Arrhythmia-Associated Calmodulin E105A Mutation Alters the Binding Affinity of CaM to a Ryanodine Receptor 2...

Calmodulin (CaM) is a small, multifunctional calcium (Ca²⁺)-binding sensor that binds and regulates the open probability of cardiac ryanodine receptor 2 (RyR2) at both low and high cytosolic Ca²⁺ concentrations. Recent isothermal titration calorimetry (ITC) studies of a number of peptides that correspond to different regions of human RyR2 showed that two regions of human RyR2 (3584-3602aa and 4255-4271aa) bind with high affinity to CaM, suggesting that these two regions might contribute to a putative RyR2 intra-subunit CaM-binding pocket. Moreover, a previously characterized de novo long QT syndrome (LQTS)-associated missense CaM mutation (E105A) which was identified in a 6-year-old boy, who experienced an aborted first episode of cardiac arrest revealed that this mutation dysregulates normal cardiac function in zebrafish by a complex mechanism that involves alterations in both CaM-Ca²⁺ and CaM-RyR2 interactions. Herein, to gain further insight into how the CaM E105A mutation leads to severe cardiac arrhythmia, we generated large quantities of recombinant CaM^WT and CaM^E105A proteins. We then performed ITC experiments to investigate and compare the interactions of CaM^WT and CaM^E105A mutant protein with two synthetic peptides that correspond to the two aforementioned human RyR2 regions, which we have proposed to contribute to the RyR2 CaM-binding pocket. Our data reveal that the E105A mutation has a significant negative effect on the interaction of CaM with both RyR2 regions in the presence and absence of Ca²⁺, highlighting the potential contribution of these two human RyR2 regions to an RyR2 CaM-binding pocket, which may be essential for physiological CaM/RyR2 association and thus channel regulation.