Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC) - Identification of cysteines essential for channel expression at the cell surface

One of the characteristic features of the structure of the epithelial sodiu m channel family (ENaC) is the presence of two highly conserved cysteine-ri ch 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 alph a beta gamma ENaC subunits by systematically mutating cysteine residues (si ngly or in combinations) into either serine or alanine, In the Xenopus oocy te expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the cha nnel. In CRD1, one of the cysteines of the rat alpha ENaC subunit (Cys(158) ) is homologous to Cys(133) Of the corresponding human subunit causing, whe n mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe sal t-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependen t inactivation of the channel. The main features of the mutant cysteine cha nnels are: (i) a decrease in cell surface expression of channel molecules t hat parallels the decrease in channel activity and (ii) a normal assembly o r rate of degradation as assessed by nondenaturing co-immunoprecipitation o f [S-35]methionine-labeled channel protein. These data indicate that the tw o cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly an d/or intrinsic channel activity. We propose that they play an essential rol e in the efficient transport of assembled channels to the plasma membrane.