Oxidation and Pharmacologic Activation of IK/SK Channels

IK/SK 通道的氧化和药理学激活

基本信息

批准号：
7568223
负责人：
DANIEL C DEVOR
金额：
$ 35.5万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7568223
关键词：
Agonist Atherosclerosis Balloon Angioplasty Behavior Blood Pressure Blood Vessels Bradykinin Calcium-Activated Potassium Channel Cardiovascular Diseases Cholinergic Agonists Clinical Coronary Arteriosclerosis Development Disease Endothelial Cells Endothelium Environment Feedback Fluorescence Resonance Energy Transfer Goals Homeostasis Hydrogen Peroxide Hypertension Inflammatory Inflammatory Response Ion Channel Ischemia Laboratories Mediating Molecular Molecular Mechanisms of Action Muscle relaxation phase Mutagenesis Oxidants Physiological Potassium Channel Process Qualifying Reactive Oxygen Species Regulation Relaxation Reperfusion Therapy Research Personnel Role Series Smooth Muscle Structure System Techniques Therapeutic Transgenic Mice Universities Vascular Diseases Vascular Smooth Muscle Vasodilation innovation oxidation patch clamp response

项目摘要

The role of intermediate (IK)and small (SK) conductance, Ca -activated K+ channels have been unequivocally demonstrated in the endothelial-dependent relaxation of vascular smooth muscle. Both IK and SK channels are activated during agonist- and flow-induced vasodilation as well as in the presence of increased reactive oxygen species (ROS), which are associated with virtually all cardiovascular disease. The expression of these channels in endothelia has also been shown to be compromised following balloon angioplasty. Finally, these channels are known to be critical to EDHF-mediated vasodilation, which is compromised in a host of cardiovascular diseases. These results have led to the proposal that the pharmacological activation of endothelial IK and SK channels would be of clinical benefit in a wide array of cardiovascular diseases. Our laboratory was the first to identify a series of structurally similar pharmacological openers of IK and SK channels. To further define the role of IK and SK channels in endothelial function and how they may be pharmacologically manipulated for clinical benefit requires us to answer two critical unknowns. First, how do ROS alter IK and SK channel function and therefore endothelial function? Second, what is the molecular mechanism of action for the known openers of IK and SK channels? Thus, we propose the following aims: (i) Define the mechanisms involved in the reactive oxygen species-dependent regulation of IK and SK channels. We will utilize a combination of patch-clamp and mutagenesis techniques to define the mechanisms whereby oxidizing agents activate IK and SK channels. These studies will be carried out on both heterologously expressed channels as well as on primary cultures of endothelial cells, (ii) We will define the molecular mechanism whereby pharmacological activators of IK and SK channels increase channel activity. These studies will be carried out utilizing a combination of patch-clamp and mutagenesis techniques, (iii)We will utilize FRET to define inter- and intra-subunit domain interactions in IK and SK channels and how physiological and pharmacological regulators of channel function modify these interactions. Defining how these interactions are altered is critical to our understanding of how these channels are regulated during the inflammatory process and how they may be manipulated pharmacologically. The results of these studies will clearly define the mechanism whereby ROS activate endothelial IK and SK channels, and thus alter vascular tone, as well as define the molecular mechanism underlying pharmacological activation of these channels; thereby furthering our understanding of how these channels may be manipulated for therapeutic benefit.

中电导 (IK) 和小电导 (SK)、Ca 激活的 K+ 通道的作用已明确在血管平滑肌的内皮依赖性松弛中得到证实。 IK 和 SK 通道均在激动剂和血流诱导的血管舒张以及活性氧增加的情况下被激活（ROS），几乎与所有心血管疾病有关。这些通道在内皮细胞中的表达球囊血管成形术后也已被证明受到损害。最后，众所周知，这些渠道至关重要 EDHF 介导的血管舒张，在许多心血管疾病中都会受到损害。这些结果导致内皮 IK 和 SK 通道的药理学激活将在以下方面具有临床益处：广泛的心血管疾病。我们的实验室是第一个鉴定出一系列结构相似的 IK 和 SK 通道的药理学开放剂。进一步明确 IK 和 SK 通道在内皮细胞中的作用功能以及如何通过药理学操纵它们以获得临床益处需要我们回答两个关键问题未知数。首先，ROS 如何改变 IK 和 SK 通道功能，从而改变内皮功能？二、什么是已知 IK 和 SK 通道开放剂的分子作用机制？因此，我们提出以下建议目标： (i) 定义 IK 和 SK 通道活性氧依赖性调节所涉及的机制。我们将利用膜片钳和诱变技术的组合来定义氧化机制代理激活 IK 和 SK 通道。这些研究将在两个异源表达通道上进行：以及内皮细胞的原代培养物，（ii）我们将定义药理学作用的分子机制 IK 和 SK 通道的激活剂可增加通道活性。这些研究将综合利用膜片钳和诱变技术，(iii)我们将利用 FRET 来定义亚基间和亚基内结构域 IK 和 SK 通道的相互作用以及通道功能的生理和药理学调节剂如何改变这些相互作用。定义这些相互作用如何改变对于我们理解这些渠道的运作方式至关重要炎症过程中的调节以及如何通过药理学操纵它们。这些结果研究将明确定义 ROS 激活内皮 IK 和 SK 通道的机制，从而改变血管张力，以及定义这些通道的药理学激活的分子机制；从而进一步加深我们对如何操纵这些通道以获得治疗效果的理解。