Knuckle subdomain of the epithelial sodium channel and sodium self‐inhibition

Abstract

Epithelial Na+ channel (ENaC) gating is regulated by the binding of extracellular Na+ to the channel, a process referred to as Na+ self-inhibition. The finger domains of the extracellular regions of the α and γ subunits contain structural elements that are critical for Na+ self-inhibition. The resolved ASIC1 structure suggests that the finger domain has contacts with the knuckle domain of an adjacent subunit, which may mediate subunit-subunit interactions. We investigated the functional role of the knuckle domain of the α subunit in Na+ self-inhibition. Fifteen knuckle domain residues were individually mutated to Cys. Na+ self-inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two-electrode voltage clamp. Three mutations (αP521C, αI529C and αL533C) greatly suppressed the Na+ self-inhibition response. αP521C nearly eliminated Na+ self-inhibition with the steady state and peak current ratio of 0.87 ± 0.02 (n=7, p < 0.001 versus 0.63 ± 0.03, n=7 (wild type)). The changes in Na+ self-inhibition by αI529C and αL533 are consistent with a helical structure. Cys substitutions at twelve other residues including six polar residues did not significantly alter the response. Our results suggest that specific residues in the α knuckle domain have a role in Na+ self-inhibition. (Supported by NIH ES14701, DK54354 and DK079307)