| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In order to clarify signal transduction mechanins between T tubule and sarcoplasmic reticulum (SR) in excitation-contraction coupling and CaィイD12+ィエD1 release mechanism from the SR, identification of the instinsic factors and analysis of their role were carried out to understand as a chain reaction in molecular complex system. It was found that CaィイD12+ィエD1 channel of SR was activated by DIDS, a stilbene derivative, and DIDS binded to 30k-Da protein of SR. As a result of biochemical analysis, 30k-Da protein was found to make molecular complex with calsequestrin (CSQ) and junctin. Partial amino acid sequence of 30k-Da was similar to that of ADP/ATP translocase (AAT) of mitochondra. From the analysis of marker enzymes and antibody, it was shown that this 30k-Da protein existed in SR. Antibody of AAT, atractyroside (inhibitor of AAT), and Cu-0-phenanthroline affected on CaィイD12+ィエD1 release from SR. Importance of this protein in the excitation-contraction coupling was recognized. It was succeeded in the cloning of the cDNA of rabbit skeletal muscle AAT and the expressed AAT in E. coli was bound to CSQ. From the above fact, it was confirmed that 30k-Da protein was identical with AAT. Secondly, in order to analyse the contribution of intrinsic factors in excitation-contraction coupling, the system which measured CaィイD12+ィエD1 release from SR by depolarization of T tubule (DICR) was established by using a membrane complex of T-tubule and SR (triad). Using this system, it was found that the CaィイD12+ィエD1 release was enhanced under the depolarization by cyclic ADP-ribose (cADPR) and IP3 which were intracellular messenger of CaィイD12+ィエD1 mobilization in other organs, but only cADPR enhanced CaィイD12+ィエD1 release by caffeine. Thus, cADPR and lP3 enhanced to open channel, but the activation site was different. In addition, it was found that cADPR worked cooperatively with calmodulin.
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