Differential modulation of a polymorphism in the COOH terminus of the α-subunit of the human epithelial sodium channel by protein kinase Cδ

The A663T polymorphism of the alpha-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of alpha beta gamma-hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of alpha-hENaC is important for this effect, as a homologous change in murine ENaC (mENaC), A692T, does not alter functional and surface expression of mENaC. Query of a phosphoprotein database suggested that the alpha-T663 residue might be phosphorylated by PKC delta. General inhibition of PKC with calphostin C decreased the functional and surface expression of alpha T663-hENaC and not alpha A663-hENaC, and was without effect on alpha 692-mENaC, alpha T692-mENaC, and a chimeric m(1-678)/h(650-669)alpha T663, m beta gamma-ENaC. These data suggest that residues outside of the alpha-hENaC COOH terminus are important for modulation of alpha T663-hENaC trafficking by PKC. In contrast, expression of PKC delta decreased the functional and surface expression of alpha T663-hENaC and the functional expression of m(1-678)/h( 650-669) alpha T663, m beta gamma-ENaC, and was without effect on alpha A663-hENaC, alpha A692-mENaC, or alpha T692-mENaC. PKC delta did not phosphorylate the COOH terminus of either alpha T663-hENaC or alpha A663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. alpha T663-hENaC was retrieved from the oocyte membrane more slowly than alpha A663-hENaC, and calphostin C increased the rate of alpha T663-hENaC removal from the oocyte membrane to a rate similar to that of alpha A663-hENaC. In contrast, PKC delta did not alter the rate of removal of alpha T663-hENaC from the oocyte membrane, suggesting that PKC delta altered rates of alpha T663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of alpha T663- vs. alpha A663-hENaC.