Epithelial Sodium Channel Gating is Modulated by Cysteine‐Palmitoylation

Abstract

The epithelial Na+ channel (ENaC) serves as the final site of renal Na+ absorption in the distal nephron, where the fine tuning of its activity regulates extracellular fluid volume and blood pressure. The three homologous subunits of ENaC (alpha, beta and gamma) have a similar topology with two transmembrane domains, a large extracellular loop, and cytoplasmic N‐ and C‐termini. While various lipids are known regulators of ENaC activity, cytoplasmic Cys‐palmitoylation of ENaC has not been previously described. Palmitoylation of individual epitope‐tagged subunits was assessed with fatty acid‐exchange chemistry, whereby palmitate is replaced by biotin and biotinylated subunits were analyzed by immunoblotting. We observed that only the beta and gamma subunits were modified. The function of palmitoylation adjacent to the membrane was studied by expressing ENaC containing a beta C43,557A mutant in Xenopus oocytes. These mutant channels exhibited significantly enhanced Na+ self inhibition and reduced open probability, when compared with wild type ENaC. In contrast, normal membrane trafficking was observed. Our results indicate that palmitoylation of the beta subunit enhances ENaC gating. (Funding from DK065161, DK065521 and AHA).