Molecular Basis of the Two Kinds of Opening Mode of Ryanodine Receptor / Calcium Relase Channel of the Sarcoplasmic Reticulum of Skeletal Muscle

骨骼肌肌浆网瑞尼定受体/钙释放通道两种开放方式的分子基础

• 批准号: 11670099
• 负责人: ENDO Makoto
• 金额: $ 2.37万
• 依托单位: Saitama Medical 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-11670099/
• 关键词: ryanodine receptor; Ca-induced Ca release (CICR); clofibric acid; dantrolene; photoaffinity labeling; reticulon 2C; calcium ion; excitation-contraction coupling; 筋小胞体; リアノジン受容体; Ca induced Ca release; ダントロレン; clofibric acid; アデニン化合物; プロカイン /

Ryanodine receptor(RyR)/Calcium release channel of the sarcoplasmic reticulum of skeletal muscle opens in two different modes, physiological mode and calcium-induced calcium release (CICR) mode. In order to find out the molecular basis of the two kinds of mode, we searched an agent that specifically affects one of the two modes, (1) We found that clofibric acid opens RyR in the mode similar to the physiological one. This is the first example of such an agent. (2) Since we previously found that dantrolene can discriminate the two kinds of mode under a certain temperature condition, we examined various derivatives of dantrolene and found that GIF-0082 specifically inhibits without interacting CICR mechanism. (3) Since GIF-0082 contains iodine in it and has a radical with which photoaffinity labeling can be carried out, we prepared radioactive GIF-0082 with <125>^I and tried to find out GIF-0082 binding protein(s). The redioligand did not bind RyR molecules, but it bound 23 kDa protein in the fragmented sarcoplasmic reticulum. We isolated and purified this protein and determined its amino acid sequence that was found to be similar to sequence of reticulon 2C of mouse and human. (4) There is also GIF-0082 binding protein in the soluble fraction of skeletal muscle cells, a 45 kDa protein. The binding of [<125>^I]-GIF-0082 to 23 kDa protein was inhibited by dantrolene but only at its high concentration (20-5O /μM), but that to 45 kDa protein was effectively inhibited by a low concentration of dantrolene. The 4S kDa protein, although soluble, can bind RyR under an appropriate condition. These results suggest that the above-mentioned proteins, especially the 45 kDa protein, might play an important role in the physiological calcium release process. We are currently investigating in this direction

骨骼肌肌浆网Ryanodine受体/钙释放通道（RyR/Calcium release channel）以两种不同的方式开放：生理模式和钙诱导钙释放（CICR）模式。为了找出这两种模式的分子基础，我们寻找了一种特异性影响其中一种模式的药物：（1）我们发现氯贝酸以类似于生理模式的方式打开RyR。这是这种代理的第一个例子。(2)由于我们先前发现丹曲林可以在一定温度条件下区分这两种模式，我们检查了丹曲林的各种衍生物，发现GIF-0082特异性抑制而不相互作用CICR机制。(3)由于GIF-0082中含有碘，并且具有可以进行光亲和标记的基团，因此我们用13 I制备放射性GIF-0082<125>，并试图找出GIF-0082结合蛋白。放射配体不结合RyR分子，但它结合23 kDa的蛋白质在破碎的肌浆网。我们分离纯化了该蛋白，并测定了其氨基酸序列，发现其与小鼠和人的reticulon 2C的序列相似。(4)在骨骼肌细胞的可溶性部分中也存在GIF-0082结合蛋白，一种45 kDa的蛋白。[<125>^I]-GIF-0082与23 kDa蛋白的结合被丹曲林抑制，但仅在其高浓度（20- 50/μM）下，但与45 kDa蛋白的结合被低浓度的丹曲林有效抑制。4S kDa蛋白虽然是可溶的，但在适当的条件下可以结合RyR。这些结果表明，上述蛋白，特别是45 kDa蛋白，可能在生理性钙释放过程中起重要作用。我们目前正在朝这个方向调查

项目成果

Endo, M., Ikemoto, T.: "Handbook of Experimental Pharmacology. 147. Pharmacology of Ionic channel Function : Activators and Inhibitors(分担分)"Springer-Verlag. 20 (2000)

Endo, M., Ikemoto, T.：“实验药理学手册。147。离子通道功能的药理学：激活剂和抑制剂”Springer-Verlag 20 (2000)。

Ikemoto, T., Endo, M.: "Properties of Ca^<2+> release induced by clofibric acid in the sarcoplasmic reticulum of mouse skeletal muscle fibres"British Journal of Pharmacology. 134. 719-728 (2000)

Ikemoto，T.，Endo，M.：“小鼠骨骼肌纤维肌浆网中氯贝酸诱导的Ca ^ 2 释放的特性”英国药理学杂志。

Ikemoto T., Hosoya T., Aoyama H., Kihara Y., Suzuki M. & Endo M: "Effects of dantrolene and its derivatives on the Ca^<2+> release in the sarcoplasmic reticulum of mouse skeletal muscle fibres"British Jounrnal of Pharmacology. 134. 729-736 (2001)

池本 T.、细谷 T.、青山 H.、木原 Y.、铃木 M.

Endo,M.& Ikemoto,T.(分担執筆): "Handbook of Experimental Pharmacology Pharmacology of Ionic Channel Function : Activators and Inhibitors"Springer-Verlag.. 662 (2000)

Endo, M. 和 Ikemoto, T.（撰稿人）：“离子通道功能实验药理学药理学手册：激活剂和抑制剂” Springer-Verlag.. 662 (2000)

Ikemoto T. & Endo M: "Properties of Ca^<2+> release induced by clofibric acid in the sarcoplasmic reticulum of mouse skeletal muscle fibres"British Jounrnal of Pharmacology. 134. 719-728 (2001)

池本 T.