title: Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
abstract: The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, School of Medicine, University of Pittsburgh, 3550 Terrace Street, Pittsburgh, PA 15261, USA. shaohu@pitt.edu
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
authors.forename: Ahmad B
authors.initials: AB
authors.lastname: Maarouf
authors.forename: James B
authors.initials: JB
authors.lastname: Bruns
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D007202:Indicators and Reagents / Q000494:pharmacology; D017136:Ion Transport / Q000235:genetics; D008564:Membrane Potentials / Q000502:physiology*; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D020125:Mutation, Missense; D009865:Oocytes / Q000166:cytology; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D004220:Disulfides; D053503:Epithelial Sodium Channels; D007202:Indicators and Reagents; D008698:Mesylates; C074502:methanethiosulfonate; D012964:Sodium; D003545:Cysteine