COMMUNICATION
Electrophysiological and Biochemical Evidence That DEG/ENaC Cation Channels Are Composed of Nine Subunits*

Peter M. SnyderDagger , Chun Cheng, Lawrence S. Prince§, John C. Rogers, and Michael J. Welsh

From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha , beta , and gamma ) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha , three beta , and three gamma  subunits. Sucrose gradient sedimentation of alpha hENaC translated in vitro, as well as alpha -, beta -, and gamma hENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The DEG/ENaC protein family includes channels with diverse physiologic and pathophysiologic functions. Epithelial Na+ channels (ENaC) absorb Na+ in kidney, lung, and intestine (1, 2), and mutations in human ENaC (hENaC) cause disease (3-6). Several family members from Caenorhabditis elegans, including MEC-4, MEC-10, and DEG-1, play a role in mechanotransduction, and some gain-of-function mutations cause neurodegeneration (7). In Helix aspersa, the FMRFamide-gated channel (FaNaCh) functions as a neurotransmitter receptor (8). Three family members have recently been identified in the mammalian nervous system, BNC1 (MDEG, BNaC1) (9-11), BNaC2 (ASIC) (11, 12), and DRASIC (13).

All members of the DEG/ENaC family appear to function as multimers. ENaC contains three homologous subunits, alpha , beta , and gamma  (14-18). Functional studies show that simultaneous expression of all three subunits is required to generate maximal Na+ current, although expression of alpha ENaC alone can produce small currents. In addition, biochemical data show that the three human ENaC (hENaC) subunits associate (19). Genetic evidence suggests that MEC-4, MEC-10, and DEG-1 also function as heteromultimers (7, 20). However, the subunit stoichiometry is not known for any DEG/ENaC channel.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

cDNAs and mutations were generated as described previously (9, 17, 18). FaNaCh was amplified by polymerase chain reaction following reverse transcription of RNA from H. aspersa. We tagged the C terminus of alpha hENaC with the sequence DYKDDDDK (alpha Flag) for immunoprecipitation by anti-Flag M2 monoclonal antibody. This did not alter the function of the alpha  subunit in Xenopus oocytes or epithelia or its ability to associate with other subunits (19).

Wild-type or mutant alpha -, beta -, and gamma hENaC (0.2 ng each) were expressed in Xenopus oocytes by nuclear injection of cDNA (18). When a mixture of wild-type and mutant cDNAs for a subunit was coinjected, the total amount of cDNA for the subunit remained constant. 16-24 h after injection, whole-cell Na+ current was measured by two-electrode voltage clamp at -60 mV (bathing solution, 116 mM NaCl, 2 mM KCl, 0.4 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4). To increase the affinity for amiloride of channels containing gamma G536C, we decreased the Na+ concentration for experiments in Fig. 1 (25 mM NaCl, 93 mM KCl). Under these conditions, 3 mM amiloride completely inhibits hENaC current. The fraction of Na+ current inhibited by MTS1 reagents, Inh, was determined by measuring current blocked by a maximal concentration of amiloride before and after addition of MTSET (Toronto Research Chemicals) or MTSEA to the bathing solution for 100 s. Following coexpression of wild-type and mutant hENaC subunits, we determined the number of alpha , beta , or gamma  subunits in the channel complex, n, as described previously by MacKinnon (21) using the equation Inh=f <SUP>n</SUP><SUB><UP>wt</UP></SUB>Inh<SUB><UP>wt</UP></SUB>+(1−f <SUP>n</SUP><SUB><UP>wt</UP></SUB>)Inh<SUB><UP>mut</UP></SUB> where Inhwt is the fraction of current inhibited in channels containing only wild-type subunits, Inhmut is the fraction of current inhibited in channels containing n mutant subunits, and f is the fraction of subunits expressed that are wild-type or mutant, as indicated.

