Analyses of epithelial Na+ channel variants reveal that an extracellular ß-ball domain critically regulates ENaC gating.

ABSTRACT

Epithelial Na+ channel (ENaC)-mediated Na+ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K+]. Among the thousands of human ENaC variants, only a few exist whose functional consequences have been experimentally tested. Here, we used the Xenopus oocyte expression system to investigate the functional roles of four nonsynonymous human ENaC variants located within the ß7-strand and its adjacent loop of the α-subunit extracellular ß-ball domain. αR350Wßγ and αG355Rßγ channels exhibited 2.5- and 1.8-fold greater amiloride-sensitive currents than WT αßγ human ENaCs, respectively, whereas αV351Aßγ channels conducted significantly less current than WT. Currents in αH354Rßγ-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (αR350Wßγ, αV351Aßγ, and αG355Rßγ) were similar to that of WT. However, three mutant channels (αR350Wßγ, αH354Rßγ, and αG355Rßγ) exhibited a reduced Na+ self-inhibition response. Open probability of αR350Wßγ was significantly greater than that of WT. Moreover, other Arg-350 variants, including αR350G, αR350L, and αR350Q, also had significantly increased channel activity. A direct comparison of αR350W and two previously reported gain-of-function variants revealed that αR350W increases ENaC activity similarly to αW493R, but to a much greater degree than does αC479R. Our results indicate that αR350W along with αR350G, αR350L, and αR350Q, and αG355R are novel gain-of-function variants that function as gating modifiers. The location of these multiple functional variants suggests that the αENaC ß-ball domain portion that interfaces with the palm domain of ßENaC critically regulates ENaC gating.