Involvement of the H₃O⁺-Lys-164-Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H⁺,K ⁺-ATPase

Gastric H⁺,K⁺-ATPase is shown to transport 2 mol of H⁺/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H⁺ transport mechanism is due to charge transfer and/or transfer of hydronium ion (H₃O⁺). From transport of [ ¹⁸O]H₂O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H⁺,K⁺-ATPase α-subunit at E₁ conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H₃O⁺-Lys-164-Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H₃O⁺-Gln-161-Glu-345) in mutant K164L and (H₃O⁺-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H⁺,K⁺-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H₃O⁺-Asn-794-Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H₂O in sites 2 and 1, the H₂O comes from cytosolic medium, and H ₃O⁺ in the sites are transported into lumen during the conformational transition from E₁P to E₂P.