Endothelial BK Channel Regulation by Caveolae Targeting

描述（由申请人提供）：我们研究项目的总体目标是了解心血管系统中离子通道的调节。我们在牛主动脉内皮细胞（BAEC）上发现了大电导钙激活钾通道（BK）靶向细胞膜小窝的新发现。小窝是作为整合特定细胞信号转导过程的平台的专门的膜微结构域。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，以及这种相互作用的生理相关性。待检验的假设是：1）BK通道靶向血管内皮细胞中的小窝，并直接与小窝蛋白-1相互作用。2)BK通道功能通过靶向血管内皮细胞中的小窝来调节。3)β 2-肾上腺素能受体（β 2AR）靶向内皮细胞小窝，在调节BK通道功能方面比β 1AR更有效。4)切应力调节小窝功能并上调内皮BK通道功能.提出了三个具体目标。目的1是确定BK通道和小窝蛋白-1之间的物理和分子相互作用。这些将通过细胞分级分离、密度梯度离心、免疫沉淀、免疫荧光成像、hSIo和小窝蛋白-1的诱变以及GST融合蛋白结合测定来评估。目的二是确定BK通道与细胞膜小窝之间的生理相互作用。将进行野生型和突变体hSIo和小窝蛋白-1的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、Ca 2+依赖性、单通道开放概率和动力学的影响。将探讨三种可能的机制。这些战略包括：A）合成cavtratin，一种细胞可渗透的小窝蛋白-1支架结构域肽，以确定小窝蛋白-1是否是BK通道的负调节剂。B）BAEC中BK通道的表面生物素化以确定BK通道活化是否涉及BK通道从细胞内位点运输至膜。C）通过siRNA敲低小窝蛋白-1以确定通过信号传导途径激活BK通道是否需要正常小窝结构。目的3是确定BK通道-小窝蛋白-1相互作用的生理相关性。我们计划确定小窝在血管内皮细胞BK通道β-肾上腺素能激活中的作用，以及剪切力对小窝和BK功能的影响。将分析BAEC中的β AR亚型及其激活BK通道的机制。这些研究结果将有助于我们更好地理解内皮细胞中BK通道的调节，以及调节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.