Abstract 576: Defective Domain-Domain Interaction Between C-Terminal and Central Regions of Ryanodine Receptor as a Critical Cause of Diastolic Ca2+ Spark and Delayed Afterdepolarization
Ryanodine receptor-2 (RyR2) central domain peptide, DPc10 (Gly2460-Pro2495), was found to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between N-terminal1– 600 and central domains2000 –2500. Using the domain peptide approach, here, we investigated how single point mutation at C-terminal region in CPVT influences on intracellular Ca2+ handling.
Methods and Results: Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (n= 5), then fluorescently labeled with methylcoumarin acetate (MCA) using DP4090 – 4123, a synthetic domain peptide corresponding to Pro-Glu (mutable domain in CPVT), as a site-directing carrier; the carrier was removed after MCA labeling. The DP4090 – 4123 mediated a specific MCA fluorescence labeling of RyR2. After tryptic digestion, the major MCA-fluorescently labeled fragment of RyR2 (150 kD) was detected by an antibody (Ab) against central region (Ab2163), but not by Ab2808, N-terminal (Ab10) and C-terminal region (Ab5030). Addition of DP4090 – 4123 to the MCA-labeled SR induced domain unzipping between C-terminal and its putative counter domains (EC50=30μM), as confirmed by a marked quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP4090 – 4123 induced a prominent SR Ca2+ leak (EC50=30μM). In DP4090 – 4123-incoporated cardiomyocytes (by protein delivery kit), diastolic Ca2+ spark frequency was markedly increased (4.0±1.0 s-1 100ìm-1, p<0.01), compared to normal cardiomyocytes (1.4±0.4 s-1 100ìm-1), and after addition of 100nM forskolin both Ca2+ wave and delayed afterdepolarization-mediated Ca2+ transient (DAD-CaT) were also frequently observed (but not in normal cardiomyocytes). All of these effects of DP4090 – 4123 were abolished either by addition of 10 μM tetracaine or by Asn-to-Lys mutation made in DP4090 – 4123, mimicking human CPVT mutation.
Conclusions: Domain-domain interaction between C-terminal and central domains (within 1200 –2850) seems to play an important role in channel gating of RyR2, and its defectiveness by point mutation may induce diastolic Ca2+ spark and delayed afterdepolarization that triggers lethal arrhythmia.