Arrhythmogenic Mutation-Linked Defects in Ryanodine Receptor Autoregulation Reveal a Novel Mechanism of Ca Release Channel Dysfunction

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 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 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 regulation was maintained. In contrast, after channel activation, the abnormal Ca release via mutant RyR2 was intrinsically linked to altered interdomain interaction that was equivalent with all mutations and exhibited threshold characteristics (caffeine 2.5 mmol/L; Ca 150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca 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 release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca release channel dysfunction in arrhythmia and SCD. (Circ Res. 2006;98:88-97.)