Oxidation and Pharmacologic Activation of IK/SK Channels

IK/SK 通道的氧化和药理学激活

• 批准号: 7018115
• 负责人: DANIEL C DEVOR
• 金额: $ 36.6万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7018115
• 关键词: binding sites; calcium flux; fluorescence resonance energy transfer; free radical oxygen; gene mutation; glutathione; muscle pharmacology; oxidation; potassium channel; protein protein interaction; protein structure function; tissue /cell culture; vascular endothelium; vascular smooth muscle; vasodilators; voltage /patch clamp

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+ 通道的作用已在血管平滑肌的内皮依赖性松弛中得到明确证明。 IK 和 SK 通道在激动剂和血流诱导的血管舒张以及活性氧 (ROS) 增加的情况下都会被激活，而活性氧 (ROS) 几乎与所有心血管疾病相关。这些通道在内皮细胞中的表达也已被证明在球囊血管成形术后受到损害。 最后，已知这些通道对于 EDHF 介导的血管舒张至关重要，而血管舒张会在许多心血管疾病中受到损害。这些结果表明，内皮 IK 和 SK 通道的药理学激活对于多种心血管疾病具有临床益处。我们的实验室是第一个鉴定出一系列结构相似的 IK 和 SK 通道药理学开放剂的实验室。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作它们以获得临床益处需要我们回答两个关键的未知数。首先，ROS 如何改变 IK 和 SK 通道功能，从而改变内皮功能？其次，已知的 IK 和 SK 通道开放剂的分子作用机制是什么？因此，我们提出以下目标：（i）定义 IK 和 SK 通道的活性氧依赖性调节所涉及的机制。 我们将结合膜片钳和诱变技术来定义氧化剂激活 IK 和 SK 通道的机制。这些研究将在异源表达通道以及内皮细胞的原代培养物上进行，（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。这些研究将结合膜片钳和诱变技术进行，（iii）我们将利用 FRET 来定义 IK 和 SK 通道中亚基结构域间和亚基内的相互作用，以及通道功能的生理和药理学调节剂如何修改这些互动。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。这些研究的结果将清楚地阐明ROS激活内皮IK和SK通道从而改变血管张力的机制，并确定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。