Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation.

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the alpha subunit and at a single extracellular site within the gamma subunit. Channels lacking furin processing of the alpha subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK(186)) in the gamma subunit. We also demonstrated that the tract alpha D206-R231, between the two furin cleavage sites in the alpha subunit, as well as the tract gamma E144-K186, between the furin and prostasin cleavage sites in the gamma subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the alpha and gamma subunit inhibitory tracts, respectively. We examined whether release of either the alpha or gamma inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the alpha subunit (alphaR205A,R208A,R231Abetagamma) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of the alpha subunit, amiloride-sensitive currents in oocytes expressing channels carrying gamma subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (alphabetagammaR143A,DeltaE144-K186 and alphaR205A,R208A,R231AbetagammaR143A, DeltaE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the alpha and gamma subunit inhibitory tracts, alpha subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in the gamma subunit (alphabetagammaR143A,DeltaE144-K186) showed a significant reduction in the efficacy of block by the synthetic alpha-26 inhibitory peptide representing the tract alphaD206-R231. Our data indicate that removal of the inhibitory tract from the gamma subunit, in the absence of alpha subunit cleavage, results in nearly full activation of the channel.