Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the beta and gamma subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant beta subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a beta C43A, C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na+ self-inhibition, and reduced single channel P-o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that beta Cys-43 is in proximity to the first transmembrane alpha helix, whereas beta Cys-557 is within an amphipathic alpha-helix contiguous with the second transmembrane domain. We propose that beta subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.