Cysteine Palmitoylation of the γ Subunit Has a Dominant Role in Modulating Activity of the Epithelial Sodium Channel

Background: Epithelial sodium channel (ENaC) and subunits are modified by Cys palmitoylation. Results: Palmitoylation of the subunit activates ENaCs by increasing channel open probability. Conclusion: subunit palmitoylation has a dominant role in activating ENaCs compared with subunit palmitoylation. Significance: ENaC palmitoylation is an important post-translational mechanism of channel regulation. The epithelial sodium channel (ENaC) is composed of three homologous subunits (, , and ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the subunit, Cys-43 and Cys-557, are Cys-palmitoylated. ENaCs with mutant C43A/C557A exhibit normal surface expression but enhanced Na+ self-inhibition and reduced channel open probability. Although the subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the subunit are palmitoylated. ENaCs with mutant C33A, C41A, or C33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of and subunit-activating cleavage. These mutant channels have significantly enhanced Na+ self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the subunit places Cys-33 within an -helix and Cys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the and subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that subunit palmitoylation has a dominant role over subunit palmitoylation in modulating ENaC gating.