title: External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of alpha and gamma subunits and reducing channel open probability.
abstract: Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni(2+) on cloned mouse alpha-beta-gamma ENaC expressed in Xenopus oocytes. Ni(2+) reduced amiloride-sensitive Na(+) currents of the wild type mouse ENaC in a dose-dependent manner. The Ni(2+) block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni(2+) was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni(2+) inhibition of the remaining current. Mutations at alphaHis(282) and gammaHis(239) located within the extracellular loops significantly decreased Ni(2+) inhibition of ENaC currents. The mutation alphaH282D or double mutations alphaH282R/gammaH239R eliminated Ni(2+) block. All mutations at gammaHis(239) eliminated Ni(2+)-induced inward current rectification. Ni(2+) block was significantly enhanced by introduction of a histidine at alphaArg(280). Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni(2+) block. Although alphaH282C-beta-gamma channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), alpha-beta-gamma H239C channels were insensitive to MTSET. From patch clamp studies, Ni(2+) did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni(2+) reduces ENaC open probability by binding to a site consisting of alphaHis(282) and gammaHis(239) and that these histidine residues may participate in ENaC gating.
authors.affiliation: Renal-Electrolyte Division, the Department of Medicine, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261, USA. shaohu@pitt.edu
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
authors.forename: Clint J
authors.initials: CJ
authors.lastname: Perry
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D053503:Epithelial Sodium Channels; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; D015222:Sodium Channels; D006639:Histidine; D000584:Amiloride; D009532:Nickel; D012964:Sodium; D008274:Magnesium