Persistent Calcium Sparklets in Arterial Smooth Muscle

描述（由申请人提供）：本申请中的实验将测试电压门控l型CaV1.2通道簇能够进行协调打开（“耦合门控”）的假设，放大Ca2+流入动脉平滑肌。一个关键的发现是Ca2+内流通过偶联CaV1.2通道在动脉肌细胞的兴奋-收缩（EC）偶联和兴奋-转录（ET）偶联中起关键作用。初步数据表明，CaV1.2通道与锚定蛋白AKAP150的关联对于这些细胞中的偶联门控活性是必要的。观察到高血压期间动脉平滑肌中偶联CaV1.2门控事件的频率增加，以及AKAP150的缺失可以防止偶联门控和高血压，这些发现的重要性得到了强调。该项目有两个具体目的，旨在研究这些发现的机制和生理意义。具体目的1是验证CaV1.2通道的偶联门控放大动脉肌细胞Ca2+内流的假设。具体目的2是验证AKAP150在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假设。将用于实现这些目标的方法包括膜片钳电生理学，光学钳，光和化学诱导的激酶到细胞膜的动态靶向，光诱导的肾上腺素能信号的激活(即，

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.