Proton Current through Stalled Na/K-ATPase Pumps: Three Residues Point to a Possible Route and Mechanism

Na/K pumps export 3 Nai ions for every 2 Ko ions imported, and so generate outward current. But Na/K pumps stalled in Ko-free and Nao-free solution pass an inward current that is greatly augmented by lowering pHo to 6. We studied this inward current in Xenopus oocytes expressing Xenopus α1 Na/K pumps made relatively ouabain-resistant by C113Y or Q120R/N131D mutations. The inward current was abolished by arresting pumps with 10 mM ouabain or by activating them with Ko, but not by obstructing either end of the principal ion transport pathway - by BeFx action at the cytoplasmic side, or by modification of an introduced target Cys at the extracellular side. We explored whether, instead, a conformational equilibrium might expose one or more protonatable side chains alternately to extracellular and cytoplasmic solutions, allowing proton current flow down a proton electrochemical gradient. One at a time, we neutralized (E>Q, D>N) carboxylate side chains in cation coordination sites I and II (E336 in TM4; E788 in TM5; D813 and D817 in TM6) and in the proposed third-Na binding site (D935 in TM8; E963 in TM9), and mutated (H>A/N) a nearby His (H921 in TM8). Despite these mutations, low pHo robustly activated inward current in all but D935N, E963Q, and D935N/E963Q stalled pumps. D935 and E963 are 9-10 A apart in the E2P.2K Na/K-ATPase crystal structures, with a Tyr (Y780 in TM5) equidistant between them with its side chain pointing toward E963. The mutation Y780F almost abolished low-pHo-activated inward current in stalled Na/K pumps. We suggest that, in stalled Na/K pumps, E963 recruits an extracellular proton and, after a conformational change, delivers it (via Y780) to D935, which releases it to the cytoplasm, generating inward current. [NIH HL36783]