Molecular Mechanism of Function of Cation Antiporters.

阳离子逆向转运蛋白功能的分子机制。

• 批准号: 07457015
• 负责人: SHIGEKAWA Munekazu
• 金额: $ 4.16万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457015/
• 关键词: Cation exchanger; Na^+; H^+ exchanger; Ca^<2+> exchanger

We have investigated the molecular mechanisms of the functions of Na^+/H^+ and Na^+/Ca^<2+> exchangers. We used cells transfected with their mutated cDNAs and obtained the follwoing results : 1) Previously, we showed that the human NHE1 isoform of Na^+/H^+ exchanger is activated by calmodulin (CaM), which binds to the cytoplasmic autoinhibitory domain of NHE1, thus preventing the expression of its inhibitory function, when intracellular Ca^<2+> is increased. We sytematically introduced mutations into the autoinhibitory domain of NHE1 and found that the interaction of this domain with its putative acceptor within NHE1 is highly sequence-specific. We also characterized other portions of the cytoplasmic domain of NHE1 and provided evidence that the most N-terminal subdomain (aa515-595) is essential for the maintenance of high pH_i sensitivity of NHE1 and that specific segments within this subdomain are involved in the NHE1 regulation by growth factors and cell ATP.In addition, we construc … More ted chimera among NHEs 1 to 4 and examined their responses to growth factors and hyperosmotic stress. 2) We have shown that the Na^+/Ca^<2+> exchanger (NCX1) in cardiac and smooth muscle is activated by vasoactive substances such as endothelin by a mechanism involving protein kinase C.We showed that the NCX1 protein is phosphorylated under such conditions and then explored whether phosphorylation of the NCX1 protein is required for the activation of Na^+/Ca^<2+> exchange. In addition, we newly developed an isothiourea derivative (KB-R7943) that specifically inhibits NCX activity. Interestingly, this agent preferentially inhibited the reverse mode ofNa^+/Ca^<2+> exchange. We then found that both this inhibitor and a classic cation inhibitor Ni^i extert differential effects on activities of different isoforms of NCX.We constructed chimera between NCX1 and NCX3 and used them for the determination of the site of action for these inhibitors. Since our kinetical study indicated that Ni^i competes with Ca^<2+> for the external Ca^<2+> transport site, this approach sould give important structural informatin about the ion transport pathway in NCX. Less

我们研究了Na^+/H^+和Na^+/Ca^<2+>交换剂作用的分子机理。我们使用转染了它们突变的cDNA的细胞，获得了以下结果：1）以前，我们发现当细胞内Ca^2+增加时，钙调素（CaM）可以激活Na^+/H^+交换器的人NHE 1亚型，CaM可以与NHE 1的胞质自抑制结构域结合，从而阻止其抑制功能的表达。我们系统地将突变引入NHE 1的自抑制结构域，发现该结构域与NHE 1内假定的受体的相互作用是高度序列特异性的。我们还鉴定了NHE 1胞质结构域的其他部分，并提供了证据，证明NHE 1的最N端亚结构域（aa 515 -595）是维持其高pH_i敏感性所必需的，并且该亚结构域中的特定片段参与了生长因子和细胞ATP对NHE 1的调节。 ...更多信息 在NHEs 1至4中检测嵌合体，并检测它们对生长因子和高渗应激的反应。2)我们已经证明，心脏和平滑肌中的Na^+/Ca^2+交换器（NCX 1）可被血管活性物质（如内皮素）激活，激活机制涉及蛋白激酶C。我们证明了NCX 1蛋白在这种条件下被磷酸化，然后探索了NCX 1蛋白的磷酸化是否是激活Na^+/Ca^2+交换所必需的。此外，我们新开发了一种异噻唑烷衍生物（KB-R7943），可特异性抑制NCX活性。有趣的是，该试剂优先抑制Na ^+/Ca^<2+>交换的反向模式。然后我们发现这种抑制剂和经典的阳离子抑制剂Ni^i对NCX不同亚型的活性有不同的影响，我们构建了NCX 1和NCX 3之间的嵌合体，并用它们来确定这些抑制剂的作用位点。由于我们的动力学研究表明Ni^i与Ca^<2 +>竞争外部Ca^<2+>转运位点，这种方法可能提供NCX中离子转运途径的重要结构信息。少

项目成果

Iwamoto, T.et al.: "Na^+/Ca^<2+> txchanger overexpreasion impairs calcuim signaling in fibribrasts:Inhibiton of〔Ca^<2+>〕rise at cell periphery and retadation of cell adbesion" Eur.J.Cell.Biol. (in press). (1998)

Iwamoto, T. 等人：“Na^+/Ca^<2+> txchanger 过度表达损害成纤维细胞中的钙信号传导：抑制细胞外周〔Ca^<2+>〕上升和细胞粘附的恢复”Eur.J .细胞.生物学（正在出版）（1998）。

Maeda, A., et al.: "Generatinof cell transfectants expressing cardiac calcium ion channel and calcium indicator protein aequorin." J.Biol.Chem.Anal.Biochem.242. 31-39 (1996)

Maeda, A. 等人：“表达心脏钙离子通道和钙指示蛋白水母发光蛋白的细胞转染子的生成。”

Uehara, A.et al.: "Whole-cell currents from a cloned cannine coridial Na^+/Ca^<2+> exchanger NCX1 overexpressed in a fiprobrast cell CCL39" Pflugers Arch.434. 335-338 (1997)

Uehara，A.等人：“来自在fiprobrast细胞CCL39中过表达的克隆犬科瑞迪尔Na ^ /Ca ^ 2 交换器NCX1的全细胞电流”Pflugers Arch.434。

Iwamoto,T.et al.: "Na^+/Ca^<2+> exchanger overexpression impairs Calcium sigmaling in fibribrasts:Inhibition of〔Ca^<2+>〕rise at cell periphery and retardation of cell adhesion" Eur.J.Cell Biol. (in press).

Iwamoto,T.et al.：“Na^+/Ca^<2+> 交换器过度表达损害成纤维细胞中的钙信号：抑制细胞外周〔Ca^<2+>〕上升并延迟细胞粘附”Eur.J .细胞生物学（正在出版）。

Wakabayashi, S.et al.: "Molecular tiysiology of vertebrate Na^+/H^+ exchangers" Physiol.Rev.77. 51-74 (1997)

Wakabayashi, S.et al.：“脊椎动物 Na^ /H^ 交换器的分子生物学”Physiol.Rev.77。