Endothelial BK Channel Regulation by Caveolae Targeting

描述(由申请者提供)：我们研究计划的总体目标是了解心血管系统离子通道的调节。我们发现大电导钙激活的钾(BK)通道定位于牛主动脉内皮细胞(BAEC)的小窝。小窝是一种特殊的膜微域，作为整合特定细胞信号转导过程的平台。本研究的目的是确定小窝蛋白-1和BK通道之间的物理、分子和生理相互作用，以及这些相互作用的生理相关性。需要检验的假设是：1)BK通道靶向血管内皮细胞内的小窝，并与小窝蛋白-1直接相互作用。2)BK通道功能受血管内皮细胞小凹靶向的调节。3)β2-肾上腺素能受体(Beta2AR)靶向于内皮细胞小窝，在调节BK通道功能方面比Beta1AR更有效。4)切应力调节血管小窝功能，上调内皮细胞BK通道功能。提出了三个具体目标。目的1确定BK通道和小窝蛋白-1之间的物理和分子相互作用。这些将通过细胞分离、密度梯度离心、免疫沉淀、免疫荧光成像、hSIO和Caveoline-1的突变以及GST-融合蛋白结合试验进行评估。目的2确定BK通道和小窝之间的生理相互作用。将对野生型和突变型hSIO和小窝蛋白-1进行全细胞和单通道膜片钳记录。将确定小窝蛋白-1对BK电流密度、通道电压依赖性、钙离子依赖性、单通道开放概率和动力学的影响。我们将探索三种看似合理的机制。这些策略包括：a)合成细胞可通透性的小窝蛋白-1支架结构域肽，以确定小窝蛋白-1是否是BK通道的负性调节因子。B)BAEC中BK通道的表面生物素化，以确定BK通道的激活是否涉及BK通道从细胞内位置到膜的运输。C)用siRNA敲除小窝蛋白-1，以确定信号通路激活BK通道是否需要正常的小窝结构。目的3是确定BK通道-小窝蛋白-1相互作用的生理学相关性。我们计划确定小凹在血管内皮细胞β-肾上腺素能激活BK通道中的作用，以及切应力对小窝和BK功能的影响。我们将分析BAEC中的β-AR亚型及其激活BK通道的机制。这项研究的结果不仅有助于我们更好地了解BK通道在内皮细胞中的调节，而且有助于我们更好地了解调控内皮功能的基本机制。

DESCRIPTION (provided by applicant): The overall objective of our research program is to understand the regulation of ion channels in the cardiovascular system. We made a novel observation that the large conductance Ca2+-activated K+ (BK) channels are targeted to caveolae in bovine aortic endothelial cells (BAEC). Caveolae are specialized membrane microdomains that serve as platforms for integrating specific cellular signal transduction processes. The goal of this study is to determine the physical, molecular, and physiological interactions between caveolin-1 and BK channels, as well as the physiological relevance of such interactions. The hypotheses to be tested are: 1) BK channels are targeted to caveolae in vascular endothelial cells and interact directly with caveolin-1. 2) BK channel function is modulated by caveolae targeting in vascular endothelial cells. 3) beta2-Adrenergic receptors (beta2AR) are targeted to endothelial caveolae and are more efficient than beta1AR in regulating BK channel function. 4) Shear stress modulates caveolae function and up- regulates endothelial BK channel function. Three specific aims are proposed. Aim 1 is to determine the physical and molecular interactions between BK channels and caveolin-1. These will be assessed by cell fractionation, density gradient centrifugation, immunoprecipitation, immunofluorescence imaging, mutagenesis of hSIo and caveoline-1, and GST-fusion protein binding assays. Aim 2 is to determine the physiological interaction between BK channels and caveolae. Whole-cell and single channel patch clamp recordings of wild-type and mutant hSIo and caveolin-1 will be performed. The effects of caveolin-1 on BK current densities, channel voltage dependence, Ca2+ dependence, single channel opening probability and kinetics will be determined. Three plausible mechanisms will be explored. The strategies include: A) Synthesis of cavtratin, a cell permeable caveolin-1 scaffolding domain peptide to determine whether caveolin-1 is a negative regulator of BK channel. B) Surface biotinylation of BK channels in BAEC to determine whether BK channel activation involves the trafficking of BK channels from intracellular sites to the membrane. C) Knockdown of caveolin-1 by siRNA to determine whether activation of BK channels by signaling pathways requires normal caveolae structure. Aim 3 is to determine the physiological relevance of BK channel- caveolin-1 interaction. We plan to determine the role of caveolae in beta-adrenergic activation of BK channels in vascular endothelial cells and the effect of shear stress on caveolae and BK function. The betaAR subtype in BAEC and the mechanism through which they activate BK channels will be analyzed. Results of the proposed research will help us better understand not only the regulation of BK channels in endothelial cells, but also fundamental mechanisms that modulate endothelial function.