Abstract 11212: Coupled Conformational Changes in the Local and Global Inter-Domain Interactions Trigger Diastolic Ca2+ Release and Lethal Arrhythmia in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
Background: CPVT is caused by single point mutation in cardiac ryanodine receptor (RyR2). However, the underlying mechanism, by which a single mutation causes drastic effects on the channel function, remains elusive. Here, we examined the role of inter-domain interactions within RyR2 in the mutation-linked channel disorder in human CPVT-associated RyR2S2246L /+ knock-in (KI) mice model.
Methods and Results: In all KI mice (n=6), VT was observed after exercise with treadmill. In saponin-permeabilized KI cardiomyocytes, cAMP (1µM)-dependent increase in the frequency of Ca2+ sparks (SpF: s-1•100µm-1) was more pronounced than in WT cardiomyocytes (KI:18.3±2.4 vs WT:12.6±1.8, p<0.01), even though SR Ca2+ content was significantly lower in the KI cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232-2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741-2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding in saponin-permeabilized WT cardiomyocytes (Kd: 0.09µM for DP2246mut, 0.15µM for DP2246). However, neither DP2246 nor 2246mut showed specific binding in KI cardiomyocytes, suggesting a tight interaction of the 2246 domain/K201-binding domain in KI RyR2. Fluorescence quench assays of inter-domain interactions within RyR2 further showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1-600)/central (2000-2500) domain pair (domain switch) in an allosteric manner. In KI mice, dantrolene (1µM) (but not K201: unable to bind domain1741-2270 in KI), by binding domain 601-620 within RyR2, corrected the mutation-caused domain unzipping, reduced the cAMP (1µM)-dependent increase in SpF (13.1±1.5, p<0.01), stopped the exercise-induced VT.
Conclusions: The CPVT-linked mutation of RyR2, S2246L, causes an abnormally tight local sub-domain/sub-domain interaction within the central domain involving the mutation site, which produces more global conformational change: namely, an aberrant domain unzipping of domain switch. This triggers diastolic Ca2+ release and lethal arrhythmia.
- © 2011 by American Heart Association, Inc.