Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation
O. Prakash, Nitika Gupta, Amy Milburn, Liam McCormick, Vishvangi Deugi, Pauline Fisch, Jacob Wyles, N. Lowri Thomas, S.V. Antonyuk, Caroline Dart, Nordine Helassa
Abstract
Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca 2+ -sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKII kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first highresolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKII peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Ca v 1.2 Ca 2+ /CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca 2+ -binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKII, Ca v 1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKII and Ca v 1.2. This provides a crucial insight into the molecular factors contributing to CaMmediated arrhythmias with a central role for CaMKII.