Disease-associated calmodulin mutations disrupt L-type Ca2+ channel (Cav1.2) activity and CaMKIIdelta phosphorylation in long QT syndrome

Abstract

Abstract Introduction Long QT Syndrome (LQTS) is a major inherited arrhythmia syndrome that can cause sudden cardiac death. Using genome sequencing in human patients, mutations in the ubiquitous calcium (Ca2+) sensor protein calmodulin (CaM) have been associated to LQTS. CaM is an ion channel regulator and can modulate the activity of the voltage-gated calcium channel (Cav1.2) and Ca2+/CaM-dependent protein kinase II (CaMKIIδ), involved in cardiac muscle contraction. However the molecular mechanism by which CaM mutations contribute to irregular heartbeats remains unclear. Methods Interaction of CaM proteins with Cav1.2 and CaMKIId synthetic peptides (Cav1.2-NSCaTE51–68, Cav1.2-IQ1665–1685, Cav1.2-C1627–1652, CaMKIIδ294–315,) was investigated using Isothermal Titration Calorimetry (ITC) and X-ray crystallography. Whole-cell patch clamp electrophysiology was used to determine the effect of CaM mutations on L-type Ca2+ currents and Ca2+-dependent inactivation (CDI). CaMKIIδ phosphorylation activity was determined by western blot and fluorescence kinase assay. Results Binding affinity of CaMKIId and Cav1.2 peptides to the LQTS-associated CaM variants was significantly reduced, up to 7-fold. Interestingly, the Cav1.2-IQ1665–1685 peptide showed a stronger binding, up to 2-fold, towards LQTS-CaM mutants. Crystal structures of Ca2+-CaM:CaMKIId294–315 showed structural alterations induced by LQTS associated mutations. In addition, we demonstrated that CaMKIIδ autophosphorylation and kinase activity can be significantly reduced by LQTS-associated CaM mutants. Electrophysiological examination of Cav1.2 function revealed that CaM mutations significantly impaired channel CDI, without affecting the voltage dependence of activation and inactivation. Conclusions These data demonstrate a strong correlation between LQTS-associated CaM mutations and Cav1.2 activity. We provide molecular insights into the diverse factors contributing to CaM-mediated arrhythmias. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship