Interactions between subunits of the human epithelial sodium channel.
The human epithelial sodium channel (hENaC) mediates Na+ transport across the apical membrane of epithelia, and mutations in hENaC result in hypertensive and salt-wasting diseases. In heterologous expression systems, maximal hENaC function requires co-expression of three homologous proteins, the alpha, beta, and gammahENaC subunits, suggesting that hENaC subunits interact to form a multimeric channel complex. Using a co-immunoprecipitation assay, we found that hENaC subunits associated tightly to form homo- and heteromeric complexes and that the association between subunits occurred early in channel biosynthesis. Deletion analysis of gammahENaC revealed that the N terminus was sufficient but not necessary for co-precipitation of alphahENaC, and that both the N terminus and the second transmembrane segment (M2) were required for gamma subunit function. The biochemical studies were supported by functional studies. Co-expression of gamma subunits lacking M2 with full-length hENaC subunits revealed an inhibitory effect on hENaC channel function that appeared to be mediated by the cytoplasmic N terminus of gamma, and was consistent with the assembly of nonfunctional subunits into the channel complex. We conclude that the N terminus of gammahENaC is involved in channel assembly.
Adams
C M
CM
Howard Hughes Medical Institute and Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Snyder
P M
PM
Welsh
M J
MJ
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D019281:Dimerization; D053503:Epithelial Sodium Channels; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D009865:Oocytes / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D016324:Sequence Tagged Sites; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015222:Sodium Channels