Persistent Calcium Sparklets in Arterial Smooth Muscle

描述（由申请人提供）：本申请中的实验将检验电压门控L型CaV1.2通道簇能够进行协调开放（“偶联门控”），放大Ca 2+流入动脉平滑肌的假设。一个关键的发现是，通过耦合CaV1.2通道的Ca 2+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，协会的CaV1.2通道与锚定蛋白AKAP 150是必要的耦合门控活动在这些细胞。这些发现的重要性通过以下观察结果得到强调：在高血压期间，动脉平滑肌中偶联CaV1.2门控事件的频率增加，并且AKAP 150的缺失保护免于偶联门控和高血压。该项目有两个具体目标，旨在研究这些发现的机制和生理意义。具体目标1是检验CaV1.2通道的偶联门控放大动脉肌细胞中的Ca 2+内流的假设。具体目标2是检验以下假设：AKAP 150是高血压发展过程中增加偶联CaV1.2通道活性和诱导动脉功能障碍所必需的。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。实验将涉及新的转基因，敲入，敲出小鼠。这项工作将产生AKAP 150和CaV1.2通道控制的兴奋性，基因表达和EC耦合在血管平滑肌在生理和病理条件下的机制的基本信息。

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.