J Biol Chem 2002 Dec 20;27751:50098-111. doi: 10.1074/jbc.M209975200.

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.

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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.

???displayArticle.pubmedLink??? 12397059
???displayArticle.link??? J Biol Chem
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