Browsing by Author "Tian, Xixi"
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Item Open Access Molecular Mechanism of Calcium Release Activation and Termination(2013-04-15) Tian, Xixi; Chen, S. R. WayneIt 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.