Coupled Gating of L-type Calcium Channels in Heart

心脏 L 型钙通道的耦合门控

基本信息

批准号：
8645692
负责人：
Luis F Santana
金额：
$ 38.22万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-20 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8645692
关键词：
1,2-diacylglycerol A kinase anchoring protein Action Potentials Address Adrenergic Agents Amino Acid Substitution Arrhythmia Autistic Disorder Calcineurin Calmodulin Cells Cellular Membrane Coupled Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Development Diglycerides Disease Electrophysiology (science)Event Frequencies Funding Gene Expression Genetic Transcription Goals Health Heart Heart failure Human Image Individual Knock-in Mouse Knockout Mice L-Type Calcium Channels Label Lead Light Location Long QT Syndrome Membrane Methods Modality Modeling Modification Molecular Biology Mus Muscle Cells Optics Phosphorylation Site Phosphotransferases Physiological Play Population Probability Public Health Research Resolution Role Sarcolemma Scaffolding Protein Shapes Signal Transduction Surface System Techniques Testing Timothy syndrome Total Internal Reflection Fluorescent Transgenic Mice Transgenic Organisms Translating Ventricular Wild Type Mouse Work adrenergic calcineurin phosphatase design heart function innovation interest mutant novel optogenetics patch clamp red fluorescent protein research study response transcription factor transcription factor NF-AT c3 treatment strategy

项目摘要

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.

描述（由申请人提供）：本申请中提出的工作将测试以下假设：由于这些通道的亚群与支架相互作用产生的信号微域，L-型 Cav1.2 通道活性沿着心室肌细胞的肌膜发生变化。蛋白质 AKAP150。初步数据表明，AKAP150 相关的 Cav1.2 通道在心室肌细胞动作电位波形、心律失常发生、兴奋-收缩 (EC) 耦合和兴奋-转录 (ET) 耦合方面发挥着关键作用。一个关键发现是 AKAP150 相关 Cav1.2 通道的小簇能够协调打开和关闭（“耦合门控”）。在表达突变型 Cav1.2 通道的细胞中，耦合门控事件的频率增加，该通道会导致患有长 QT 综合征 8 (LQT8) 的人类出现心律失常和自闭症。该项目有三个具体目标，旨在研究这些发现的生理和病理生理学意义。具体目标 1 是测试野生型 (WT) 和 Cav1.2-LQT8 通道的耦合门控需要 AKAP150 的假设。具体目标 2 是检验 WT 和 Cav1.2-LQT8 通道的耦合门控调节心室肌细胞中 EC 耦合的假设。最后，具体目标 3 是检验 AKAP150 缺失可防止 WT 和 LQT8 小鼠出现心律失常的假设。用于实现这些目标的方法包括膜片钳电生理学、光钳、光和化学诱导的激酶动态靶向细胞膜、光诱导的肾上腺素信号传导激活（即光遗传学）、共聚焦和 TIRF显微镜。实验将涉及专门为此项目创建的转基因小鼠，该小鼠在心室肌细胞中表达荧光标记的 Cav1.2-LQT8 通道，以及合作者创建的其他转基因、敲入和敲除小鼠。这项工作将产生有关生理和病理条件下 AKAP150 和 Cav1.2 通道控制心脏兴奋性、基因表达和 EC 耦合的机制的基本信息。