The long term goal of the proposed project is to elucidate the molecular properties of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor), purified as a 30S protein complex comprised of four large (ryanodine receptor, Mr approximately 560,000) and four small (FK506 binding protein, Mr 12,600 subunits), and shown to be regulated by various endogenous effector molecules including Ca2+, Mg2+, ATP, calmodulin, protein kinases, NO and reactive oxygen species. The proposed research will use biochemical, electrophysiological and molecular biological methods to further characterize the structure and function of the cardiac CRC. An arterially perfused rabbit interventricular septal and papillary muscle preparation will be used to assess CRC post-translational modifications (phosphorylation, poly S-nitrosylation, oxidation) during myocardial ischemia and reflow. Functional consequences of these modifications will be assessed by determining, at the single channel level and in sarcoplasmic reticulum vesicles, the regulation of the cardiac CRC by protein phosphorylation, S-nitrosylation and oxidation, under normal and simulated ischemic conditions. Other effectors to be studied will include Ca2+ and calmodulin. These studies will test the hypothesis that CRC activity can be regulated. by sarcoplasmic reticulum luminal Ca2+ activation and Ca2+ inactivations sites and as such may play a role in cardiac ionotropy. The relationship between CRC protein structure and regulatory mechanisms will be addressed by expressing full-length normal and mutant and chimeric proteins transiently and stably. As our experiments progress we expect to gain new insights into the complex interaction of the cardiac CRC with its regulatory ligands and how these regulatory processes are altered in ischemic and post-ischemic heart. This information should suggest new directions of research to investigators attempting to minimize and overcome the risks of cardiac arrest in humans.
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