Molecular mechanism of skeletal muscle excitation-contraction coupling

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We carried out a series of experiments in order to elucidate molecular mechanism of excitationcontraction coupling,in particular the coupling between T-tubule and sarcoplasmic reticulum(SR). We have developed a two-dimensional native gel electrophoresis method of SR proteins for the analysis of chemical modification of ryanodine receptors(RyR). Using chemically cleavable cross-linking agents,we cross-linked partially purified RyR samples and were able to detect cross-linked products corresponding to dimer,trimer and tetramer of RyR. Furthermore,there seems to be a larger cross-linked product which might contain RyR-associated proteins. Limited proteolysis of RyR by m-calpain in situ in skinned muscle fibers resulted in the cleavage-of-1300 amino acids from the N-terminus. This caused potentiation of Ca^<2+> release without obvious effect on the Ca^<2+> sensitivity,suggesting the presence of functional domains within the molecule. As another way to locate functional domains on the molecule,we carried out cross-linking between RyR and doxorubicin,a potentiator of the Ca^<2+> release channel. So far the binding site of doxorubicin seems to be located on the C-terminus half of the molecule. To gain further insight into E-C coupling,both biochemical and physiological experiments have to be performed on the same preparation. Since physiological experiments on mammalian fibers are difficult,we now stated determination of the sequence of RyR complementary DNA from frog skeletal muscle.