Investigation of Regulation Mechanisms of Gastric Proton Pump

胃质子泵调节机制的研究

• 批准号: 11672165
• 负责人: ASANO Shinji
• 金额: $ 2.3万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672165/
• 关键词: proton pump; H^+, K^+-ATPase; gastric acid secretion; active transport; ion recognition; site-directed mutagenesis

H^+, K^+-ATPase is the proton pump responsible for gastric acid secretion. This pump consists of a catalytic α-subunit and a non-catalytic β-subunit. We studied the functional sites on the α- and β-subunits by using site-directed mutagenesis and chimera construction.(1) We identified Glu-345 on the 4th transmembrane (M4) segment of the α-subunit as the K^+-recognition site which is involved in the K^+-dependent dephosphorylation step of H^+, K^+-ATPase.(2) We found that the lysine/glycine cluster structure located on the N-terminus of the α-subunit is not directly involved in the function of pump activity as an ion filter.(3) We found that the first extracellular loop (loop 1) of the α-subunit is not the binding site of proton pump inhibitor, SCH 28080. We newly prepared a series of chimeric pumps between H^+, K^+-ATPase and Na^+, K^+-ATPase α-subunits, and found that one of the chimeras was inhibited by both SCH 28080 and ouabain, indicating that the binding sites of these two inhibitors are separate.(4) We constructed chimeric β-subunits between H^+, K^+-ATPase and Na^+, K^+-ATPase, and studied the compatibility of these β-subunits for the functional expression of proton pump. The whole cytoplasmic and transmembrane segments were interchangeable between H^+, K^+- and Na^+, K^+-ATPases. We newly identified the "^<76>QLK^<79>S" motif in the extracellular segment of H^+, K^+-ATPase β-subunit which is specific for the functional expression of H^+, K^+-ATPase.(5) H^+, K^+-ATPase β-subunit contains seven suger chains. We progressively removed these sugar chains from the β-subunit, and studied the roles of sugar chains on the function of proton pump. We found that each sugar chain is not involved in the catalytic activity, α/β assembly and the cell surface delivery of the α- and β-subunits. We also found that the cell surface delivery mechanism is more dependent on the sugar chains than the expression of H^+, K^+-ATPase activity and α/β assembly.

H^+，K^+-ATP酶是负责胃酸分泌的质子泵。该泵由催化α亚基和非催化β亚基组成。我们通过定点突变和嵌合体构建研究了α-和β-亚基上的功能位点。(1)我们鉴定了α亚基第4跨膜（M4）片段上的Glu-345作为K^+识别位点，参与H^+，K^+-ATP酶的K^+依赖性去磷酸化步骤。(2)我们发现位于α亚基N端的赖氨酸/甘氨酸簇结构并不直接参与作为离子过滤器的泵活性功能。(3)我们发现α-亚基的第一个胞外环（loop 1）不是质子泵抑制剂SCH 28080的结合位点。我们新制备了一系列H^+，K^+-ATP酶和Na^+，K^+-ATP酶α亚基之间的嵌合泵，发现其中一个嵌合泵同时被SCH 28080和哇巴因抑制，表明这两种抑制剂的结合位点是分开的。(4)我们构建了H^+，K^+-ATP酶和Na^+，K^+-ATP酶的嵌合β亚基，并研究了这些β亚基对质子泵功能表达的相容性。H^+，K^+-ATP酶和Na^+，K^+-ATP酶的整个胞质和跨膜片段可以互换。我们<76><79>在H^+，K^+-ATP酶β亚基的胞外片段中新发现了“^ QLK^ S”基序，该基序对H^+，K^+-ATP酶的功能表达具有特异性。(5)H^+，K^+-ATP酶β亚基含有7条糖链。我们将这些糖链从β-亚基中逐步去除，并研究了糖链对质子泵功能的作用。我们发现每个糖链不参与α和β亚基的催化活性、α/β组装和细胞表面递送。我们还发现，细胞表面的转运机制更多地依赖于糖链，而不是H^+，K^+-ATP酶活性和α/β组装的表达。

项目成果

Shinji Asano: "Chimeric Domain Analysis of the Compatibility between H^+, K^+-ATPase and Na^+, K^+-ATPase β-Subunits for the Functional Expression of Gastric H^+, K^+-ATPase."Journal of Biological Chemistry. 274(32). 22257-22265 (1999)

Shinji Asano：“针对胃 H^+、K^+-ATP 酶功能表达的 H^+、K^+-ATP 酶与 Na^+、K^+-ATP 酶 β 亚基之间的相容性嵌合结构域分析。”生物化学杂志274(32)。

Asano, S.et al.: "The Role of Carbohydrate Chains of the β-subunit on the Functional Expression of Gastric H^+, K^+-ATPase."J.Biol.Chem.. 275. 6848-6854 (2000)

Asano, S. 等人：“β 亚基碳水化合物链对胃 H^+、K^+-ATP 酶功能表达的作用。J.Biol.Chem.. 275. 6848-6854 (2000) ）

浅野真司: "胃プロトンポンプの構造と機能"生化学. 72(1). 48-51 (2000)

Shinji Asano：“胃质子泵的结构和功能”生物化学72（1）（2000）。

Asano, S.et al.: "A chimeric H^+, K^+-ATPase Inhibitable with Both Ouabain and SCH 28080."Elsevier Science, Na/K-ATPase and Related ATPases. 163-168 (2000)

Asano, S.et al.：“A chimeric H^ , K^ -ATPase Inhibit with Both Ouabain and SCH 28080.”Elsevier Science，Na/K-ATPase 和相关 ATPase。

浅野真司: "胃プロトンポンプの構造と機能"生化学. 72. 48-51 (2000)

浅野真司：“胃质子泵的结构和功能”生物化学72. 48-51 (2000)。