Abstract
Gastric H+,K(+)-ATPase was functionally expressed in the human kidney HEK293 cell line. The expressed enzyme catalyzed ouabain-resistant K(+)-dependent ATP hydrolysis. The K(+)-ATPase activity was inhibited by SCH 28090, a specific inhibitor of gastric proton pump, in a dose-dependent manner. By using this functional expression system in combination with site-directed mutagenesis, we investigated effects of mutations in the putative cation binding site and the catalytic center of the gastric H+,K(+)-ATPase. In Na+,K(+)-ATPase, the glutamic acid residue in the 4th transmembrane segment is regarded as one of the residues responsible for the K(+)-induced conformational change (Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 2993-3000). When the corresponding glutamic acid (Glu-345) of H+,K(+)-ATPase was mutated to aspartic acid, lysine, or valine, the SCH 28080-sensitive K(+)-ATPase activity was abolished. However, when this residue was replaced by glutamine, about 50% of the activity was retained. This mutant showed a 10-fold lower affinity for K+ (Km = 2.6 mM) compared with the wild-type enzyme (Km = 0.24 mm). Thus, Glu-345 is important in determining the K+ affinity of H+,K(+)-ATPase. When the aspartic acid residue in the phosphorylation site was mutated to glutamic acid, this mutant showed no SCH 28080-sensitive K(+)-ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K(+)-ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.
Highlights
Hϩ,Kϩ-ATPase is the proton pump responsible for gastric acid secretion (1, 2)
The activity was inhibited by SCH 28080 and scopadulcic acid B, specific inhibitors of the gastric Hϩ,Kϩ-ATPase (9, 10). By using this functional expression system, we investigated the role of amino acid residues of the putative cation binding site and the catalytic center
When the cells were transfected with the ␣-subunit cDNA, a single weak band was detected around 95 kDa
Summary
Hϩ,Kϩ-ATPase is the proton pump responsible for gastric acid secretion (1, 2). It consists of ␣- and -subunits. We report the functional expression of rabbit gastric Hϩ,Kϩ-ATPase in human HEK293 cells. We replaced Asp-387 of the phosphorylation site and Lys-519 of the FITC binding site of the Hϩ,Kϩ-ATPase ␣-subunit, measured the enzyme activity of the
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