Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase

A compound, SCH 28080 (2-methyl-8-(phenylmethoxy) imidazo[1,2-a]pyridine-3-acetonitrile) reversibly inhibits gastric and renal ouabain-insensitive H+,K+-ATPase, but not colonic ouabain-sensitive H+,H+-ATPase, By using the functional expression system and site-directed mutagenesis, we analyzed the putative binding sites of SCH 28080 in gastric H+,K+-ATPase alpha-subunit, It was previously reported that the binding site of SCH 28080, which is a K+-site inhibitor specific for gastric H+,K+-ATPase, was in the first extracellular loop between the first and second transmembrane segments of the alpha-subunit; Phe-126 and Asp-138 were putative binding sites, However, we found that all the mutants in the first extracellular loop including Phe-126 and Asp-ISS retained H+,K+-ATPase activity and sensitivity to SCH 28080, Therefore, amino acid residues in the first extracellular loop are not directly involved in the SCH 28080 binding nor indispensable for the H+,K+-ATPase activity. Here rye propose a candidate residue that is important for the binding with SCH 28080, Glu-822 in the sixth transmembrane segment. Mutations of Glu-822 to Asp and Ala (mutants termed E822D and E822A, respectively) decreased the ATPase activity to about 45% and 35% of the wild-type enzyme, respectively, while the mutations to Gin and Leu abolished the activity, Mutant E822A showed a significantly lower affinity for K+ than the wild-type enzyme, indicating that Glu-822 is involved in determining the affinity for K+, The sensitivity of mutant E822D to SCH 28080 was 8 times lower than that of the wild-type enzyme, The counterpart of Glu-822 in gastric H+,K+-ATPase is Asp in Na+,K+-ATPase and other colonic ouabain-sensitive H+,K+-ATPase, which are insensitive to SCN 28080, These results suggest that Glu-822 is one of important sites that bind with SCH 28080.