Identification of Epithelial Na+ Channel (ENaC) Intersubunit Cl- Inhibitory Residues Suggests a Trimeric αγβ Channel Architecture

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 sites involved in Cl- inhibition, we mutated residues in the extracellular domain of alpha-, beta-, and gamma ENaC that are homologous to the Cl- binding site in acid-sensing ion channel 1a and tested the effect of Cl- on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl- inhibitory sites in ENaC. One is formed by residues in the thumb domain of alpha ENaC and the palm domain of beta ENaC. Mutation of residues at this interface decreased Cl- inhibition and decreased Na+ self-inhibition. The second site is formed by residues at the interface of the thumb domain of beta ENaC and the palm domain of gamma ENaC. Mutation of these residues also decreased Cl- inhibition yet had no effect on Na+ self-inhibition. In contrast, mutations in the thumb domain of gamma ENaC and palm of alpha ENaC had little or no effect on Cl- inhibition or Na+ self-inhibition. The data demonstrate that Cl- inhibits ENaC activity by two distinct Na+-dependent and Na+-independent mechanisms that correspond to the two functional Cl- inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl- inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl- inhibition, the data support a model in which ENaC subunits assemble in an alpha gamma beta orientation (listed clockwise when viewed from the top).