Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains

Activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na+. We searched for determinants of Na+ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at gamma Met(438) altered the Na+ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na+ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na+ self-inhibition. Mutations at the homologous site in the alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na+ self-inhibition. Mutations at the homologous beta Ala(422) resulted in minimal or no change in Na+ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alpha G481M beta gamma, alpha beta gamma M438V, and alpha G481M/beta gamma M438V, respectively. This pair of residues map to the alpha 5 helixin the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both alpha G481C beta gamma and alpha beta gamma M438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alpha Gly(481) and gamma Met(438) are functional determinants of Na+ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.