INTRODUCTION

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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, , , and  (14-18). Functional studies show that simultaneous expression of all three subunits is required to generate maximal Na⁺ current, although expression of 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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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 hENaC with the sequence DYKDDDDK (_Flag) for immunoprecipitation by anti-Flag M2 monoclonal antibody. This did not alter the function of the  subunit in Xenopus oocytes or epithelia or its ability to associate with other subunits (19).

Wild-type or mutant -, -, and 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 _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 , , or  subunits in the channel complex, n, as described previously by MacKinnon (21) using the equation where Inh_wt is the fraction of current inhibited in channels containing only wild-type subunits, Inh_mut 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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Stoichiometry of hENaC Using the G536C Mutation-- To investigate the stoichiometry of hENaC, we first asked how many  subunits contribute to the channel complex. Our strategy, similar to that described by MacKinnon (21), was to coexpress mixtures of wild-type and mutant  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  subunit lines the channel pore (23).³ When we replaced Gly⁵³⁶ with cysteine (_G536C), MTSET irreversibly decreased current 87% (Fig. 1B) by covalently modifying the introduced cysteine. Wild-type  (_wt) and _G536C produced equal Na⁺ currents (Fig. 1E). Interestingly, when we coexpressed a 0.5:0.5 mixture of _wt and _G536C (with wild-type  and hENaC), most of the Na⁺ current (77%) was inhibited by MTSET (Fig. 1C). When we increased the contribution of _wt (0.8:0.2 _wt:_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  subunit and the _G536C phenotype is dominant.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Stoichiometry of hENaC using G536C.  and hENaC coexpressed with wt, 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  subunits expressed that was wild-type (remainder was 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  subunits (0.5:0.5 ratio) predicted from binomial distribution if n = 1-3.

Stoichiometry of -, -, and 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  subunit. As a second independent test of the number of  subunits and to determine the number of  and  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 (_S549C, _S520C, and _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 -cysteine(s) and inhibit current.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Stoichiometry of hENaC using the Deg mutation. A, current (nA) in oocytes expressing wild-type - and hENaC with wt, 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 (S549C, S520C, 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).

Sucrose Gradient Analysis of Channel Mass-- To further test stoichiometry, we determined the molecular mass of hENaC by sucrose gradient sedimentation. Because hENaC can form a homomeric channel, we first determined the mass of channels containing only the  subunit. We translated 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  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  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  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.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Sucrose gradient sedimentation of DEG/ENaC channels expressed in vitro. A, autoradiogram and quantitation of hENaC, following sedimentation on 10% (fraction 1) to 45% (fraction 15) sucrose gradient and SDS-PAGE. Glycosylated and unglycosylated hENaC and sedimentation of standard protein markers (kDa) are indicated. As previously shown (22), glycosylated and unglycosylated forms of ENaC each migrate as doublets on SDS-PAGE. B, quantitation by phosphorimaging (ImageQuant, Molecular Dynamics) of glycosylated forms of hENaC, FaNaCh, and BNC1, following sedimentation on a 10-45% sucrose gradient.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Sucrose gradient sedimentation of  and  expressed with hENaC in COS-7 cells. Following sedimentation on 10-45% gradient, hENaC was immunoprecipitated and separated by SDS-PAGE, and the glycosylated form in each fraction was quantitated by phosphorimaging. hENaC that coprecipitated with hENaC was also quantitated (at 0 h, insufficient  was present for quantitation).

	DISCUSSION

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Our functional and our biochemical data support a model of hENaC containing nine subunits; three , three , and three . 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 , , and  subunits in the channel complex, rather than a ratio of one subunit relative to another. Third, use of two independent electrophysiological assays for the  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 , three , and three  subunits. In their model, the pore is formed by 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.