trp582. C, an illustration of the arrangement of amino acids within the pore and M2 regions of ENaC. This model was generated based on evidence suggesting that the N-terminal portion of the pore may form an -helix (51) and that the carboxyl-terminal portion of the pore containing the selectivity filter exists as an extended or random-coiled region. The pore domain enters and then exits the outer face of the membrane, similar in structure to the KcsA K+ channel (15). M2 is shown as an -helical structure that begins at the outer face of the plasma membrane. -Trp582, -Gly587, and -Ser589 are placed within the proposed selectivity filter as in model A. D and E, alternative topologies of the pore region and M2 are illustrated. In these structures, the pore and M2 regions span the membrane from its outer to its inner face. -Trp582, -Gly587, and -Ser589 are placed within the proposed selectivity filter region as in model B. The membrane-spanning pore region is ~33 residues in length (-Ser580--Leu612). To accommodate these 33 residues within the membrane, we speculate that the M2 domain must reside at an angle to the membrane normal. Two models are presented (D and E), with different orientations of M2 within the membrane. The placement of the amino-terminal portion of the pore region in D and E was arbitrary, given the limited available information that would predict its location within the pore.

Our results and recent analyses of the putative pore region of the channel suggest several models of how the ENaC pore is arranged (Fig. 9). One model, proposed by Kellenberger et al. and Snyder et al. (11, 12, 14), has the pore region extending from an extracellular site directly into the -helical second membrane-spanning domain, analogous to two funnels joined together by their spouts (Fig. 9D). In this proposed structure, the pore enters the membrane and narrows to a region containing the selectivity filter that is an extended structure of up to 10 residues in length (-Ser⁵⁸⁰--Ser⁵⁸⁹). We propose that the pore transitions to an -helical second membrane-spanning domain and that the pore widens as it undergoes this transition to facilitate high rates of throughput in a putative aqueous environment.

Our data suggest that selectivity does not reside at single site but that multiple sites are involved in this process. We propose that residues -Ser⁵⁸⁰ through -Ser⁵⁸⁹ form an extended selectivity filter and that residues -Leu⁵⁹¹ (or -Ser⁵⁹²) through -Leu⁶¹² (or -Arg⁶¹⁶) form an -helical membrane-spanning domain. This is contrast to the pore model proposed by Kellenberger et al. (11) and Snyder et al. (14), who suggested that the selectivity filter is formed solely by the (G/S)XS tract. Amiloride is proposed to interact with a portion of the channel within the membrane-spanning domain and with specific residues within the - and -subunits in positions analogous to -Ser⁵⁸³ (10). If all residues within the proposed selectivity filter and -helical M2 domain (-Ser⁵⁸⁰ through -Leu⁶¹²) are present within the region of the channel spanning the membrane (or lipid bilayer), the putative membrane-spanning region would extend for a minimum of 33 residues in length. This would comprise an extraordinarily long membrane-spanning tract if the residues are arranged in a somewhat linear or -helical fashion (Fig. 9, D and E). For example, the second membrane-spanning domain of the KcsA channel is 28 residues in length and is tilted with respect to the membrane normal by 25° (15).

An alternative pore region structure would also be consistent with our data. This structure, analogous to the pore of the KcsA K⁺ channel, is formed by a pore -helix that enters the membrane followed by an extended region directed toward the ectodomain of the channel (Fig. 9C). This extended region forms the selectivity filter. We propose that this region within mENaC is ~10 residues in length and is similar in length to the selectivity filter of the KcsA K⁺ channel, which has 7 residues (15). A turn region (or loop) is then followed by the -helical second membrane-spanning domain. This model is not consistent with the notion that amiloride interacts directly with -Gly⁵²⁵ and -Gly⁵⁴². However, mutations at these residues might result in changes in the structure of the pore that indirectly alter amiloride's K_i (see above). Our observation that substitution of the GSS tract (residues -Gly⁵⁸⁷--Ser⁵⁸⁹) in mENaC with GYG led to a K⁺-selective cation channel is also consistent with the notion that the ENaC pore may be similar in structure to the pore of the KscA K⁺ channel (Fig. 9C).

We and others have identified residues in homologous regions within the -, -, and -subunits that probably participate in forming the pore of the channel (11, 12, 14). However, our results do not exclude the possibility that other residues participate in permeation of cations through ENaC. Other residues could be involved in the permeation process by helping to maintain the structure of the selectivity filter. For example, we observed a modest but significant reduction in the Li⁺/Na⁺ current ratio with the S573C mutation. If the channel pore has a structure similar to the KcsA channel, -Ser⁵⁷³ might be present within the pore helix and interact through hydrogen bonding with residues within the selectivity filter. Mutation of Ser⁵⁷³ might affect pore helix-selectivity filter interactions and alter both the overall conformation of the selectivity filter and the channel's ionic selectivity. Mutation at residue -Ser⁵⁹² altered Li⁺/Na⁺ selectivity (Figs. 4 and 5), in agreement with previous observations of Waldmann et al. (13), although a mutation of the corresponding residue within human ENaC to alanine (C541A) resulted in no significant change in either Li⁺/Na⁺ or K⁺/Na⁺ selectivity (14). This residue (Ser⁵⁹² within mouse and rat ENaC) might be located at the transition site where the extended C-terminal portion of the pore region of the -subunit transitions to the -helical M2 domain (Fig. 9C). We propose that mutations at this site alter Li⁺/Na⁺ selectivity by changing the conformation of the adjacent selectivity filter, although we cannot exclude the possibility that -Ser⁵⁹² is within the selectivity filter.

Mutations within the amino-terminal putative cytoplasmic domain of the related cation channel ASIC2 altered the channel's cation selectivity (42), and residues preceding the pore region altered the cation (Na⁺/K⁺) selectivity of bovine -subunit channels expressed in planar lipid bilayers (31). These data suggest that additional sites may have a role in determining the cation selectivity of ENaC.

In summary, our results suggest that the selectivity filter within the -subunit of ENaC is formed by an extended region (C-terminal half of the pore region), including residues -Ser⁵⁸⁰ through -Ser⁵⁸⁹. Our data suggest that there are at least two regions within this domain that have sites where cation discrimination occurs and are consistent with the hypothesis that that the ENaC pore has multiple barriers that a Na⁺ ion must pass (43, 44).

	ACKNOWLEDGEMENTS

We thank Drs. Thomas Kieber-Emmons and Farhad Kosari for helpful discussions during the course of these studies and Juliette Watts for the refinement of Fig. 9.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK54354 and a grant from 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.

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

^§ To whom correspondence should be addressed: Renal Division, 700 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144. Tel.: 215-573-1848; Fax: 215-898-0189; E-mail: kleyman@mail.med.upenn.edu.

	ABBREVIATIONS

The abbreviations used are: ENaC, epithelial sodium channel; mENaC, mouse ENaC.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				H.-L. Ji, S. Parker, A. L. B. Langloh, C. M. Fuller, and D. J. Benos Point mutations in the post-M2 region of human {alpha}-ENaC regulate cation selectivity Am J Physiol Cell Physiol, July 1, 2001; 281(1): C64 - C74. [Abstract] [Full Text] [PDF]

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