title: Arrhythmogenic mutation-linked defects in ryanodine receptor autoregulation reveal a novel mechanism of Ca2+ release channel dysfunction.
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 Ca2+ 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 Ca2+ 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 Ca2+ regulation was maintained. In contrast, after channel activation, the abnormal Ca2+ 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; Ca2+ >150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca2+ 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 Ca2+ release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca2+ release channel dysfunction in arrhythmia and SCD.
authors.affiliation: Department of Cardiology, Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff, UK. georgech@cf.ac.uk
authors.forename: Christopher H
authors.initials: CH
authors.lastname: George
authors.forename: Hala
authors.initials: H
authors.lastname: Jundi
authors.forename: Nicola
authors.initials: N
authors.lastname: Walters
authors.forename: N Lowri
authors.initials: NL
authors.lastname: Thomas
authors.forename: Robert R
authors.initials: RR
authors.lastname: West
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D000818:Animals; D001145:Arrhythmias, Cardiac / Q000209:etiology*; D016466:CHO Cells; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D016757:Death, Sudden, Cardiac / Q000209:etiology; D031541:Fluorescence Resonance Energy Transfer; D006706:Homeostasis; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D017180:Tachycardia, Ventricular / Q000209:etiology
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium