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Ryanodine受体(RyR)/钙释放通道以生理模式和钙诱导钙释放（CICR）模式打开。为了找出这两种模式的分子基础，我们寻找了一种专门影响这两种模式之一的药物，(1)我们发现纤维酸打开RyR的模式与生理模式相似。这是此类代理的第一个示例。(2)由于我们之前发现丹曲洛烯在一定的温度条件下可以区分这两种模式，所以我们检测了丹曲洛烯的各种衍生物，发现GIF-0082特异性抑制CICR机制，但不相互作用。(3)由于GIF-0082中含有碘，并且有可以进行光亲和标记的自由基，因此我们制备了<125>^I的放射性GIF-0082，并试图寻找GIF-0082的结合蛋白。该再配体不结合RyR分子，但它结合了碎片化肌浆网中的23kda蛋白。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网状蛋白2C序列相似。(4)骨骼肌细胞的可溶部分还存在一个45 kDa的GIF-0082结合蛋白。[<125>^I]-GIF-0082与23 kDa蛋白的结合受到丹曲洛烯的抑制，但仅在高浓度（20- 50 μM）下，而对45 kDa蛋白的结合受到低浓度丹曲洛烯的有效抑制。4S kDa蛋白虽然是可溶性的，但在适当的条件下可以与RyR结合。以上结果提示，上述蛋白，尤其是45 kDa蛋白，可能在生理性钙释放过程中发挥重要作用。我们正在朝这个方向调查

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