External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of α and γ subunits and reducing channel open probability

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni2+ on cloned mouse alpha-beta-gamma ENaC expressed in Xenopus oocytes. Ni2+ reduced amiloride-sensitive Na+ currents of the wild type mouse ENaC in a dose-dependent manner. The Ni2+ block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni2+ 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 Ni2+ inhibition of the remaining current. Mutations at alphaHis(282) and gammaHis(239) located within the extracellular loops significantly decreased Ni2+ inhibition of ENaC currents. The mutation alphaH282D or double mutations alphaH282R/gammaH239R eliminated Ni2+ block. All mutations at gammaHis(239) eliminated Ni2+-induced inward current rectification. Ni2+ block was significantly enhanced by introduction of a histidine at alphaArg(280). Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni2+ 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, Ni2+ did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni2+ 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.