Abstract 2727: Mutation-linked Channel Disorder in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is Caused by Abnormally Tight Domain-Domain Interaction in RyR2
CPVT is known to be 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, has remained unresolved. Here, we examined the pathogenic role of domain-domain interaction 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 = 8), VT was observed after exercise with treadmill (Ex:8/8). In saponin-permeabilized KI cardiomyocytes, the frequency of Ca2+ sparks (SpF: s−1. 100μm−1) was more increased in response to cAMP (1μM) than in WT cardiomyocytes (KI:18.2±2.5 vs WT:12.3±1.3, p < 0.01), even though SR Ca2+ content was rather decreased in KI cardiomyocytes. Using canine cardiac SR, we could fluorescently label RyR2 with methylcoumarin acetamido (MCA), using either domain peptide (DP)2232–2266 or DP2232–2266mut; Ser is mutated to Leu (S2246L) like KI, as a site-directing carrier. After tryptic digestion of each MCA-labeled RyR2, the MCA-labeled fragment of RyR2 (49kD) was commonly identified as the fragment derived from the central region of RyR2, as detected by an antibody (Ab) raised against central region (Ab2132), but not by C-terminal Ab4963 and N-terminal Ab12. To further identify the partner domain of DP2232–2266 or DP2232–2266mut, we performed site-directed MCA labeling of RyR2 recombinant fragments (1– 610, 741–1260, 1245–1768, 1741–2270, 2234 –2750) using DP2232–2266 or DP2232–2266mut, as a site-directing carrier. Both DP2232–2266 and DP2232–2266mut specifically mediated MCA labeling only to the fragment 1741–2270. A quartz crystal microbalance technique (a highly sensitive mass-measuring technique) indeed showed specific binding of DP2232–2266 and DP2232–2266mut only to the fragment 1741–2270, and interestingly DP2232–2266mut (Kd = 0.16μM) showed much higher binding affinity than DP2232–2266 (Kd = 0.88μM).
Conclusions. One of the CPVT-type mutations in RyR2 (S2246L) may form abnormally tight domain-domain interaction between domain2232–2266 and another central domain (1741–2270), causatively induce hyper-activated channel gating, and hence trigger diastolic Ca2+ release and hence lethal arrhythmia.