Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-Mediated Ca2+ Release

The intracellular Ca2+ sensor calmodulin (CaM) regulates the cardiac Ca2+ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long-QT syndrome (LQTS). Here, we investigated the effect of CaM mutations causing CPVT (N53I), LQTS (D95V and D129G) or both (CaM N97S) on RyR2-mediated Ca2+-release. All mutations increased Ca2+-release and rendered RyR2 more susceptible to store-overload induced Ca2+-release (SOICR) by lowering the threshold of store Ca2+-content at which SOICR occurred, and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca2+-binding of the N-terminal and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM binding domain (CaMBD) of RyR2. The N53I mutation decreased the affinity of Ca2+ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V and D129G had little or no effect on Ca2+ binding to the N- domain, but markedly decreased the affinity of the C-domain for Ca2+. These results suggest that mutations D95V, N97S and D129G alter the interaction between CaM and the CaMBD, and thus RyR2 regulation. Since the N53I mutation minimally affected Ca2+ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations, and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca2+-saturated C-domain is constitutively bound to RyR2, and the N-domain senses increases in Ca2+ concentration, is proposed.