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)模式开放。为了找出这两种模式的分子基础，我们寻找了一种特异性地影响这两种模式之一的药物：(1)我们发现氯纤酸以类似于生理模式的模式开放RyR。这是此类代理的第一个例子。(2)由于我们以前发现丹曲烯在一定的温度条件下可以区分这两种模式，所以我们研究了丹曲烯的各种衍生物，发现GIF-0082特异性地抑制了这两种模式，而没有相互作用的CICR机制。(3)由于GIF-0082中含有碘，并且含有可进行光亲和标记的自由基，因此我们用~lt；125&gt；^I制备了放射性GIF-0082，并试图找出GIF-0082结合蛋白(S)。该配体不能结合RyR分子，但能结合碎裂肌浆网中的23 kDa蛋白。我们分离纯化了该蛋白，并测定了其氨基酸序列，发现其与人和小鼠的网状2C序列相似。(4)骨骼肌细胞的可溶性部分也含有GIF-0082结合蛋白，约为45 kDa。丹曲烯仅在高浓度(20~(-50)O/μM)时抑制[-lt；125&gt；i]-GIF-0082与23 kDa蛋白的结合，而低浓度的丹曲烯能有效地抑制其与45 kDa蛋白的结合。4S kDa蛋白虽然是可溶的，但在适当的条件下可以与RyR结合。这些结果表明，上述蛋白，尤其是45 kDa蛋白，可能在生理钙释放过程中发挥重要作用。我们目前正在朝这个方向进行调查

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