Chen, S. R. WayneTian, Xixi2013-04-152015-04-162013-04-152013Tian, X. (2013). Molecular Mechanism of Calcium Release Activation and Termination (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/26989http://hdl.handle.net/11023/602It is now well established that sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle is triggered via a mechanism termed Ca2+-induced Ca2+ release (CICR). It is unknown, however, how the SR Ca2+ release is terminated. The overall objective of the current study is to understand the mechanisms of activation and termination of SR Ca2+ release mediated by the cardiac ryanodine receptor (RyR2) and their roles in the pathogenesis of cardiac disease. A simple HEK293 cell expression system was established to assess the impact of RyR2 mutations on both Ca2+ release activation and termination. We found that mutations in the pore-forming region of RyR2 affected either the activation or the termination of Ca2+ release or both, indicating that the pore-forming region is a major determinant of Ca2+ release termination. Two additional regions, the N-terminal region and the calmodulin binding domain (CaMBD) of RyR2, were also found to be important for the termination of Ca2+ release. These results demonstrate that RyR2 itself controls the termination of Ca2+ release. RyR2 and its numerous modulators together form a macromolecular complex. Whether these modulators regulate the activation or termination of Ca2+ release is largely unknown. Our results show that both the 12.6 kDa FK506 binding protein (FKBP12.6) and calmodulin (CaM) facilitate the termination of Ca2+ release, but have little effect on Ca2+ release activation. On the other hand, cytosolic Ca2+ affects both the activation and termination of Ca2+ release, whereas CaM-dependent protein kinase II (CaMKII) only alters the activation of Ca2+ release. These data indicate that Ca2+ release termination is a common target of RyR2 regulation. RyR2 modulators are believed to exert their impact on channel function by inducing conformational changes in the channel, but these ligand-induced conformational changes and their functional correlation have yet to be demonstrated. Using a novel fluorescence resonance energy transfer (FRET)-based conformational probe, we assessed conformational changes in the “clamp” region near the corners of the square-shaped three-dimensional structure of RyR2 upon activation by a number of ligands. Our data demonstrate that conformational changes in the clamp region of RyR2 are ligand dependent, and suggest that RyR2 possesses multiple ligand-dependent gating mechanisms associated with distinct conformational changes. Enhanced luminal Ca2+ activation has been recognized as a common defect of RyR2 mutations linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). However, why some CPVT mutations are also associated with cardiomyopathies is unknown. Single cell luminal Ca2+ imaging revealed that RyR2 mutations that are associated with dilated cardiomyopathy (DCM) or arrhythmogenic right ventricular dysplasia type 2 (ARVD2) markedly reduced the threshold for Ca2+ release termination and increased the fractional Ca2+ release. In contrast, a RyR2 mutation associated with hypertrophic cardiomyopathy (HCM) increased the threshold for Ca2+ release termination and reduced the fractional Ca2+ release. These results provide the first evidence that abnormal fractional Ca2+ release attributable to aberrant termination of Ca2+ release is a common defect in RyR2-associated cardiomyopathies. Overall, these findings provide novel and important insights into the molecular basis and regulation of Ca2+ release activation and termination, and their roles in the genesis of cardiac arrhythmias and cardiomyopathies.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Biology--CellBiology--MolecularPhysiologyBiology--MolecularCardiac ryanodine receptor (RyR2)Conformational changesFluorescence resonance energy transfer (FRET)Intracellular calcium releaseCardiac arrhythmiasCardiomyopathiesdoctoral thesishttp://dx.doi.org/10.11575/PRISM/26989