The overall goal of this project is to continue the Investigator's work on the molecular mechanism of excitation- contraction coupling in normal and diseased cardiac muscle focusing on the role of the ryanodine receptor. The importance of the ryanodine receptor (RyR) is shown by the fact that mutations in the RyR lead to various cardiac disorders. In congenital cardiac diseases, mutations are localized in the four regions of each subunit of the RyR tetramer. The Investigator has shown that two of these regions form a Domain Switch. Their zipping closes the calcium channel, while unzipping opens it; incomplete domain zipping in the resting state causes calcium channel dysfunctions, resulting in heart failure (arrhythmias and hypertrophy). The investigator's recent data suggest that the same type of channel regulation is operating also within the other two regions, pointing to their involvement in the channel gating mechanism, serving as Gate Manager. It appears that the Domain Switch and the Gate Manager are coupled by mediation of yet another set of domains, Transmitter. Thus, defect of the inter-domain interaction within each of these regions of the receptor will cause RyR-linked cardiac diseases. To elucidate the spatial relationship of interacting domains in each of these three key regions, the Investigator plans to perform site-directed fluorescence labeling of the target domain using peptides as a site- direction carrier and identify the sites of domain-domain interaction using the peptide mapping and mass spectroscopic techniques. To establish the role of these domains in channel regulation, (i) a local conformational change induced by domain peptides or site-specific antibodies in these domains will be detected using the protein-bound fluorescence probe, and (ii) the resultant activation of the channel activity will be followed by assays of [3H]ryanodine binding, single channel conductance and intra-cellular Ca2+ transients, to determine correlation between (i) and (ii). Further, in collaboration with researchers working on model systems of cardiac arrhythmias and hypertrophy, the Investigator will examine how the defective domain- domain interaction in the in vivo RyR causes disease states, and how one can pharmacologically control the domain interaction and channel function. The new information derived from this program will permit a better understanding of the pathogenic mechanisms of calcium channel dysfunction occurring in some cardiac diseases and will offer new opportunities for therapeutic interventions. The ryanodine receptor is a calcium channel protein playing a central role in the beat to beat regulation of the cellular calcium level in cardiac muscle. Defective channel function causes various heart diseases, such as ventricular tachycardia (sudden cardiac death) and hypertrophy. This program is designed to identify molecular mechanisms (inter-domain interactions in particular), whose defect causes channel dysfunction, and to develop new methods of therapeutic treatment of these disorders. ? ? ? ? ?
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