Identification of epithelial Na+ channel (ENaC) inter‐subunit Cl‐ coordinating residues suggests a trimeric alpha, gamma, beta channel architecture

Abstract

The extracellular domain of the epithelial Na+ channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl− inhibits ENaC activity. To identify residues involved in Cl− inhibition, we mutated residues in the extracellular domain of α‐, β‐ and γENaC that are homologous to the Cl− binding site in ASIC1 and tested the effect of Cl− on the activity of ENaC expressed in Xenopus oocytes. We identified two functionally significant Cl− sites in αβγENaC. One site is formed by residues in the thumb domain of αENaC and palm domain of βENaC. Mutation of residues in this domain decreased Cl− inhibition and decreased Na+ self‐inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl− inhibition, yet had no effect on Na+ self‐inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had minimal effect on Cl− inhibition or Na+ self‐inhibition. The data demonstrate that Cl− inhibits ENaC activity by two discrete Na+ dependent and Na+ independent mechanisms. Furthermore, our results suggest that ENaC adopts an αγβ rather than αβγ confirmation, providing additional insight into the functional significance of α, β and γENaC subunit interactions.PMS is supported by NIH HL072256