Mutational Analysis of Cysteine-rich Domains of the Epithelium Sodium Channel (ENaC)

Abstract

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat ␣␤␥ ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of ␣, ␤, or ␥ ENaC subunits led to a temperaturedependent inactivation of the channel. In CRD1, one of the cysteines of the rat ␣ENaC subunit (Cys 158 ) is homologous to Cys 133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in ␣ and ␤ but not in the ␥ subunit also produced a temperaturedependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35 S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.The amiloride-sensitive epithelial sodium channel (ENaC) 1 belongs to the rapidly growing ENaC/DEG (degenerins) family of ion channels implicated in a variety of physiological functions. The specific tissue and species expression patterns of the channels allow us to classify them in four subfamilies: (i) ␣, ␤, and ␥ ENaC subunits, mainly expressed in epithelia; (ii) MDEG1, MDEG2, ASIC, and DRASIC genes, recently identified in the nervous system of mammals; (iii) FaNaCh involved in synaptic transmission in snail; (iv) MEC-4, MEC-10, DEG-1 (degenerins), and UNC 105 of the Caenorhabditis elegans nematode expressed in sensory neurons and muscles, respectively (1). All proteins of this supergene family share a common membrane topology with two transmembrane domains, short intracellular N and C termini and a large extracellular loop (2). An heterotetrameric structure has been recently proposed for ENaC (3, 4), whereas an homo-tetrameric structure has been proposed for FaNaCh (5). It is therefore likely that this gene family of cation channel is tetrameric, despite a study suggesting a nonameric architecture for ENaC (6).Structure-function studies performed on ENaC subunits have indicated some specific functional roles of different domains of the proteins. Schild et al. , an important mechanism controlling Na ϩ reabsorption (12). In intact cells (i.e. Xenopus oocytes) phosphorylation studies have also demonstrated a possible role of the C termini, as the target sequences for hormone-stimulated protein kinases A-and C-...

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

“…no disulfide bond). Because we observed a similar reduction in ENaC Na ϩ self-inhibition with either the ␣Cys-1 or ␣Cys-6 single mutation, which was not additive in the double mutant, these data are consistent with the presence of a disulfide bond between ␣Cys-1 and ␣Cys-6, in accordance with previous findings (14). Similarly, the Na ϩ self-inhibition response of mENaCs with double mutations at ␣Cys-4/␣Cys-5, ␣C7/␣Cys-16, and ␣Cys-10/␣Cys-13, showed nonadditive effects compared with ENaC with the corresponding single mutations (Fig.…”

“…The gray circles identify Cys residues whose mutations significantly reduced the Na ϩ self-inhibition response, and the circle with a thick circumference identifies ␣Cys-11, whose substitution enhanced Na ϩ self-inhibition. Proposed disulfide bonds based on this study are shown as gray lines (14). The dashed line identifies a potential disulfide bond between ␣Cys-8 and ␣Cys-15 that was not demonstrated in this study.…”

“…Firsov et al (14) suggested that ␣Cys-1 and ␣Cys-6 in CRD-I as well as ␣Cys-11 and ␣Cys-12 in CRD-II form disulfide bonds, based on a comparison of changes in ENaC currents in oocytes expressing subunits with single or double Cys mutations. Using a similar approach, we tested whether those Cys residues whose substitutions significantly suppressed Na ϩ self-inhibition represented disulfide-linked residues by construction of double Cys mutants in the ␣ or ␥ subunits.…”

“…The authors concluded that the Cys-1 and Cys-6 residues of CRD-I in all three subunits and the Cys-11 and Cys-12 of CRD-II in ␣ and ␤ (but not in ␥) ECLs are required for efficient expression of ENaC at the plasma membrane. However, the authors also reported that individual mutation of six of ten CRD-II Cys residues significantly increased Na ϩ currents without a proportional change in the number of channels expressed at the cell surface (14), suggesting that these Cys mutations increase the single channel activity. It has been known for many years that specific extracellular sulfhydryl reagents activate ENaC currents by interfering with Na ϩ self-inhibition (4,21).…”

Five novel mutations in the SCNN1A gene causing autosomal recessive pseudohypoaldosteronism type 1

“…Previous studies suggest that all stop mutations within the extracellular loop affect ENaC function alike (23,25,26). Only missense mutations in the SCNN1A were found to result in retained channel function (12,27,28).…”