Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase
Mutational analysis of putative SCH 28080 binding sites of the gastric H+,K+-ATPase
复制标题
DOI：
10.1074/jbc.272.28.17668
复制
发表时间：
1997-07-11
影响因子：
4.8
通讯作者：
Takeguchi, N
中科院分区：
文献类型：
--
作者：
Asano, S;Matsuda, S;Takeguchi, N
化合物 SCH 28080（2-甲基-8-（苯基甲氧基）咪唑并[1,2-a]吡啶-3-乙腈）可逆抑制胃和肾哇巴因不敏感的 H+,K+-ATP 酶，但不抑制结肠哇巴因敏感的 H+,H+-ATP 酶。通过使用功能表达系统和定点诱变，我们 分析了 SCH 28080 在胃 H+,K+-ATP 酶 α 亚基中的推定结合位点。之前报道，SCH 28080 是胃 H+,K+-ATP 酶特异性 K+ 位点抑制剂，其结合位点位于 α 亚基第一和第二跨膜片段之间的第一个细胞外环中； Phe-126和Asp-138是假定的结合位点，然而，我们发现第一胞外环中的所有突变体，包括Phe-126和Asp-ISS，保留了H+,K+-ATP酶活性和对SCH 28080的敏感性，因此，第一胞外环中的氨基酸残基不直接参与SCH 28080结合，也不是H+,K+-ATP酶不可缺少的 活动。黑麦在此提出了一个候选残基，该残基对于与第六跨膜片段中的 SCH 28080、Glu-822 的结合非常重要。 Glu-822 突变为 Asp 和 Ala（突变体分别称为 E822D 和 E822A）使 ATPase 活性分别降低至野生型酶的约 45% 和 35%，而 Gln 和 Leu 突变则消除了该活性，突变体 E822A 对 K+ 的亲和力明显低于野生型酶，表明 Glu-822 参与决定 K+ 的亲和力，突变型 E822D 对 SCH 28080 的敏感性比野生型酶低 8 倍，胃 H+,K+-ATPase 中 Glu-822 的对应物是 Na+,K+-ATPase 中的 Asp 和其他结肠哇巴因敏感的 H+,K+-ATPase，而对 SCN 28080，这些结果表明 Glu-822 是与 SCH 28080 结合的重要位点之一。
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.