Abstract 944: Tight Binding Of Calmodulin To The Cardiac Ryanodine Receptor May Reduce The Spontaneous Ca2+ Release Events Induced By Mutation-linked, Defective Inter-domain Interaction
Interaction between N-terminal1– 600 and central domains2000 –2500 of ryanodine receptor (RyR2), where many mutations have been found in patients with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2) or catecholaminergic polymorphic ventricular tachycardia (CPVT), was recently found to play a critical role in channel regulation. Using the domain peptide approach, here, we investigated the role of calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), on Ca2+ release kinetics. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using DP163–195, which harbors a human mutation site in ARVC (R176Q), as a site-directing carrier. DP163–195 mediated a specific MCA fluorescence labeling of central domain (60Kd) of RyR2. Addition of DP163–195 to the MCA-labeled SR competitively induced the domain unzipping between N-terminal and central domains, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In saponin-permeabilized cardiomyocytes, the addition of DP163–195 markedly increased the frequency of Ca2+ sparks (SpF; s−1·100μm−1:13.1±0.9, p<0.01), compared with normal cells (6.9±0.3). Addition of recombinant CaM (100nM), in the presence of KN-93 (CaMKII inhibitor), inhibited the DP163–195-induced increase in SpF (7.2±0.5, p<0.01). This effect of CaM was, however, abolished by co-addition of the antibody against the binding site of CaM within RyR2 (3583–3603) (SpF:12.9±0.7, p=ns), strongly suggesting that the binding of CaM to RyR2 corrects the abnormal Ca2+ release induced by DP163–195. The mutation made in the domain peptide, mimicking the same human mutation in ARVC (R176Q) abolished all of the effects that would have been produced by DP163–195. In conclusion, the mutation-linked defective inter-domain interaction between N-terminal and central domains within RyR2 (viz. domain unzipping) may increase spontaneous Ca2+ release, perhaps by weakening CaM-binding to RyR2. Restored binding of CaM to RyR2 may correct defective channel gating of the mutant RyR2.