cDNAs were transcribed and translated in vitro in the presence of canine pancreatic microsomal membranes, as described previously (22). COS-7 cells were electroporated as described previously (19), pulse-labeled with 100 µCi/ml [35S]methionine (NEN Life Science Products) at 37 °C for 30 min, and then chased for 0-7 h at 15 °C. We have found that hENaC subunits assemble in the ER (19), then become insoluble to detergents prior to transport to the golgi.2 To minimize insolubility, we incubated cells at 15 °C to inhibit transport out of the ER. Proteins were solubilized in cold Tris-buffered saline (50 mM Tris, 150 mM NaCl) containing 1% digitonin, 0.4 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotonin, 20 µg/ml leupeptin, and 10 µg/ml pepstatin A and sedimented on sucrose gradients as described in figure legends.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Stoichiometry of gamma hENaC Using the G536C Mutation-- To investigate the stoichiometry of hENaC, we first asked how many gamma  subunits contribute to the channel complex. Our strategy, similar to that described by MacKinnon (21), was to coexpress mixtures of wild-type and mutant gamma  subunits in Xenopus oocytes and then determine the sensitivity to an inhibitor. We used MTSET,3 an agent that covalently modifies cysteines. Wild-type hENaC is relatively insensitive to MTSET (Fig. 1A). Previous studies indicate that Gly536 in the gamma  subunit lines the channel pore (23).3 When we replaced Gly536 with cysteine (gamma G536C), MTSET irreversibly decreased current 87% (Fig. 1B) by covalently modifying the introduced cysteine. Wild-type gamma  (gamma wt) and gamma G536C produced equal Na+ currents (Fig. 1E). Interestingly, when we coexpressed a 0.5:0.5 mixture of gamma wt and gamma G536C (with wild-type alpha  and beta hENaC), most of the Na+ current (77%) was inhibited by MTSET (Fig. 1C). When we increased the contribution of gamma wt (0.8:0.2 gamma wt:gamma G536C), MTSET decreased Na+ current 48% (Fig. 1D). The finding that MTSET decreased Na+ current out of proportion to the fraction of mutant subunits suggested that hENaC contains more than one gamma  subunit and the gamma G536C phenotype is dominant.


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Fig. 1.   Stoichiometry of gamma hENaC using gamma G536C. alpha  and beta hENaC coexpressed with gamma wt, gamma G536C, or mixtures, as indicated. A-D, representative time course of current in Xenopus oocytes. Amiloride (3 mM) and MTSET (3 mM) were added as indicated (bars). In D, 1 mM and 3 mM amiloride were used. E, amiloride-sensitive Na+ current (mean ± S.E., n = 9-12 for each). F, fraction of amiloride-sensitive current inhibited by MTSET (Inh) versus fraction (fwt) of gamma  subunits expressed that was wild-type (remainder was gamma G536C); mean ± S.E. (n = 7-13 for each). Values calculated for n when fwt = 0.5, 0.65, and 0.8 are shown. G, combinations of wild-type and mutant gamma  subunits (0.5:0.5 ratio) predicted from binomial distribution if n = 1-3.

To determine the number of gamma  subunits in hENaC, we make two assumptions. First, we assume that gamma wt and gamma G536C express equally and associate randomly in the channel complex. This seems reasonable since expression of gamma wt or gamma G536C with alpha - and beta hENaC produced equal amounts of Na+ current. If this is correct, then channel composition will be determined by a binomial distribution. Second, we assume that a single mutant gamma  subunit is sufficient to make a channel sensitive to MTSET. The dominant effect of gamma G536C suggests this is correct.

Consider the examples illustrated in Fig. 1G (expression of 0.5:0.5 gamma wt:gamma G536C). If hENaC has one gamma  subunit, there will be two channel populations; one will contain a wild-type gamma  subunit and the other will contain gamma G536C. In contrast, if hENaC has three gamma  subunits, then one-eighth of the channels will have three wild-type gamma  subunits and one-eighth will have three mutant subunits. The remainder (six-eighths) will contain both wild-type and mutant gamma  subunits. If a single mutant gamma  makes a channel sensitive to MTSET, then the fraction of Na+ current sensitive to MTSET will be determined by the fraction of channels that have at least one mutant gamma  subunit. Thus, we can calculate the number of gamma  subunits in the hENaC complex (n) by measuring the fraction of Na+ current inhibited by MTSET (Inh). Fig. 1F shows a plot of the values measured for Inh when we expressed gamma wt (fwt = 1.0), gamma G536C (fwt = 0), or mixtures of both. Curves for expected values of Inh if n is 1-4 are superimposed. The data suggest hENaC has three gamma  subunits.

