Substitutions of a pair of histidine residues within the extracellular loop of alpha subunit of the epithelial sodium channel suppress sodium self‐inhibition

Abstract

Epithelial Na+ channels (ENaC) mediate apical Na+ transport and play an essential role in body fluid volume homeostasis. Recent studies have demonstrated a key role of the extracellular Na+ concentration in controlling the open probability of ENaC through Na+ self‐inhibition. Here we investigated the role of a pair of adjacent His residues within the extracellular loop in Na+ self‐inhibition. The His325 and His326 of mouse αENaC were mutated to di‐Ala residues. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. A significantly suppressed (50%) and slowed (65%) Na+ self‐inhibition was observed in oocytes expressing αH325A‐H326Aβγ, compared to the wild type. Based on the recently published structure of the chicken acid sensing ion channel 1 (cASIC1) and a sequence alignment with αENaC, the di‐His residues are predicted to be located at the C‐terminus of β4 within the β‐ball domain. Interestingly, residue Arg191 of cASIC1, which is adjacent to the sequence homologous to HH of αENaC, directly interacts with a residue (Asp350) within the thumb domain that is predicted to bind proton. Based on our results and the cASIC1 structure, we propose that the di‐His residues interact with the thumb domain and the intrasubunit interaction is involved in the conformational changes induced by the binding of extracellular Na+. (Supported by NIH DK54354)