Side-Chain Carboxylate Needed for Nonstoichiometric Proton Leak through Na/K-ATPase Pumps
Cyclic, stoichiometric exchange of 3 Na ions for 2 K ions per ATP hydrolyzed generates outward Na/K-ATPase pump current. With no external Na (Nao) and K (Ko), a nonstoichiometric inwardly-rectifying current flows through Na/K-pumps, proposed carried by protons because it is increased when pH is lowered. Stoichiometric and nonstoichiometric currents are both abolished by the Na/K-pump inhibitor ouabain. In Xenopus pumps with ouabain-resistance mutation C113Y, nonstoichiometric current appeared even at pH 7.6 in the absence of Nao and Ko, was augmented ∼4 fold at pH 6, was abolished by Ko, and was diminished by high Nao. We suggested that alternate-side exposure of a protonatable side chain in E2P-like conformations might allow shuttling of protons across the membrane. We therefore evaluated the role of three key carboxylates, E336 (TM4), E788 (TM5), and D935 (TM8), by mutating C113Y Xenopus pumps. Outward Na/K-pump current was practically abolished in E336Q(C113Y) or E336C(C113Y) pumps, but nonstoichiometric inward current at zero Ko and Nao (replaced by TMAo) was little altered, and was greatly augmented in high Nao. E788C(C113Y) pumps, on the other hand, like parent C113Y pumps, generated robust stoichiometric outward Na/K transport currents (voltage dependent in Nao, but not in TMAo), and little nonstoichiometric inward current in high Nao; but nonstoichiometric current in TMAo was diminished ∼3-fold compared to parent C113Y pumps. In contrast, D935N(C113Y) pumps generated stoichiometric outward Na/K transport currents with altered voltage dependence that was similar in Nao or in TMAo, but inward nonstoichiometric current was nearly absent both in Nao and in TMAo, and was not augmented by lowering pH to 6 either in Nao and in TMAo. The D935 carboxylate thus seems uniquely required for the nonstoichiometric inward flow of protons through the Na/K-pump. [NIH HL36783].