Localization of Na^+ and K^+ pathways in Na^+, K^+-ATPase

Na^ 和 K^ 途径在 Na^ 、K^ -ATPase 中的定位

• 批准号: 10672053
• 负责人: HOMAREDA Haruo
• 金额: $ 2.05万
• 依托单位: Kyorin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672053/
• 关键词: Na^+, K^+-ATPase; Oligomycin; Active transport; Na^+ transport; K^+ transport; Ion pump; p-nitrophenyl phosphatase; pNPPase活性; Na^+輸送路; Ca^<2+>-ATPase; H^+,K^+-ATPase; キメラATPase

I have investigated the ion transport mechanism of Na^+, K^+-ATPase using oligomycin, which inhibits the Na^+ transport and the ATPase activity. I obtained the following findings. 1) Oligomycin inhibited only the Na^+ transport among the typical P-type-ATPases, i.e., Na^+, K^+-ATPase, H^+, K^+-ATPase and Ca^<2+>-ATPase. 2) Oligomycin had a binding domain in the N-terminal 200 amino acids of Na^+, K^+-ATPase α subunit, . This domain included the first and second transmembrane segments. The hydrophobic oligomycin, therefore, should bind somewhere in these segments, though I cannot identify its fine binding sites. From these results and other findings, which are an assumptive model of Na^+, K^+-ATPase and localization of the amino acids required for ion transport, I propose the following hypothesis ; the conformational change of the first-sixth transmembrane segments is accompanied with Na^+ transport. When Na^+ is transported, oligomycin disturb the change of the first and second segments, so that the transport is inhibited. When the conformation comes back to the original state, K^+ is transported. Oligomycin does not inhibit K^+ transport. According to this hypothesis, I have investigated oligomycin and Na^+ on K^+-dependent p-nitrophenyl phosphatase (pNPPase) activity, which is a partial reaction of Na^+, K^+-ATPase. When Na^+ : K^+ ratio is over10 : 1, oligomycin stimulated pNPPase activity. This result is unexpected, because it is expected that oligomycin increases the inhibitory effect of Na^+ on pNPPase activity. Under this experimental conditions, oligomycin increased the affinity for K^+ to Na^+, K^+-ATPase. This finding suggested that oligomycin affected the interaction between ions and K^+ binding (and/or Na^+ releasing site) at extracellular side.

本研究利用寡霉素抑制Na^+，K^+-ATP酶的Na^+转运和ATP酶活性，探讨了Na^+，K^+-ATP酶的离子转运机制。我获得了以下发现。1)寡霉素只抑制典型的P型ATP酶之间的Na^+转运，Na^+，K^+-ATP酶、H^+，K^+-ATP酶和Ca^<2+>-ATP酶。2)寡霉素在Na^+，K^+-ATP酶α亚基N端200个氨基酸处有一个结合域。该结构域包括第一和第二跨膜区段。因此，疏水性寡霉素应该结合在这些节段的某个地方，尽管我不能确定它的精细结合位点。根据这些结果和其他发现（即Na^+，K^+-ATP酶的假设模型和离子转运所需氨基酸的定位），我提出了以下假设：第一至第六跨膜片段的构象变化伴随着Na^+转运。在Na^+转运过程中，寡霉素干扰了第一、第二节段的变化，从而抑制了Na ^+的转运。当构象恢复到初始状态时，K^+被转运。寡霉素不抑制K^+转运。根据这一假设，我研究了寡霉素和Na^+对K^+依赖的对硝基苯基磷酸酶（pNPPase）活性的影响，pNPPase是Na^+，K^+-ATP酶的部分反应。当Na^+：K^+比值大于10：1时，寡霉素对pNPPase活性有促进作用。这一结果是出乎意料的，因为预计寡霉素会增加Na^+对pNPPase活性的抑制作用。在此实验条件下，寡霉素增加了Na^+，K^+-ATP酶对K^+的亲和力。这一结果表明寡霉素影响了细胞外离子与K^+结合位点（和/或Na^+释放位点）的相互作用。

项目成果

Homareda Haruo: "Oligomycin in “The Encyclopedia of Molecular Biology (未定)" John Willey & Sons,Inc., (1999)

Homareda Haruo：“《分子生物学百科全书 (TBD)》中的寡霉素”John Willey & Sons, Inc.，(1999)

Homareda, H: "Effect of oligomycin on interaction of Na^+ with Na^+, K^+-ATPase"Na/K-ATPase and Related ATPases. (Taniguchi, K and Kaya C., eds.). 451-454 (2000)

Homareda，H：“寡霉素对 Na+ 与 Na+、K+-ATP 酶相互作用的影响”Na/K-ATP 酶和相关 ATP 酶。

Haruo Homareda: "Effect of Oligomycin on Interaction of Na^+ with Na^+,K^+-ATPase in Na/K-ATPase and Related ATPases"Kazuya Taniguchi, Shunji Kaya. 771 (2000)

Haruo Homareda：“寡霉素对 Na/K-ATP 酶和相关 ATP 酶中 Na^ 与 Na^ ,K^ -ATP 酶相互作用的影响”Kazuya Taniguchi，Shunji Kaya。

Haruo Homareda: "Effect of oligomycin on interaction of Na^+ with Na^+ ,K^+-ATPase in "Na/K Pump & Related Pump""Elsevier Science B. V.. 4 (2000)

Haruo Homareda：“寡霉素对“Na/K 泵中 Na^ 与 Na^ ,K^ -ATPase 相互作用的影响”

Haruo Homareda: "Oligomycin in "Encyclopedia of Molecular Biology""Thomas E.Creighton. 3(2855) (1999)

Haruo Homareda：“《分子生物学百科全书》中的寡霉素”Thomas E.Creighton。