Abstract
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 (H(3)O(+)). From transport of [(18)O]H(2)O, 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 alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-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 (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.
Highlights
In P-type ATPases such as Naϩ,Kϩ-ATPase, Ca2ϩ-ATPase, and Hϩ,Kϩ-ATPase, at least two conformation states exist, E1 and E2, with conformational changes being accompanied by ion transportation [1]
Water Transport Activity of Hog Gastric Vesicles—If the pump transports hydronium ions (H3Oϩ) from the cytosolic medium into gastric vesicles or if charge transfer couples with water transport (H2O), [18O]H2O in the cytosolic medium will be transported into the vesicles
If only the charge transfer is involved in Hϩ pumping, [18O]H2O in the cytosolic medium will not be transported
Summary
In P-type ATPases such as Naϩ,Kϩ-ATPase, Ca2ϩ-ATPase, and Hϩ,Kϩ-ATPase, at least two conformation states exist, E1 and E2, with conformational changes being accompanied by ion transportation [1]. One ml of gastric vesicle solution containing 1 mg/ml proteins, 130 mM KCl, 4 mM MgCl2, 10 g/ml valinomycin, 8 atom% [18O]H2O, and 40 mM PIPES/Tris (pH 6.8) was incubated in the presence (ϩATP) or absence (ϪATP) of 4 mM ATP for 20 min at 25 °C.
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