Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances

The epithelial Na+ channel (ENaC) is a multimeric membrane protein consisting of three subunits, alpha, beta, and gamma. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2 alpha:1 beta:1 gamma or a higher-ordered stoichiometry of 3 alpha:3 beta:3 gamma. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC alpha, beta, or gamma subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of alpha, beta, and gamma showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant alpha-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant alpha subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant beta G529A or gamma G534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2 alpha 1 beta, and 1 gamma come together around central pore.