Arrhythmogenic calmodulin variants D131E and Q135P disrupt interaction with the L‐type voltage‐gated Ca 2+ channel (Ca v 1.2) and reduce Ca 2+ ‐dependent inactivation

Abstract Aim Long QT syndrome (LQTS) and catecholaminergic polymorphism ventricular tachycardia (CPVT) are inherited cardiac disorders often caused by mutations in ion channels. These arrhythmia syndromes have recently been associated with calmodulin (CaM) variants. Here, we investigate the impact of the arrhythmogenic variants D131E and Q135P on CaM's structure–function relationship. Our study focuses on the L‐type calcium channel Ca v 1.2, a crucial component of the ventricular action potential and excitation–contraction coupling. Methods We used circular dichroism (CD), 1 H‐ 15 N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca 2+ and interaction of Ca 2+ /CaM with Ca v 1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Ca v 1.2 activity was determined using HEK293‐Ca v 1.2 cells (B'SYS) and whole‐cell patch‐clamp electrophysiology. Results Using a combination of protein biophysics and structural biology, we show that the disease‐associated mutations D131E and Q135P mutations alter apo/CaM structure and stability. In the Ca 2+ ‐bound state, D131E and Q135P exhibited reduced Ca 2+ binding affinity, significant structural changes, and altered interaction with Ca v 1.2 domains (increased affinity for Ca v 1.2‐IQ and decreased affinity for Ca v 1.2‐NSCaTE). We show that the mutations dramatically impair Ca 2+ ‐dependent inactivation (CDI) of Ca v 1.2, which would contribute to abnormal Ca 2+ influx, leading to disrupted Ca 2+ handling, characteristic of cardiac arrhythmia syndromes. Conclusions These findings provide insights into the molecular mechanisms behind arrhythmia caused by calmodulin mutations, contributing to our understanding of cardiac syndromes at a molecular and cellular level.