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 Na⁺. 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 α-, β-, and γ-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the β- and γ-subunits, but not the α-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 β-ENaC, the interacting motif included βSer ⁶³¹, βAsp⁶³², and βSer⁶³³. In the C terminus of mouse γ-ENaC, it included γSer⁶⁴⁰. Once these motifs were deleted, we were able to use the C termini of β- and γ-ENaC to prevent Nedd4-mediated down-regulation of Na⁺ channel activity. The C terminus of the α-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na⁺ channels. We conclude that mouse Nedd4 interacts with the β- and β-subunits of ENaC.