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Volume 272, Number 28, Issue of July 11, 1997 pp. 17668-17674
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutational Analysis of Putative SCH 28080 Binding Sites of the Gastric H⁺,K⁺-ATPase

(Received for publication, March 14, 1997, and in revised form, April 15, 1997)

Shinji Asano , Saiko Matsuda ^¶ , Yasuhiro Tega ^¶ , Kanae Shimizu ^¶ , Shinya Sakamoto ^¶ and Noriaki Takeguchi ^¶

From the  Molecular Genetics Research Center and the ^¶ Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan

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⁺,K⁺-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 -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 -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-138 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 we 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 Gln 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 SCH 28080. These results suggest that Glu-822 is one of important sites that bind with SCH 28080.

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