The overall goal of this project is to resolve the molecular mechanism of Excitation (E)-Contraction (C) Relaxation (R) coupling in normal and diseased muscles. Skeletal muscle-type E-C-R coupling appears to occur in several sequential steps. The proposed experiments aim to elucidate the mechanism for each of these steps. (1) Upon depolarization of the surface membrane (excitation of muscle cell), the activator domain of the dihydropyridine receptor II-III loop binds to, and its blocker domain dissociates from, the ryanodine receptor (RyR)/calcium release channel protein; T-tubule polarization reverses these processes (hypothesis). The investigator will test this model (and alternative models as well) by examining how the peptides corresponding to these domains (activator and blocker) compete with their in vivo counterparts during E-C coupling in triads and skinned or permeabilized fibers. To further define the mechanism, the pattern of peptide activation/inhibition will be correlated with the pattern of peptide binding. (2) The binding of these II-III loop domains to their specific binding sites on the RyR produces local conformational changes in the signal reception region. The investigator will localize the binding sites of these loop domains, and will monitor the dynamic conformational changes occurring in the signal reception region during E-C coupling using the site-specific fluorescence probe. (3) The conformational change in the signal reception region is coupled with a global conformational change in the RyR and calcium release (contraction). This process seems to involve interactions of a number of regulatory sub-domains within the RyR. Using a novel peptide probe technique, this investigator has uncovered several sub-domains involved in the regulation of the RyR calcium channel. Efforts will be made to uncover a sufficient number of sub-domains to deduce the global structure of the intra-molecular communication network. (4) Soon after the induction of calcium release (contraction), the calcium ATPase is activated to facilitate re-uptake of the released calcium (relaxation). The investigator hypothesizes that the communication between the RyR and the calcium ATPase is mediated by the transient changes occurring in the luminal calcium. This will be tested by correlating the time course of the changes in the activity of the calcium ATPase with those in the luminal calcium concentration. This program will likely resolve the basic mechanisms governing individual steps of E-C coupling, and will provide a better understanding of abnormal channel regulation in skeletal and cardiac muscles.
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