Stoichiometry of alpha -, beta -, and gamma hENaC Using the "Deg" Mutation-- In contrast to MTSET, MTSEA inhibited current in cells expressing wild-type subunits by 54% (Fig. 2A). Our unpublished data suggest that MTSEA inhibits by covalently modifying one or more pore-lining cysteines in the gamma  subunit. As a second independent test of the number of gamma  subunits and to determine the number of alpha  and beta  subunits, we took advantage of a mutation in the Deg residue that prevents inhibition. In MEC-4, mutation of Ala442 to bulky amino acids causes neurodegeneration (20), and mutation of the equivalent residue in BNC1 activates the channel (10). In hENaC subunits, the Deg residues are serines. We found that mutation of a Deg serine to cysteine in any of the subunits (alpha S549C, beta S520C, and gamma S529C) abolished inhibition by MTSEA. Instead, MTSEA and MTSET (Fig. 2A) stimulated a small increase in Na+ current. In this regard, modification of this residue in hENaC may be similar to the activation that occurs with a bulky residue at the Deg position in MEC-4 and BNC1. Perhaps after the cysteine is modified, it provides a steric barrier that prevents MTSEA from entering the pore where it could alter the gamma -cysteine(s) and inhibit current.


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Fig. 2.   Stoichiometry of hENaC using the Deg mutation. A, current (nA) in oocytes expressing wild-type beta - and gamma hENaC with alpha wt, alpha S549C, or a mixture, as indicated. Amiloride (100 µM), MTSET (1 mM), and MTSEA (1 mM), added as indicated (bars). B-D, plots of Inh (mean ± S.E., n = 6-17) versus fwt and calculated values for n. Wild-type and mutant (alpha S549C, beta S520C, gamma S529C) subunits were coexpressed, as in the legend for Fig. 1. E, amiloride-sensitive Na+ current (relative to wild-type) in oocytes expressing two wild-type subunits with the indicated mutant subunit (mean ± S.E., n = 7-8).

We expressed mixtures of these mutant and wild-type subunits and measured current inhibition by MTSEA. Because MTSEA stimulates channels containing the Deg Ser-to-Cys mutation, it complicates the quantitative assessment of MTSEA's inhibition of wild-type channels. To circumvent this problem, we took advantage of the fact that MTSET also stimulates these mutant channels (Fig. 2A), but it does not inhibit wild-type channels (Figs. 1A and 2A). We performed the experiments in two steps as shown in Fig. 2A. First, we added MTSET. It stimulated channels containing alpha S549C, but more importantly, it prevented stimulation on subsequent addition of MTSEA. Second, after washing out MTSET, we applied MTSEA and measured the inhibitory effect on amiloride-sensitive Na+ current (Fig. 2A). In contrast to wild-type hENaC, when we expressed alpha S549C, MTSEA produced a minimal decrease in current (6.9%). Studies with mixtures suggest that alpha S549C prevents channel inhibition by MTSEA in a dominant manner, since Na+ current was inhibited less than the proportion of wild-type alpha subunits. Similar results were obtained with beta S520C and gamma S529C, although MTSET stimulated gamma S529C less than the other mutants. Each mutant produced Na+ currents equal to wild-type (Fig. 2E), supporting the assumption that wild-type and mutant subunits express equally.

Fig. 2, B-D, shows the fraction of Na+ current inhibited by MTSEA versus the fraction of subunits that were wild-type. The results suggest that hENaC contains three alpha , three beta , and three gamma  subunits.

