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

点击翻译按钮获取中文摘要

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