Study on the Structure and Mechanism Involved in Functional regulation of Gastric Proton Pump

胃质子泵功能调节的结构与机制研究

• 批准号: 13142202
• 负责人: ASANO Shinji
• 金额: $ 38.98万
• 依托单位: Ritsumeikan University (2004-2005)Toyama Medical and Pharmaceutical University (2001-2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13142202/
• 关键词: nanomachine; proton pump; active transport; gastric acid secretion; modeling; 能動輸送; モデリング; プロテアソーム; ユビキチン; ユビチキン

The gastric H^+,K^<+->ATPase is the proton pump responsible fbr gastric acid secretion. This pump consists of the catalytic α-and non-catalytic β-subunits. In this research project, we constructed stable cell lines expressing the gastric proton pump, and studied the finctional regulation mechanisms by site-directed mutagenesis in combination with homology modeling method.(1) We constructed three kinds of stable cell lines; the a-expressing cells, the [3-expressing cells, and the α+β-expressing cells. The α-subunit was retained in the intracellular compartment, and no cell surface expression was observed in the absence of the β-subunit. On the other hand, cell surface expression of the β-subunit was observed even in the absence of the α-subunit. Cell surface expression of the α-and β-subunits was observed in the α+β-expressing cells. The α+β-expressing cells represented rubidium (^<86>Rb) and proton transport activities, which were inhibited by inhibitors of prothn pump.(2) We studied t … More he binding site of acid pump antagonist (reversible inhibitor of the gastric proton pump), SCH 28080 by site-directed mutagenesis in combination with homology modeling method. When Tyr-801 on the M5 transmembrane segment of the a-subunit was replaced by alanine, the mutant (Y801A) showed 60 times lower sensitivity to SCH 28080 compared with that of wild type. The sensitivity to SCH 28080 was dependent on the bulkiness of this residue, indicating that the side chain of this residue is important for the interaction with this inhibitor. In the 3-D models of the E_2 and E_2P conformations of α-subunit, Tyr-801 is located at a top surface of a docking pocket of the luminal cavity, putative binding site of SCH 28080, surrounded by the TM1, TM4, TMS, TM6 and TM8 segments and the M5/M6, M7/M8 and M9/M10 luminal loops.(3) We studied the molecular mechanisms of the proton transfer by the gastric proton pump. Homology modeling and molecular dynamics calculation of the α-subunit indicate that protons are transferred from the basic amino acid residue in the cytoplasmic side to the acidic amino acid residue in the cation binding site via the polar amino acid residues. On the other hand, in the estimation of water transport activity in the hog GI gastric vesicles, we found that 1.8 mol of water were transported by 1 mol of ATP hydrolysis, and that this water transport was completely inhibited by an inhibitor ofprotonpump, SCH 28080. From these findings, we concluded that proton transport is carried out by charge movement between the polar amino acid residues in the cytoplamsic half, and that the oxonium ions (H_3O+) formed in the cation binding sites are transported in the luminal half of the membrane. Less

胃H^+，K^+-ATP酶是负责胃酸分泌的质子泵。该泵由催化性α亚基和非催化性β亚基组成。本研究构建了稳定表达胃质子泵的细胞系，并通过定点突变结合同源模建的方法研究了胃质子泵的功能调控机制。(1)我们构建了三种稳定的细胞系：α-表达细胞、β-表达细胞和α+β-表达细胞。α-亚基保留在细胞内区室中，在β-亚基不存在的情况下未观察到细胞表面表达。另一方面，即使在不存在α亚基的情况下也观察到β亚基的细胞表面表达。在α+β表达细胞中观察到α和β亚基的细胞表面表达。表达α+β的细胞表现出铷（<86>Rb）和质子转运活性，这些活性被蛋白泵抑制剂抑制。(2)我们研究了T ...更多信息 通过定点突变结合同源模建方法获得酸泵拮抗剂（胃质子泵可逆抑制剂）SCH 28080的结合位点。当α-亚基M5跨膜片段上的Tyr-801被丙氨酸取代时，突变体（Y801 A）对SCH 28080的敏感性比野生型低60倍。对SCH 28080的敏感性取决于该残基的体积，表明该残基的侧链对于与该抑制剂的相互作用很重要。在α亚基E_2和E_2P构象的三维模型中，Tyr-801位于SCH 28080的结合位点- (3)我们研究了胃质子泵质子转移的分子机制。同源模建和分子动力学计算表明，质子从胞质侧的碱性氨基酸残基通过极性氨基酸残基转移到阳离子结合位点的酸性氨基酸残基。另一方面，在估算猪胃肠道胃囊泡中的水转运活性时，我们发现1 mol ATP水解可转运1.8 mol水，而质子泵抑制剂SCH 28080可完全抑制这种水转运。根据这些结果，我们认为质子的运输是通过细胞质中极性氨基酸残基之间的电荷移动来进行的，而在阳离子结合位点形成的氧离子（H_3O+）则是在细胞膜的腔半部分进行的。少

项目成果

Kimura, T.: "Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system."Biochemistry. 42. 4771-4779 (2003)

Kimura, T.：“通过泛素/蛋白酶体系统对内质网中胃质子泵的数量和质量进行控制。”生物化学。

Kimura, T. et al.: "Mutational study on the roles of disulfide bonds in the β-subunit of gastric H^+, K^+-ATPase"Journal of Biological Chemistry. 277. 20671-20677 (2002)

Kimura, T. 等人：“胃 H^+、K^+-ATP 酶 β 亚基中二硫键作用的突变研究”生物化学杂志 277. 20671-20677 (2002)

Takahashi, Y. et al.: "Expression of ATPIALI, a non-gastric proton pump, in human colorectum"Japanese Journal of Physiology. 52. 317-321 (2002)

Takahashi, Y. 等人：“非胃质子泵 ATPIALI 在人结肠直肠中的表达”日本生理学杂志。

胃酸分泌プロトンポンプの機能と膜ダイナミクス

胃酸分泌质子泵的功能和膜动力学

• 发表时间: 2006
• 期刊: 膜 31(印刷中)
• 作者: Guo C.;et al.;浅野 真司
• 通讯作者: 浅野 真司

胃ベシクルにおける膜リン脂質転移の新規分子機構

胃囊泡膜磷脂转移的新分子机制

• 发表时间: 2005
• 期刊: 膜 30(6)
• 作者: Sakaya;N.;et al.;K.Taniguchi;Toyama-Sorimachi N et al.;森井 孫俊
• 通讯作者: 森井 孫俊