Identification of ENaC intersubunit interfacing residues provides insight into conformational changes associated with channel gating

Abstract

The extracellular domain of human ENaC functions as a sensor that fine tunes channel activity in response to changes in pH. In previous work, we determined that mutations at γE455 and βE446 decreased the response to acidic pH while αK477A increased the response to acidic pH. Here, we investigated potential intersubunit interactions. Based on the ASIC1a structure, we predict that αK477 and γE455 interact with βV85 and αL120, respectively. To test this, we substituted these residues with Cys and measured mutant channel activity in Xenopus oocytes. We found that covalent modification of Cys mutations at βV85 and αL120 with MTSET+ and MTSESaltered ENaC activity in a manner that was charge dependent. Modification with like charges (i.e. αK477 and MTSET+) increased current while opposite charges decreased current (i.e γE455 and MTSET+) – suggesting an electrostatic interaction between the two residues. To further test for an interaction, we cross‐linked the subunits. In cells expressing αK477CβV85CγENaC, treatment with long cysteine reactive cross‐linking reagents increased current, but short reagents did not. Intriguingly, treatment with either long or short reagents disrupted channel gating, reducing Na+ self‐inhibition. The data provide a potential link between pH sensing, extracellular domain conformational changes, and channel gating. PMS is supported by NIH HL072256, DMC is supported by AHA 10PRE2610282