title: Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory 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 sites 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 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 αENaC and the palm domain of βENaC. 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 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 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 αγβ orientation (listed clockwise when viewed from the top).
authors.affiliation: Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Collier
authors.forename: Peter M
authors.initials: PM
authors.lastname: Snyder
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D002712:Chlorides / Q000378:metabolism*; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000838:Anions; D002712:Chlorides; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D021122:Protein Subunits