Arrhythmogenic calmodulin variants D131E and Q135P disrupt interaction with the L-type voltage-gated Ca2+ channel (Cav1.2) and reduce Ca2+-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_v1.2, a crucial component of the ventricular action potential and excitation–contraction coupling.

Methods

We used circular dichroism (CD), ¹H-¹⁵N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca²⁺ and interaction of Ca²⁺/CaM with Ca_v1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Ca_v1.2 activity was determined using HEK293-Ca_v1.2 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 Ca²⁺-bound state, D131E and Q135P exhibited reduced Ca²⁺ binding affinity, significant structural changes, and altered interaction with Ca_v1.2 domains (increased affinity for Ca_v1.2-IQ and decreased affinity for Ca_v1.2-NSCaTE). We show that the mutations dramatically impair Ca²⁺-dependent inactivation (CDI) of Ca_v1.2, which would contribute to abnormal Ca²⁺ influx, leading to disrupted Ca²⁺ 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.