title: Cys palmitoylation of the beta subunit modulates gating of the epithelial sodium channel.
abstract: The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (α, β, and γ) 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 β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β 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 β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Gunhild M
authors.initials: GM
authors.lastname: Mueller
authors.forename: Ahmad B
authors.initials: AB
authors.lastname: Maarouf
authors.forename: Carol L
authors.initials: CL
authors.lastname: Kinlough
authors.forename: Nan
authors.initials: N
authors.lastname: Sheng
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.forename: Sora
authors.initials: S
authors.lastname: Okumura
authors.forename: Sarah
authors.initials: S
authors.lastname: Luthy
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D004285:Dogs; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D054878:Lipoylation / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D009865:Oocytes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D026941:Sodium Channel Blockers; D000584:Amiloride; D012964:Sodium