Kang Po Wei, Woodbury Lucy, Angsutararux Paweorn, Sambare Namit, Shi Jingyi, Marras Martina, Abella Carlota, Bedi Anish, Zinn DeShawn, Cui Jianmin, Silva Jonathan R
bioRxiv. 2023 Jan 29:2023.01.28.526031. doi: 10.1101/2023.01.28.526031.
Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates. As CaM regulates several key cardiac ion channels, a mechanistic understanding of CaM variant-associated arrhythmias requires elucidating individual CaM variant effect on distinct channels. One key CaM regulatory target is the KCNQ1 (K 7.1) voltage-gated potassium channel that underlie the I current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function.
To observe how arrhythmia-associated CaM variants affect binding to KCNQ1, channel membrane trafficking, and KCNQ1 function.
We combine a live-cell FRET binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants effect on KCNQ1. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared to CaM wild-type over physiological Ca ranges. We further identify several CaM variants that affect KCNQ1 and I membrane trafficking and/or baseline current activation kinetics, thereby contextualizing KCNQ1 dysfunction in calmodulinopathy. Lastly, we delineate CaM variants with no effect on KCNQ1 function.
This study provides comprehensive functional data that reveal how CaM variants contribute to creating a pro-arrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function. This study provides a new approach to collecting details of CaM binding that are key for understanding how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels.
钙调蛋白（CaM）中的错义变异使患者易患死亡率高的心律失常。由于CaM调节多个关键的心脏离子通道，因此要从机制上理解与CaM变异相关的心律失常，就需要阐明单个CaM变异对不同通道的影响。一个关键的CaM调节靶点是构成I 电流基础的KCNQ1（K 7.1）电压门控钾通道。然而，关于CaM变异如何与KCNQ1相互作用或影响其功能，我们了解得相对较少。
观察与心律失常相关的CaM变异如何影响与KCNQ1的结合、通道膜运输以及KCNQ1功能。
我们结合活细胞荧光共振能量转移（FRET）结合测定、荧光运输测定和功能电生理学，来表征10多种与心律失常相关的CaM变异对KCNQ1的影响。我们鉴定出一种变异体（G114W），它与KCNQ1的结合严重减弱，但发现与野生型CaM相比，在生理钙浓度范围内，大多数其他CaM变异与KCNQ1的结合亲和力相似。我们进一步鉴定出几种影响KCNQ1和I 膜运输和/或基线电流激活动力学的CaM变异，从而将KCNQ1功能障碍与钙调蛋白病联系起来。最后，我们确定了对KCNQ1功能无影响的CaM变异。
本研究提供了全面的功能数据，揭示了CaM变异如何通过导致KCNQ1膜运输异常和电流传导异常来促成心律失常底物的形成。我们发现CaM对KCNQ1的调节并不一致，其影响从良性到功能显著丧失不等。本研究提供了一种收集CaM结合细节的新方法，这些细节对于理解CaM变异如何通过多个心脏离子通道的失调使患者易患心律失常至关重要。
