In Search of ENaC Sodium Sensors

Abstract

The epithelial Na+ channel (ENaC), which constitutes the limiting conduit for transepithelial Na+ transport in several tissues, is inhibited by extracellular Na+. Extracellular Na+ likely inhibits ENaC allosterically by binding at low affinity sites in the large extracellular regions of ENaC subunits. These sites remain unidentified. Examination of a recent comparative model of the ENaC α subunit revealed an acidic cleft, analogous to the acidic region observed in the resolved structure of acid sensing ion channel 1 (ASIC1). We hypothesized that this cleft may contain the putative low affinity Na+ effector sites. We mutated acidic residues within the defined region of the α subunit, and measured the effect of mutation on Na+ self-inhibition. We identified one site in the peripheral finger domain at which mutation reduced Na+ self-inhibition. Testing the analogous sites in the β and γ subunits produced a similar result. Inspection of our α ENaC model suggested that this site is near a poorly conserved loop emanating from a β-sheet from the central core of the structure. Scanning mutagenesis of the loop revealed marked effects on Na+ self-inhibition. Introducing cys to both the site on the finger domain and the nearby loop led to channels that were strongly stimulated by dithiothreitol. Our results suggest that conformational changes in the acidic cleft are associated with channel gating, making it a candidate for a Na+ sensor site on ENaC.