Study on Intracellular Localization and Functional Regulation of the Gastric Proton Pump.

胃质子泵的细胞内定位和功能调节研究。

• 批准号: 13672276
• 负责人: ASANO Shinji
• 金额: $ 2.62万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672276/
• 关键词: Proton pump; Structure-function relationship; Site-directed mutagenesis; Active transport; Gastric acid secretion; Inhibitor; Proteasome; Ubiqutin; H^+,K^+-ATPase; ナノマシーン

The gastric H^+,K^+-ATPase is the proton pump responsible for 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 regulation mechanisms by site-directed mutahenesis.(1) We constructed three kinds of stable cell lines ; the α-expressing cells, the β-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 were observed in the α+β-expressing cells. The α+β-expressing cells represented rubidium (^<86>Rb) and proton transport activities, which were inhibited by proton pump inhibitors.(2) We also studied the quantity control mechanism of the gastric proton pump in the stable cell lines. The α- and (β-subunits were co-translationally inserted to the ER membrane to form the functional holoenzyme with a stoichiometry of 1 : 1. In this process, unassembled α-subunits were unstable in the cells. They were retained on the ER, modified with chains of polyubiquitin, and degraded by the proteasomes. On the other hand, unassembled β-subunits were more stable, and can travel to the cell surface by itself. We found that the number of functional proton pump, the α/β holoenzyme, was strictly regulated by ubiqutin/proteasome system.(3) The β-subunit contains three conserved disulfide bond in the ectodomain. We studied the role of these disulfide bonds on the function of the gastric proton pump by site-directed mutagenesis. We found that each disulfide bond was important for the correct assembly between the α- and β-subunits, and their cell surface delivery, and the maintenance of H^+,K^+-ATPase activity.

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，并通过定点突变研究了其调控机制。(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，可被质子泵抑制剂抑制。(2)并在稳定的细胞系中研究了胃质子泵的数量调控机制。将α-和β-亚基以1：1的化学计量比共选择性地插入ER膜形成功能性全酶。在这个过程中，未组装的α亚基在细胞中是不稳定的。它们保留在ER上，用聚泛素链修饰，并被蛋白酶体降解。另一方面，未组装的β亚基更稳定，可以自行移动到细胞表面。我们发现，功能质子泵（α/β全酶）的数量受到泛素/蛋白酶体系统的严格调控。(3)β亚基在胞外域含有三个保守的二硫键。我们通过定点突变研究了这些二硫键对胃质子泵功能的作用。我们发现，每个二硫键对于α和β亚基之间的正确组装、它们的细胞表面递送以及H^+，K^+-ATP酶活性的维持都很重要。

项目成果

Tabuchi Y. et al.: "Cibenzoline, an ATP-sensitive K^+ channel blocker, binds to the K^+-binding site from the cytoplasmic side of gastric H^+,K^+-ATPase."Br.J.Pharmacol.. 134. 1655-1662 (2001)

Tabuchi Y. 等人：“西苯唑啉，一种 ATP 敏感的 K^ 通道阻断剂，从胃 H^ ,K^ -ATP 酶的细胞质侧结合到 K^ - 结合位点。”Br.J.Pharmacol.. 134

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)

浅野真司: "消化管上皮組織機能とイオンチャネル"医学のあゆみ. 201. 856-860 (2002)

Shinji Asano：“胃肠上皮组织功能和离子通道”医学史 201. 856-860 (2002)。

Kimura, T. et al.: "Stable expression of gastric proton pump activity at cell surface"Journal of Biochemistry. 131. 923-932 (2002)

Kimura, T. 等人：“细胞表面胃质子泵活性的稳定表达”生物化学杂志。

Sakai H., et al.: "Molecular and pharmacological properties of inwardly rectifying K channels of human lung cancer cells."European Journal of Pharmacology. 435. 125-133 (2002)

Sakai H.等人：“人肺癌细胞内向整流K通道的分子和药理学特性。”欧洲药理学杂志。