Coupled Gating of L-type Calcium Channels in Heart

描述（由申请人提供）：本申请中提出的工作将检验以下假设：由于这些通道的亚群与支架蛋白AKAP 150相互作用产生的信号微区，L型Cav1.2通道活性沿心室肌细胞的肌膜沿着变化。初步数据表明，AKAP 150相关的Cav1.2通道在心室肌细胞动作电位波形的形成、心室肌发生、兴奋-收缩（EC）偶联和兴奋-转录（ET）偶联中起关键作用。一个关键的发现是，AKAP 150相关的Cav1.2通道的小簇能够经历协调的打开和关闭（“耦合门控”）。在表达突变Cav1.2通道的细胞中，偶联门控事件的频率增加，该突变Cav1.2通道导致长QT综合征8（LQT 8）患者的心律失常和自闭症。该项目有三个具体目标，旨在调查这些发现的生理和病理生理影响。具体目的1是检验AKAP 150是野生型（WT）和Cav1.2-LQT 8通道的偶联门控所需的假设。具体目标2是检验WT和Cav1.2-LQT 8通道的偶联门控调节心室肌细胞中的EC偶联的假设。最后，具体目标3是检验AKAP 150的缺失在WT和LQT 8小鼠中保护免受心律失常的假设。将用于实现这些目的的方法包括膜片钳电生理学、光学钳位、光诱导和化学诱导的激酶动态靶向细胞膜、肾上腺素能信号传导的光诱导激活（即，光遗传学）、共聚焦和TIRF显微术。实验将涉及专门为此项目创建的转基因小鼠，其在心室肌细胞中表达荧光标记的Cav1.2-LQT 8通道，以及合作者创建的其他转基因、敲入和敲除小鼠。这项工作将产生AKAP 150和Cav1.2通道控制兴奋性，基因表达和EC耦合在心脏的生理和病理条件下的机制的基本信息。 公共卫生相关性：本申请中概述的实验对公共卫生具有重要意义，因为它们研究了一种新的Ca 2+信号传导模式，该模式在正常条件和疾病下调节心脏的电和收缩活动。从拟议的工作所产生的信息将有助于了解控制心脏功能的机制，并可能导致心律失常和心力衰竭治疗的合理策略的发展。

DESCRIPTION (provided by applicant): The work proposed in this application will test the hypothesis that L-type Cav1.2 channel activity varies along the sarcolemma of ventricular myocytes due to signaling micro-domains created by a subpopulation of these channels interacting with the scaffolding protein AKAP150. Preliminary data suggest the novel concept that AKAP150-associated Cav1.2 channels play a critical role in shaping action potential waveform, arrhythmogenesis, excitation-contraction (EC) coupling, and excitation-transcription (ET) coupling in ventricular myocytes. A key discovery is that small clusters of AKAP150-associated Cav1.2 channels are capable of undergoing coordinated openings and closings ("coupled gating"). The frequency of coupled gating events increases in cells expressing a mutant Cav1.2 channel that causes arrhythmias and autism in humans with long QT syndrome 8 (LQT8). The project has three specific aims designed to investigate the physiological and pathophysiological implications of these findings. Specific aim 1 is to test the hypothesis that AKAP150 is required for coupled gating of wild type (WT) and Cav1.2-LQT8 channels. Specific aim 2 is to test the hypothesis that coupled gating of WT and Cav1.2-LQT8 channels modulates EC coupling in ventricular myocytes. Finally, Specific aim 3 is to test the hypothesis that loss of AKAP150 protects against arrhythmias in WT and LQT8 mice. The methods that will be used to achieve these aims include patch-clamp electrophysiology, optical clamping, light- and chemically-induced dynamic targeting of kinases to cellular membranes, light-induced activation of adrenergic signaling (i.e., optogenetics), confocal, and TIRF microscopy. Experiments will involve a transgenic mouse specially created for this project and that expresses fluorescently labeled Cav1.2-LQT8 channels in ventricular myocytes as well as other transgenic, knock in, and knock out mice created by collaborators. This work will generate fundamental information on the mechanisms by which AKAP150 and Cav1.2 channels control of excitability, gene expression, and EC coupling in the heart under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: The experiments outlined in this application have important implications to public health because they investigate a new Ca2+ signaling modality that regulates the electrical and contractile activity of the heart under normal conditions and disease. The information resulting from the proposed work will contribute to understanding of the mechanisms controlling the function of the heart and may lead to the development of rational strategies for the treatment of arrhythmias and heart failure.