Incorporation of the -subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis

Abstract

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits ( and-), but a fourth--subunit may replace - and form atypical -ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the -- and --subunits are differentially expressed in different tissues and that --ENaC predominantly is present in the urogenital tract. Using wholecell and single-channel electrophysiology of oocytes expressing Xenopus - or -ENaC, we demonstrate that the presence of the --subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the -- and --subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike --ENaC, the --subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by -ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the --subunit prevented cleavage of --ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus --ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.