Persistent Calcium Sparklets in Vascular Smooth Muscle

描述（由申请人提供）：钙通过二氢吡啶敏感、电压门控的l型钙通道流入，在动脉平滑肌的兴奋性、收缩性和基因表达的调节中起着至关重要的作用。我们最近发现，小簇的l型钙通道可以在“持续”门控模式下运作，在平滑肌中产生几乎连续的钙内流（称为“持续钙火花”），这导致了一种范式转变，即这些细胞中的钙内流主要由这一过程控制，并结合个别l型钙通道罕见的电压依赖性开口。然而，持续钙火花在局部和整体细胞内钙调控中的作用，以及在生理和病理生理条件下平滑肌中这些钙内流事件的激活和调节的分子机制几乎是未知的。本研究将使用本小组开发的新方法来定义脑动脉平滑肌中钙微粒的生物物理特性和功能作用。我们的初步结果表明，所有提出的实验都是可行的，并将提供重要的新信息。这项提议的工作旨在测试三个新的假设。在具体目标1中，我们将验证钙通过钙火花流入有助于局部和整体细胞内钙浓度变化的假设。具体目标2的实验将验证Cav1.2通道是动脉平滑肌钙火花的基础的假设。最后，在Specific Aim 3中，我们以前两个Specific Aims的工作为基础，验证了持续钙火花活性在高血压期间增加的假设。这项工作将首次提供钙火花介导的信号传导及其在调节脑动脉功能中的作用的综合观点，并显著增强我们对动脉功能在健康和疾病中的理解。

DESCRIPTION (provided by applicant): Calcium influx via dihydropyridine-sensitive, voltage-gated L-type calcium channels plays a crucial role in the regulation of excitability, contraction, and gene expression in arterial smooth muscle. Our recent discovery that small clusters of L-type calcium channels can operate in a "persistent" gating mode that create sites of nearly continual calcium influx (called "persistent calcium sparklets") in smooth muscle has led to a paradigm shift, whereby calcium influx in these cells is predominantly controlled by this process in combination with rare voltage-dependent openings of individual L-type calcium channels. However, the role of persistent calcium sparklets on the regulation of local and global intracellular calcium as well as the molecular mechanisms underlying the activation and modulation of these calcium influx events in smooth muscle under physiological and pathophysiological conditions is virtually unknown. The proposed work will use new methods developed by our group to define the biophysical properties and functional roles of calcium sparklets in cerebral artery smooth muscle. Our preliminary results suggest that all proposed experiments are feasible and will provide important new information. The proposed work seeks to test three novel hypotheses. In Specific Aim 1, we will test the hypothesis that calcium influx via calcium sparklets contributes to changes in local and global intracellular calcium concentration. The experiments in Specific Aim 2 will test the hypothesis that Cav1.2 channels underlie calcium sparklets in arterial smooth muscle. Finally, in Specific Aim 3, we build on the work in the previous two Specific Aims and test the hypothesis that persistent calcium sparklet activity is increased during hypertension. This work should provide the first integrated view of calcium sparklet-mediated signaling and their role in modulating cerebral arteries function and significantly enhance our understanding of arterial function in health and disease.