Long QT syndrome-associated calmodulin mutations and their interactions with the Kv7.1 voltage-gated potassium channel

Abstract

Introduction Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+) dependent signalling. Its flexible structure allows CaM to bind and modulate many targets, including cardiac ion channels. Genotyping has revealed several CaM mutations associated with congenital disorders of heart rhythm, known as long QT-syndrome (LQTS). LQTS patients suffer from prolonged ventricular recovery times (QT-interval) which increases their risk of significant cardiac events. Loss of function KV7.1 mutations are the largest cause of LQTS, accounting for >50% of cases. CaM facilitates Ca2+-sensitivity to KV7.1 in producing IKs, Kv7.1 mutations which reduce CaM binding promote LQTS pathology. However, the effects of LQTS-associated CaM mutations on Kv7.1 function remain unknown. Purpose To determine the biophysical consequences of congenital LQTS-associated CaM mutations and how they alter modulation of Kv7.1 in producing the ventricular repolarising IKs current. Methods WT and mutant CaM proteins were recombinantly expressed and purified for biophysical characterisation. Using circular dichroism, secondary structures and thermostability of proteins were quantified. Isothermal titration calorimetry was used to quantitatively measure interactions between CaM proteins and binding sites of KV7.1 (Helix B). NMR was employed to study the conformations of target-bound WT and mutant proteins. Whole cell currents were determined using voltage clamp electrophysiology in HEK cells. Results Mutations significantly changed the thermostability and secondary structure distributions of CaM, and also caused site-dependent increases in susceptibility to protease digestion. CaM interacted with Helix B (KV7.1) via both Ca2+-dependent and independent mechanisms. Ca2+ dependent binding to Helix B was much higher affinity than Ca2+-independent binding, with >2000-fold reduction in dissociation constant measured. LQTS-CaM variants reduced Helix B affinity with the largest reductions found in EF-hand IV mutants. These mutants also adopted most distinct conformations when Helix B-bound. Calmodulation of the KV7.1 channel produced larger (IKs) currents without altering channel activation kinetics. IKs exhibited Ca2+-sensitivity, in response to increased cytosolic Ca2+, larger currents were generated. Modulation by CaM mutants reduced current density at systolic Ca2+-concentrations (1000 nM), within physiological time periods (0.35 s), revealing a direct QT-prolonging modulatory effect. Conclusions Provided here are mechanistic insights as to how LQTS-associated CaM variants contribute to electrical disease of the heart. Mutations in the highly conserved structure of CaM disrupt protein conformation and perturb complex formation with KV7.1. This results in aberrant Ca2+-sensitivity of Kv7.1, reducing IKs generation. This ultimately decreases the repolarisation capacity of cells and would extend the QT interval of myocytes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship

Calmodulin mutations can underlie the phenotype of long QT syndrome variant 1

“…The protein product of KCNQ1 (Kv7.1 α‐subunit), when co‐expressed with its β‐subunit, encoded by KCNE1 , forms the slowly activating channel that conducts I KS (Ackerman et al., 2011; Nerbonne & Kass, 2005). The first known subtypes of inherited LQTS, LQT1–3, which account for the majority of congenital LQTS, were distinguished by mapping to distinct chromosomes, with LQT1 mapping to chromosome 11 (Keating et al., 1991). LQT1, found to be due to mutations in the KCNQ1 gene, is the most common LQTS variant, representing 30–35% of all congenital LQTS (Ackerman et al., 2011).…”

“…In an article in this issue of The Journal of Physiology , McCormick et al. (2023) have used a multidisciplinary approach to investigate three previously reported CALM2 mutations: CALM2‐D95V, CALM2‐N97I and CALM2‐D131H. Structural, biochemical and electrophysiological experiments were carried out that showed that each of these mutations disrupts the binding of calmodulin to the KCNQ1 CTD which, in turn, resulted in a suppression of I KS channel activity, in a manner that resembles KCNQ1 loss of function due to LQT1 mutations.…”