Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe arrhythmic condition caused by mutations in the proteins involved in Ca 2+ handling in the heart. To date, more than 100 CPVT mutations have been mapped to the cardiac ryanodine receptor (RyR2). These mutations are localized in the hotspots of the RyR2, including the N-terminal/central domains and the transmembrane region. We used our system of Flp-In T-Rex-293 SERCA2a stable line recombinantly expressing wild-type or mutant RyR2s, SERCA2a, and ER Ca 2+ sensor R-Cepia1er to characterize the functional impact of the mutations localized at the B8-B9 loop of RyR2 N-terminal domain A. We investigated two intersubunit interfaces of the B8-B9 loop with the neighboring subunit domains—one involving the N-terminal domain B and another involving the central domain. We found that the disruption of the interface between the B8-B9 loop and the central domain by the artificial K174A mutation caused a severe gain-of-function phenotype for RyR2 in basal conditions, whereas the disruption of the interface between the B8-B9 loop and the N-terminal domain B by the mutation K167A caused a minimal impact. We are investigating the molecular bases of these mutations using ab initio protein structures and molecular modeling. By comparing previously discovered CPVT (R169Q, A165D) and LQTS (S166C) mutations, we found that the severity of Ca 2+ leak increased as we approached the base of the B8-B9 loop proximal to the interface with the central domain. In contrast, the mutations proximal to the interface with N-terminal domain B were less severe. Thus, we can suggest that the disruption of the intersubunit interface between the B8-B9 loop and the opposing central domain by mutations in the loop could be a major driver for the RyR2 gain-of-function phenotype during CPVT.
Nikolaienko et al. (Sun,) studied this question.