Sucrose Gradient Analysis of Channel Mass-- To further test stoichiometry, we determined the molecular mass of hENaC by sucrose gradient sedimentation. Because alpha hENaC can form a homomeric channel, we first determined the mass of channels containing only the alpha  subunit. We translated alpha hENaC in vitro with microsomal membranes. This assay allows subunit multimerization in the ER, while minimizing the possibility that other cellular proteins will associate with the hENaC channel complex and alter its molecular mass. Fig. 3A shows that unglycosylated alpha  subunits sedimented mainly in fractions 4 and 5, similar to a standard of 240 kDa. This migration suggested that most of the unglycosylated subunits were in the form of dimers or trimers, consistent with our previous finding that subunit interactions begin prior to glycosylation (19). In contrast, glycosylated subunits sedimented in the same fractions (8-10) as a 950-kDa standard (Fig. 3A), consistent with a channel complex containing at least nine subunits. Because most of the alpha  subunits in fractions 8-10 were glycosylated, the data suggest that subunits oligomerize during processing in the ER. Further, multimerization is probably very efficient, since little glycosylated alpha  was found in fractions 4 and 5. Because most multimeric proteins multimerize in the ER (24), this assay probably provides an accurate representation of the size of the complex at the plasma membrane.


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Fig. 3.   Sucrose gradient sedimentation of DEG/ENaC channels expressed in vitro. A, autoradiogram and quantitation of alpha hENaC, following sedimentation on 10% (fraction 1) to 45% (fraction 15) sucrose gradient and SDS-PAGE. Glycosylated and unglycosylated alpha hENaC and sedimentation of standard protein markers (kDa) are indicated. As previously shown (22), glycosylated and unglycosylated forms of alpha ENaC each migrate as doublets on SDS-PAGE. B, quantitation by phosphorimaging (ImageQuant, Molecular Dynamics) of glycosylated forms of alpha hENaC, FaNaCh, and BNC1, following sedimentation on a 10-45% sucrose gradient.

We asked whether other members of the DEG/ENaC family form a complex of similar size. The glycosylated form of FaNaCh sedimented in the same fractions as alpha hENaC (Fig. 3B). BNC1 sedimented in lighter fractions (peak in fraction 8) (Fig. 3B), consistent with the lower molecular mass of glycosylated BNC1 monomer (70 kDa) compared with alpha hENaC (87 kDa) and FaNaCh (90 kDa). These results are consistent with FaNaCh and BNC1 complexes that contain at least nine subunits.

To determine whether the mass of an hENaC complex containing alpha , beta , and gamma  was the same as a homomeric complex, we coexpressed the three subunits in COS-7 cells. Sedimentation was determined at times from 0 to 7 h after pulse labeling. Immediately after the 30-min pulse, immunoprecipitated alpha  subunits peaked in fraction 4, and very little alpha  was present in fractions 8 and 9 (Fig. 4). This suggests a complex containing two-three subunits. With time, the fraction of alpha  in denser fractions increased, and at 7 h, a significant amount of alpha  subunits were in a complex that sedimented in fractions 8 and 9. We also determined the sedimentation of beta  subunits that coimmunoprecipitated with alpha hENaC. At 7 h, a large fraction of beta  subunits were also in a complex sedimenting in fractions 8 and 9. Because this assay only detected beta  subunits that were associated with alpha , the results suggest that fractions 8 and 9 contain heteromeric complexes and likely represent the oligomeric state of the functional channel complex. These data suggest that the channel complex contains at least nine subunits. We cannot exclude the possibility that other cellular proteins tightly associate with hENaC and influence the density. However, this seems unlikely because sedimentation of the channel complex in cells and in vitro was similar.


