Persistent Calcium Sparklets in Vascular Smooth Muscle

描述(申请人提供)：通过二氢吡啶敏感的电压门控L型钙通道的钙内流在调节动脉平滑肌的兴奋性、收缩和基因表达方面起着至关重要的作用。我们最近发现，L型钙通道的小簇可以在一种“持续”的门控模式下工作，这种门控模式在平滑肌中创造了几乎持续的钙内流部位(称为“持续性钙小火花”)，这导致了范式的转变，即这些细胞中的钙内流主要由这一过程以及个别L型钙通道罕见的电压依赖性开口来控制。然而，在生理和病理生理条件下，持续钙离子对局部和整体细胞内钙离子的调节作用以及激活和调节这些钙离子内流事件的分子机制几乎是未知的。这项拟议的工作将使用我们团队开发的新方法来定义大脑动脉平滑肌中钙火花的生物物理性质和功能作用。我们的初步结果表明，所有拟议的实验都是可行的，并将提供重要的新信息。这项拟议的工作试图测试三个新的假设。在特定的目标1中，我们将检验这样一种假设，即通过钙小颗粒的钙内流有助于局部和整体细胞内钙浓度的变化。在特定目标2中的实验将检验Cav1.2通道存在于动脉平滑肌钙闪光下的假说。最后，在特定目标3中，我们在前两个特定目标工作的基础上，验证了高血压期间持久钙火花活性增加的假设。这项工作应该提供第一个完整的观点，钙火花介导的信号及其在调节脑动脉功能中的作用，并显著增强我们对健康和疾病中的动脉功能的理解。

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.