CPVT-associated calmodulin variants N53I and A102V dysregulate Ca2+ signalling via different mechanisms

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate development of strategies for future therapies.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

“…However, another research group showed a decreased activation of CaMKII in the presence of CaM variants N97S, D95V, and D129G ( 78 ). We previously investigated CPVT-associated CaM variants and showed that CaM-N53I did not affect the kinase activity of CaMKIIδ, whereas CaM-A102V significantly increased substrate phosphorylation levels by ∼60% ( 79 ). Interestingly, autophosphorylation levels between CaM-WT and CPVT-associated mutants were not significantly different, demonstrating that the increase in kinase activity observed for CaM-A102V cannot be attributed to enhanced autophosphorylation.…”

“…For the biophysical experiments, recombinant protein was obtained by cloning the human CaM gene into pE-SUMOPro as previously described ( 79 ). CaM variant E140G was generated by site-directed mutagenesis following the QuikChange protocol (Agilent Technologies).…”

“…CaM-WT and E140G variant were expressed and purified as previously described ( 79 ). In short, CaM pESUMOPro-Kan plasmids were transformed into Escherichia coli BL21(DE3) STAR and cultured in 2xYT media containing 100 μg/ml kanamycin.…”

Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

“…For biophysical and structural biology experiments. The sequence of human wild-type (WT) CaM was subcloned into the pE-SUMOPro-Kan vector (LifeSensors, USA) as previously described (Prakash et al, 2021(Prakash et al, , 2023. A series of site-directed mutagenesis (SDM) reactions were performed using the QuikChangeII kit (Agilent Technologies, USA), according to the manufacturer's recommendation, in order to generate LQTS-associated CaM mutants D95V, N97I and D131H.…”

Basic Research Approaches to Evaluate Cardiac Arrhythmia in Heart Failure and Beyond

“…The non-cardiac cell line human embryonic kidney 293 is used in cardiac research to explore the effect pathogenic variants have on the activity of specific ion channels. Pathogenic variants of ion channel genes can be transiently expressed in the cells to elucidate cellular mechanisms behind cardiac disease (Prakash et al, 2021). The reader is directed to Odening et al (2021) for a more detailed description on the role such cells play in cardiac electrophysiology research.…”