Persistent Calcium Sparklets in Arterial Smooth Muscle

描述(申请人提供)：本申请中的实验将测试电压门控L类型CaV1.2通道簇能够经历协调开放(“耦合门”)，放大钙离子内流到动脉平滑肌的假设。一个重要的发现是，钙离子通过CaV1.2偶联通道内流在动脉肌细胞的兴奋-收缩(EC)偶联和兴奋-转录(ET)偶联中起着关键作用。初步数据表明，CaV1.2通道与锚定蛋白AKAP150的结合对于这些细胞中的偶联门控活动是必要的。观察到高血压期间动脉平滑肌CaV1.2偶合门控事件的频率增加，而AKAP150的缺失可预防偶合门控和高血压，这一观察结果强调了这些发现的重要性。该项目有两个特定的目标，旨在调查这些发现的机制和生理意义。具体目的1是验证CaV1.2通道偶联门控增强动脉肌细胞内钙内流的假说。具体目的2是验证这一假说，即在高血压的发展过程中，偶联CaV1.2通道活性的增加和动脉功能障碍的诱导需要AKAP150。将用于实现这些目的的方法包括膜片钳电生理学、光学钳制、光和化学诱导的对细胞膜的激酶的动态靶向、光诱导的肾上腺素能信号的激活(即， 光遗传学)、共焦显微镜和TIRF显微镜。实验将涉及到新的转基因、敲入和敲除小鼠。这项工作将为AKAP150和CaV1.2通道在生理和病理条件下控制血管平滑肌兴奋性、基因表达和EC偶联的机制提供基础信息。

DESCRIPTION (provided by applicant): The experiments in this application will test the hypothesis that clusters of voltage-gated L-type CaV1.2 channels are capable of undergoing coordinated openings ("coupled gating"), amplifying Ca2+ influx into arterial smooth muscle. A key discovery is that Ca2+ influx via coupled CaV1.2 channels plays a critical role in excitation-contraction (EC) coupling and excitation-transcription (ET) coupling in arterial myocytes. Preliminary data suggest that association of CaV1.2 channels with the anchoring protein AKAP150 is necessary for coupled gating activity in these cells. The significance of these findings is underscored by the observation that the frequency of coupled CaV1.2 gating events increases in arterial smooth muscle during hypertension and that loss of AKAP150 protects against coupled gating and hypertension. The project has two specific aims designed to investigate the mechanisms and physiological implications of these findings. Specific aim 1 is to test the hypothesis that coupled gating of CaV1.2 channels amplifies Ca2+ influx in arterial myocytes. Specific aim 2 is to test the hypothesis that AKAP150 is required for increased coupled CaV1.2 channel activity and the induction of arterial dysfunction during the development of hypertension. 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 new transgenic, knock in, and knock out mice. 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 vascular smooth muscle under physiological and pathological conditions.