Long QT syndrome-associated calmodulin variants D130V and E141K alter RyR2 and Ca_v1.2 calcium channel activity

 

Kirsty Wadmore¹, Caroline Dart¹ and Nordine Helassa¹

 

¹ Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 3BX, UK.

 

Introduction

Approximately 1:2000 births are affected by the life-threatening cardiac arrhythmia long QT syndrome (LQTS). In recent years, there has been an increasing link between LQTS and mutations in the protein calmodulin (CaM), a calcium (Ca²⁺) sensing protein. However, the causative mechanisms behind irregular heartbeats from these CaM variants remains elusive. CaM primarily regulates the activity of a diverse range of proteins, including the Ca²⁺ channels involved in cardiac muscle contraction: RyR2 and Ca_v1.2. The aim of this project is to investigate the consequences of LQTS-associated mutations D130V and E141K on CaM’s structure and function, to apprehend how these variants lead to the phenotype seen in LQTS patients. 

 

Methods

Circular dichroism was used to determine thermostability and secondary structure content of CaM variants. Isothermal titration colorimetry (ITC) was utilised to investigate the binding parameters of CaM variants for RyR2 and Ca_v1.2 (IQ and NSCaTE domains). Functional characterisation of the CaM variants on Ca²⁺ ion-channel activity, was investigated using Ca²⁺ imaging (RyR2) and whole-cell patch clamp electrophysiology (Ca_v1.2). 

 

Results

Thermostability of the LQTS-associated variants D130V and E141K, was not altered in comparison to CaM-WT. a-helical content was significantly reduced to approximately 40% in the CaM variants, compared to 64% in CaM-WT. We demonstrate that CaM-WT binds to RyR2_3581-3610 both in the absence (K_{d (apo)} = 5.6 ± 0.7 mM) and presence of Ca²⁺ (K_{d (calcium)} = 125 ± 2 nM). Apo-CaM D130V, interestingly, did not bind to RyR2_3581-3610. For both variants, Ca²⁺/CaM binding to RyR2_3581-3610 was reduced (up to 3-fold for E141K). We show that LQTS-associated variants altered the kinetic parameters of RyR2-mediated Ca²⁺ oscillations, including frequency and duration, using Ca²⁺ imaging. The K_d of Ca²⁺/CaM for Ca_v1.2 binding domains was 101 ± 7 nM and 1.2 ± 0.1 mM for Ca_v1.2-IQ_1665-1685 and Ca_v1.2-NSCaTE_48-68, respectively. Affinity to Ca_v1.2-IQ_1665-1685and Ca_v1.2-NSCaTE_48-68 was reduced up to 3-fold and 2-fold respectively for the LQTS-CaM variants. Using HEK-Ca_v1.2 cells and patch-clamp electrophysiology, we demonstrated an increase in Ca_v1.2 current density at particular test potentials and significantly reduced Ca²⁺-dependent inactivation (up to 7-fold), compared to CaM-WT. 

 

Conclusions

These data help establish that the secondary structure of CaM is altered by these LQTS-associated variants, as well as CaMs ability to interact with and modulate cardiac muscle contraction relevant ion channels (RyR2 and Ca_v1.2). Prolongation of the action potential is a key feature of LQTS and would be expected due to the defects seen in RyR2 and Ca_v1.2 inactivation. 

 

Acknowledgements

This work was supported by British Heart Foundation (FS/17/56/32925, FS/EXT/22/35014 to N.H.; FS/PhD/20/29025 to N.H. and K.W.) and BBSRC (BB/V002767/1 to C.D.).