Roles of the C termini of α-, β-, and γ-subunits of epithelial Na+ channels (ENaC) in regulating ENaC and mediating its inhibition by cytosolic Na+

The amiloride-sensitive epithelial Na+ channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Nat. In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells: to investigate the role of the C termini of the alpha-, beta-, and gamma -subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the beta- and gamma -subunits, but not the alpha -subunit, inhibited the activity of the Na+ channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse beta -ENaC, the interacting motif in eluded beta Ser(631), beta Asp(632) and beta Ser(633). In the C terminus of mouse gamma -ENaC, it included gamma Ser(640). Once these motifs were deleted, we were able to use the C termini of beta- and gamma -ENaC to prevent Nedd4-mediated down-regulation of Na+ channel activity. The C terminus of the alpha -subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na+ channels. We conclude that mouse Nedd4 interacts with the beta- and gamma -subunits of ENaC.