Coupled Gating of L-type Calcium Channels in Heart

描述(申请人提供)：本申请中提出的工作将测试这样的假设，即L类型的Cav1.2通道的活性沿着心室肌细胞的肌膜变化，这是由于这些通道的亚群与支架蛋白AKAP150相互作用而产生的信号微域。初步数据表明，AKAP150相关的Cav1.2通道在形成心肌细胞动作电位波形、心律失常发生、兴奋-收缩(EC)偶联和兴奋-转录(ET)偶联中起关键作用。一项关键发现是，与AKAP150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”)。在表达突变的Cav1.2通道的细胞中，耦合门控事件的频率增加，该突变通道会导致长QT综合征8(LQT8)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学和病理生理学意义。具体目的1是验证野生型(WT)和Cav1.2-LQT8通道的耦合选通需要AKAP150的假设。具体目的2是验证WT和Cav1.2-LQT8通道的耦合门控调节心肌细胞内皮细胞偶联的假说。最后，具体目标3是在WT和LQT8小鼠身上验证AKAP150缺失对心律失常具有保护作用的假设。用于实现这些目标的方法包括膜片钳电生理学、光学钳制、光诱导和化学诱导的细胞膜激活酶的动态靶向、光诱导肾上腺素能信号的激活(即光遗传学)、共聚焦和TIRF显微镜。实验将包括一只专门为该项目创造的转基因小鼠，它在心室肌细胞中表达荧光标记的Cav1.2-LQT8通道，以及其他由合作者创造的转基因、敲入和敲除小鼠。这项工作将产生关于AKAP150和Cav1.2通道在生理和病理条件下控制心脏兴奋性、基因表达和EC偶联的机制的基本信息。

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.