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 2alpha:1beta:1gamma or a higher-ordered stoichiometry of 3alpha:3beta:3gamma. 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 betaG529A or gammaG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2alpha, 1beta, and 1gamma come together around central pore.
Anantharam
Arun
A
Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA.
Palmer
Lawrence G
LG
D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D009154:Mutation; D021122:Protein Subunits; D051381:Rats
D018076:DNA, Complementary; D053503:Epithelial Sodium Channels; D021122:Protein Subunits