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Fig. 4.   Sucrose gradient sedimentation of alpha  and beta  expressed with gamma hENaC in COS-7 cells. Following sedimentation on 10-45% gradient, alpha hENaC was immunoprecipitated and separated by SDS-PAGE, and the glycosylated form in each fraction was quantitated by phosphorimaging. beta hENaC that coprecipitated with alpha hENaC was also quantitated (at 0 h, insufficient beta  was present for quantitation).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our functional and our biochemical data support a model of hENaC containing nine subunits; three alpha , three beta , and three gamma . These results suggest a novel stoichiometry for an ion channel and contrast with the stoichiometry of voltage-gated K+ channels (four subunits) (21) and Na+/Ca2+ channels (four repeats), nicotinic acetylcholine receptors (five subunits), and gap junction hemichannels (six subunits) (25).

The finding that BNC1 and FaNaCh migrated on sucrose gradients similar to hENaC suggests that those channels may also be constructed from nine subunits. Thus, a multimeric complex composed of nine subunits may be a conserved feature of the DEG/ENaC family. In addition, genetic evidence suggests that some C. elegans family members contain more than one copy of each subunit. For example, MEC-4, MEC-10, and probably one unidentified subunit may form a functional complex. A loss-of-function mutation on one mec-4 allele suppressed a dominant neurodegeneration-associated mutation on the other mec-4 allele (26). Similar interactions appear to occur with MEC-10 subunits (27). These results suggest that the functional complex contains two or more MEC-4 and two or more MEC-10 subunits (7).

The approach we used has several advantages. First, the electrophysiological assays allowed us to selectively determine the stoichiometry of functional channels. If different combinations of subunits produced nonfunctional channels or channels not delivered to the cell surface, they would not be detected. Second, our approach allowed us to determine the absolute number of alpha , beta , and gamma  subunits in the channel complex, rather than a ratio of one subunit relative to another. Third, use of two independent electrophysiological assays for the gamma  subunit strengthen our conclusions. Fourth, we were able to avoid the use of concatameric constructs, which could disrupt the normal association of subunits, as previously reported for Shaker K+ channels (28). Finally, we used both functional and biochemical approaches. Both supported the same conclusion.

Our approach also has limitations. First, our electrophysiological assays would not detect inactive channels. Second, our sucrose gradient assay detected channels in the ER, rather than channels at the plasma membrane. However, earlier work suggested that the hENaC subunits assemble in the ER (19). Finally, our electrophysiological approach relies on two assumptions; that wild-type and mutant subunits express equally and associate randomly and that a single mutant subunit has a dominant effect on MTS sensitivity. As discussed above, these assumptions are likely valid. However, if the assumption that a single mutant subunit is dominant is incorrect, our calculations will underestimate the number of subunits. In the case of all three limitations, the concordance between biochemical and functional data support the validity of our approach.

It is interesting to speculate how nine subunits might assemble to form a highly selective channel. Based on theoretical considerations, Guy and Durell (29) independently proposed a model for ENaC that contains three alpha , three beta , and three gamma  subunits. In their model, the pore is formed by alpha beta barrels derived from the residues immediately preceding M1 and M2, and they predicted that all nine subunits contribute to the channel pore. Our experimental data are consistent with this model.

    ACKNOWLEDGEMENTS

We thank H. Robert Guy for discussions about his model of ENaC, Theresa Mayhew, Ellen Tarr, and Dan Bucher for technical assistance, and John B. Stokes, Christopher Adams, Margaret Price, Joseph Cotten, and our other laboratory colleagues for helpful discussions. We thank the University of Iowa DNA Core for assistance.

    FOOTNOTES

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

Dagger Supported by a Fellowship from the Roy J. Carver Charitable Trust and by the NHLBI and NIDDK, National Institutes of Health. To whom correspondence should be addressed: Dept. of Internal Medicine, University of Iowa College of Medicine, 200K EMRB, Iowa City, IA 52242. Tel.: 319-356-7481.

§ Supported by a National Research Service Award from the NHLBI, National Institutes of Health.

Supported by the Howard Hughes Medical Institute.

1 The abbreviations used are: MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.

2 L. S. Prince and M. J. Welsh, unpublished observations.

3 P. M. Snyder and M. J. Welsh, unpublished observations.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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