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Electrogenic Partial Reactions of the Gastric H,K-ATPase

Anna Diller ^*, Olga Vagin , George Sachs  and Hans-Jürgen Apell ^*

^* Department of Biology, University of Konstanz, 78457 Konstanz, Germany; and Department of Physiology and Medicine, University of California, Los Angeles, California 90073 USA

Correspondence: Address reprint requests to Hans-Jürgen Apell, Dept. of Biology, University of Konstanz, Fach M635, 78457 Konstanz, Germany. Tel.: 49-7531-88-2253; Fax: 49-7531-88-3183; E-mail: h-j.apell{at}uni-konstanz.de.

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K⁺ and H⁺ ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E₁ and P-E₂, are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and 4.5 in the E₁ conformation; 6.7 and 2 in the P-E₂ conformation. The equilibrium dissociation constants for K⁺ binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K⁺ concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites.