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, α, β, and γ. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2α:1β:1γ or a higher-ordered stoichiometry of 3α:3β:3γ. 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 α, β, or γ subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of α, β, and γ 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 α-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 α-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant βG529A or γG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2α, 1β, and 1γ come together around central pore.