title: Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel.
abstract: The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ 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 γ subunit places γCys-33 within an α-helix and γCys-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 β and γ 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 γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating.
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Anindit
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Gunhild M
authors.initials: GM
authors.lastname: Mueller
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Kinlough
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Nan
authors.initials: N
authors.lastname: Sheng
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Wang
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: S Atif
authors.initials: SA
authors.lastname: Mustafa
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and.
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and Department of Cell Biology, kleyman@pitt.edu.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.affiliation: From the Renal-Electrolyte Division, Department of Medicine, and Department of Cell Biology, Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
mesh_terms: D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D054878:Lipoylation; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
chemical_list: D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D012964:Sodium; D003545:Cysteine
keywords: Acid-sensing Ion Channel (ASIC); ENaC; Gating; Ion Channels; Protein Palmitoylation