Role of a PH Sensitive Site in Enac Na+ Self-Inhibition

Abstract

The epithelial Na+ channel (ENaC) is key to the regulation of extracellular fluid volume and blood pressure through its action in the aldosterone-sensitive nephron. Inhibition of ENaC by extracellular Na+, referred to as Na+ self-inhibition, likely occurs through an allosteric mechanism of Na+ binding to low affinity sites in the large extracellular regions of ENaC subunits. The effector binding sites and transduction pathways remain unidentified. 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 and analogous clefts in the and subunits host Na+ effector sites. Mutations of acidic residues in the subunit acidic cleft led us to identify an aspartate on the loop connecting the central palm and β-ball domains that reaches towards the finger domain. We also identified nearby sites on the loop and other structures that affected Na+ self-inhibition. To determine whether these sites were involved in effector coordination, we measured the ability of mutants to change the effector specificity of Na+ self-inhibition, which is Na+ > Li+ >> K+ for wild type mouse ENaC. Mutation of the palm/-ball loop aspartate and several nearby sites weakened Na+ inhibition relative to Li+ and K+ inhibition. We hypothesized that protonating any carboxylate groups involved in Na+ coordination would weaken Na+ binding. Consistent with this, lowering the pH to 4.7 increased ENaC currents and reduced Na+ self-inhibition. Mutation of the palm/β-ball loop aspartate reversed pH activation of ENaC. Our results suggest that an aspartate in the acidic cleft of the subunit may coordinate Na+ for Na+ self-inhibition.