Liverpool Centre for Cardiovascular Science, Department of Cardiovascular and Metabolic Medicine, Institute of Life Course and Medical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom.
Department of Molecular Physiology and Cell Signalling, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom.
J Biol Chem. 2023 Jan;299(1):102777. doi: 10.1016/j.jbc.2022.102777. Epub 2022 Dec 8.
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-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 CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Ca1.2 Ca/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Ca1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Ca1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.
长 QT 综合征（LQTS）是一种人类遗传性心脏病，可导致危及生命的心律失常，包括心源性猝死。普遍存在的钙感应蛋白钙调蛋白（CaM）中的突变与 LQTS 相关，但这些突变导致心律失常的分子机制尚未完全阐明。在这里，我们使用包括蛋白质生物物理学、结构生物学、共焦成像和膜片钳电生理学在内的多学科方法，确定与疾病相关的 CaM 突变 E140G 对 CaM 结构和功能的影响。我们提出了新的数据，表明突变调节的 CaMKIIδ 激酶活性受损，酶自身磷酸化速率显著降低。我们报告了第一个与 LQTS 相关的 CaM 变体与 CaMKIIδ 肽复合物的高分辨率晶体结构，与 WT 复合物相比，该结构存在显著差异。此外，我们证明 E140G 突变显著破坏了 Ca1.2 Ca/CaM 依赖性失活，而心脏兰尼碱受体（RyR2）活性不受影响。此外，我们表明，与 LQTS 相关的突变改变了 CaM 的 Ca 结合特性、二级结构含量以及与兴奋-收缩偶联（CaMKIIδ、Ca1.2、RyR2）中关键伴侣的相互作用。总之，与 LQTS 相关的 CaM 突变 E140G 严重影响 CaM 的结构-功能关系及其对 CaMKIIδ 和 Ca1.2 的调节。这为 CaM 介导的心律失常的分子因素提供了重要的见解，其中 CaMKIIδ 起着核心作用。
