Endothelial BK Channel Regulation by Caveolae Targeting

小凹靶向调节内皮 BK 通道

• 批准号: 7144408
• 负责人: Hon-Chi Lee
• 金额: $ 37万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144408
• 关键词: G protein; beta adrenergic receptor; biological signal transduction; biotinylation; calcium flux; caveolas; caveolins; cell line; chimeric proteins; density gradient ultracentrifugation; glutathione transferase; immunocytochemistry; immunoprecipitation; membrane activity; membrane structure; potassium channel; protein protein interaction; protein transport; shear stress; single cell analysis; small interfering RNA; subcellular fractionation; vascular endothelium; voltage /patch clamp

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.

描述（由申请人提供）：我们的研究计划的总体目的是了解心血管系统中离子通道的调节。我们进行了一个新的观察结果，即大型电导Ca2+激活的K+（BK）通道针对牛主动脉内皮细胞（BAEC）中的小窝。 Caveolae是专门的膜微区，可作为整合特定细胞信号转导过程的平台。这项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。要测试的假设是：1）BK通道针对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通道功能通过血管内皮细胞中的小窝靶向调节。 3）β2-肾上腺素能受体（BetA2AR）针对内皮口腔，并且在调节BK通道功能方面比beta1AR更有效。 4）剪切应力调节口腔功能，并调节内皮BK通道功能。提出了三个具体目标。目的1是确定BK通道与小窝蛋白1之间的物理和分子相互作用。这些将通过细胞分馏，密度梯度离心，免疫沉淀，免疫荧光成像，HSIO和Caveoline-1的诱变以及GST融合蛋白结合测定法进行评估。目的2是确定BK通道和小屋之间的生理相互作用。将执行野生型和突变体HSIO和Caveolin-1的全细胞和单通道贴片夹记录。小窝蛋白-1对BK电流密度，通道电压依赖性，CA2+依赖性，单个通道打开概率和动力学的影响。将探索三种合理的机制。这些策略包括：a）Cavtratin的合成，Cavtratin，一种可渗透的小窝蛋白-1脚手座肽肽的合成，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确定BK通道激活是否涉及将BK通道从细胞内部位运输到膜。 c）通过siRNA敲低小窝蛋白-1，以确定通过信号通路激活BK通道是否需要正常的小窝结构。目标3是确定BK通道 - 小窝蛋白-1相互作用的生理相关性。我们计划确定口腔在血管内皮细胞中BK通道的β-肾上腺素能激活中的作用，以及剪切应力对小窝和BK功能的影响。将分析BBAEC中的Betaar亚型及其激活BK通道的机制。拟议研究的结果将有助于我们更好地了解内皮细胞中BK通道的调节，还可以更好地了解调节内皮功能的基本机制。