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*

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 Ni²⁺ on cloned mouse α-β-γ ENaC expressed inXenopus 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 αHis²⁸²and γHis²³⁹ located within the extracellular loops significantly decreased Ni²⁺ inhibition of ENaC currents. The mutation αH282D or double mutations αH282R/γH239R eliminated Ni²⁺ block. All mutations at γHis²³⁹eliminated Ni²⁺-induced inward current rectification. Ni²⁺ block was significantly enhanced by introduction of a histidine at αArg²⁸⁰. Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni²⁺ block. Although αH282C-β-γ channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), α-β-γ 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 αHis²⁸² and γHis²³⁹ and that these histidine residues may participate in ENaC gating.