title: Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase.
abstract: 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 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-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.
authors.affiliation: Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Asano
authors.forename: S
authors.initials: S
authors.lastname: Matsuda
authors.forename: Y
authors.initials: Y
authors.lastname: Tega
authors.forename: K
authors.initials: K
authors.lastname: Shimizu
authors.forename: S
authors.initials: S
authors.lastname: Sakamoto
authors.forename: N
authors.initials: N
authors.lastname: Takeguchi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005973:Glutamine / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D054328:Proton Pump Inhibitors; D016415:Sequence Alignment; D013270:Stomach / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D054328:Proton Pump Inhibitors; C035235:Sch 28080; D005973:Glutamine; D001224:Aspartic Acid; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium