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Abstract-Arrhythmogenic cardiac ryanodine receptor (RyR2) mutations are associated with stress-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD). The causative mechanisms of RyR2 Ca 2ϩ release dysregulation are complex and remain controversial. We investigated the functional impact of clinically-severe RyR2 mutations occurring in the central domain, and the C-terminal I domain, a key locus of RyR2 autoregulation, on interdomain interactions and Ca 2ϩ release in living cells. Using high-resolution confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of interaction between fusion proteins corresponding to amino-(N-) and carboxyl-(C-) terminal RyR2 domains, we determined that in resting cells, RyR2 interdomain interaction remained unaltered after introduction of SCD-linked mutations and normal Ca 2ϩ regulation was maintained. In contrast, after channel activation, the abnormal Ca 2ϩ release via mutant RyR2 was intrinsically linked to altered interdomain interaction that was equivalent with all mutations and exhibited threshold characteristics (caffeine Ͼ2.5 mmol/L; Ca 2ϩ Ͼ150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca 2ϩ handling and interdomain interaction after channel activation that was absent in signals obtained from the central domain mutation. I domain-linked channel instability also occurred in intact RyR2 expressed in CHO cells and in HL-1 cardiomyocytes. These new insights highlight a critical role for mutation-linked defects in channel autoregulation, and may contribute to a molecular explanation for the augmented Ca 2ϩ release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca Key Words: ryanodine receptor Ⅲ mutations Ⅲ interdomain interaction Ⅲ arrhythmia T o date, 60 arrhythmogenic mutations in ryanodine receptor (RyR2) have been reported to underlie stress-or exercise-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD). 1-3 The mutations cluster in 3 discrete loci at the amino (N) terminus (15%), a central domain (25%), and at the carboxyl (C) terminus (60%). In a large number of mutations, their segregation into functionally distinct domains within the polypeptide and the complexity of the clinical phenotype may preclude a unifying mechanism of RyR2 Ca 2ϩ channel dysfunction. 1-4 Currently, our understanding of the correlation between mutation location, phenotypic manifestation, and the molecular basis of defective Ca 2ϩ release is incomplete. Several mechanisms underlying RyR2 channel dysfunction in SCD have been proposed, including altered sensitivity to luminal 5 and cytoplasmic Ca 2ϩ , 6 decreased Mg 2ϩ -dependent inhibition, 7 and FKBP12.6-dependent 7,8 and FKBP12.6-independent mechanisms. 9 -11 Despite persistent controversy surrounding the mechanistic basis of RyR2 dysregulation, there is a consensus that mutations functionally characterized to da...