Arrhythmogenic Mutation-Linked Defects in Ryanodine Receptor Autoregulation Reveal a Novel Mechanism of Ca ²⁺ Release Channel Dysfunction

Abstract

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...

Familial Evaluation in Catecholaminergic Polymorphic Ventricular Tachycardia

“…Indeed, recent in vitro experiments suggest that RYR2 mutation location may direct the mode of calcium release dysfunction and accompanying channel instability and that mutations in the C-terminal channel-forming domain may lead to more postactivation channel instability. 26 Larger multicenter studies are needed to validate this association between mutation location and disease severity. In particular, analyzing whether mutation location could play a role …”

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“…However, some RyR2 mutants displayed an increased sensitivity to cytosolic Ca 2+ and caffeine. This is the case for S2246L, N4104K and R4497C mutations expressed in HEK293 cells or HL-1 cells, where it was also shown a gain-of-function RyR2 activity, while there was no change in SR Ca 2+ load (George CH., 2006). In some cases, differences in RyR2 mutants' response to agonists are closely dependent on the mutational locus.…”

“…The central domain (domain II: amino acids 2246-2534) contains an FKBP12.6 binding domain and it is supposed to interact with the Nterminus domain (zipping-unzipping). The C-terminus domain (domain III: amino acids 3778-4201 and domain IV: amino acids 4497 to 4959) contains the transmembrane regions of the Ca 2+ channel and an hydrophobic region which it is postulated to transduce cytoplasmic events to regulate the Ca 2+ pore forming domain (George CH., 2006). Only a small number of mutations are located in regions of RyR2 outside these portions.…”

Targeting Altered Calcium Physiology in the Heart: Translational Approaches to Excitation, Contraction, and Transcription

“…Indeed, Yano et al demonstrated correction of interdoamin interaction, associated with reduced Ca leak using the small molecule antioxidant (Edaravone) (57). Changes in interdomain interaction may underly several of the previously suggested mechanisms altering RyR function and may represent another autoregulatory mechanism disturbed in pathological RyR Ca release (13).…”

“…Indeed, action on RyR require FKBP12.6. However, whether adding back FKBP12.6 either via overexpression or small molecules represents a future tool to treat CPVT mutations regardless of their position in the large RyR protein remains to be elucidated (13). Another approach in modulating the RyR function focuses on conformational changes.…”