Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na+. A His residue within the extracellular domain of gamma ENaC (gamma His(239)) was found to have a critical role in Na+ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na+ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na+ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na+ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gamma H239F mutant showed that, in the absence of Na+ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gamma His(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na+.