Searching responsible sites for the regulation of the L-type Ca channel through phosphorylation

寻找通过磷酸化调节 L 型 Ca 通道的负责位点

• 批准号: 11470011
• 负责人: YAMAOKA Kaoru
• 金额: $ 9.28万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470011/
• 关键词: L-type Ca channel; Magnesium; phosphorylation; Frog; Gunea-pig; Temperature-sensitive; 温度; 活性化酸素; 心筋

[Overview]The ultimate aim of this study is to identify responsible sites for the regulations of L-type Ca channels in the heart, such as intracellular Mg^<2+> block, or enhancement of channel activity accelerated by phsphorylation. As a result, we found that something is lacking to fully reconstruct channel activity only using L-type Ca channels with non-cardiac cells. Especcially, Mg^<2+> response was totally different from the behavior found in native cardiac cells. Depletion of intracellular Mg^<2+> led to the rapid wash out of channel activity in reconstructed systems, while in native cardiac cells, depletion of intracellular Mg^<2+> normally causes prominent increase in Ca channel current (I_<ca>). This indicates the requirement of extensive improvement of reconstruction systems regarding host cell conditions[Results]1. Increase in I_<ca> responding to reduction in intracellular Mg^<2+> was disturbed in guinea-pig cardiac cells, when temperature was below 28℃. This temperature dependency was not explained by simple thermodynamic activities.2. We identified whole length of cDNAs encoding α_1, β_<2C> and α_<2/δ> subunits of the L-type Ca channel in frog ventricular cells. They exhibited more than 80 % homology of those reported in rat or mouse. Co-expression of all subunits of α_1, β_<2C> and α_<2/δ> in BHK cells exhibited functional L-type Ca channel activity.3. Reconstructed Ca channels in BHK cells rapidly ran down, when cells were dialysed with log Mg^<2+> (below 10^<-5>M) patch solutions below 10^<-5>M. This is in contrast to data found in native cardiac cells. We need to search unkown proteins to accommodate the lacking of important regulations of the channel.

[综述]本研究的最终目的是确定心脏中L类钙通道的调控部位，如细胞内镁离子阻断或磷酸化加速的通道活性增强。结果我们发现，仅用L型钙通道与非心肌细胞结合来完全重建通道活动是不够的。具体来说，镁离子的反应与在天然心肌细胞中发现的行为完全不同。在重构的心肌细胞中，细胞内镁离子的耗竭导致通道活动的迅速消失，而在天然心肌细胞中，细胞内镁离子的耗竭通常会导致钙通道电流(I&lt；Ca&gt；)显著增加。[结果]1.当温度低于28℃时，豚鼠心肌细胞对细胞内镁离子浓度降低的反应受到干扰。这种温度依赖关系不能用简单的热力学活动来解释。我们在青蛙心室肌细胞中发现了编码L钙通道α_1、β_lt；2C&gt；和α_&lt；2/δ&gt；亚单位的全长cDNA。它们与在大鼠或小鼠身上报道的同源性超过80%。α_1、β_lt；2C&gt；和α_&lt；2/δ&gt；的所有亚基在BHK细胞中的共表达显示出功能性的L型钙通道活性。当用低于10^&lt；-5&gt；M的对数镁贴片溶液透析细胞时，BHK细胞中重建的钙通道迅速下降。这与在天然心肌细胞中发现的数据相反。我们需要寻找未知的蛋白质来适应通道中缺乏重要调控的情况。

项目成果

Yamaoka K, Yakehiro M, Yuki T, Fujii H, Seyama I: "Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes"Pflugers Arch. 440. 207-215 (2000)

Yamaoka K、Yakehiro M、Yuki T、Fujii H、Seyama I：“巯基试剂对青蛙心室肌细胞 L 型 Ca 通道调节系统的影响”Pflugers Arch。

Yamaoka K, Yuki T, Kawase K, Munemori M, Seyama I: "Temperature-sensitive intracellular Mg^<2+> block of L-type Ca"Am J Physiol. 282. H1092-H1101 (2002)

Yamaoka K、Yuki T、Kawase K、Munemori M、Seyama I：“温度敏感的细胞内 Mg^<2 > L 型 Ca 块”Am J Physiol。

Yakehiro M, Yamaoka K et al.: "Novel mechanism of blocking axonal Na^+ channels by three macrocyclic polyamine analogues and two spider toxins"Br J Pharmacol. 132. 63-72 (2001)

Yakehiro M、Yamaoka K 等人：“通过三种大环多胺类似物和两种蜘蛛毒素阻断轴突 Na 通道的新机制”Br J Pharmacol。

K. Yamaoka: "Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes"European Journal of Physiology-Pflugers Archiv. (in press).

K. Yamaoka：“巯基试剂对青蛙心室肌细胞 L 型 Ca 通道调节系统的影响”欧洲生理学杂志 - Pflugers Archive。

Yamaoka K, Yuki T, Kawase K, Munemori M, Seyama I: "Temperature-sensitive intracellular Mg^<2+> block of L-type Ca channels in cardiac myocytes"Am J Physiol. 282. H1092-H1101 (2002)

Yamaoka K、Yuki T、Kawase K、Munemori M、Seyama I：“心肌细胞中 L 型 Ca 通道的温度敏感细胞内 Mg^<2> 阻断”Am J Physiol。