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.

在血管平滑肌的内皮依赖性弛豫中，已明确证明了中间体（IK）和小（SK）电导的K+通道的作用。在激动剂和流动诱导的血管舒张期间，以及在存在增加的活性氧（ROS）的情况下，IK和SK通道都被激活，这几乎与所有心血管疾病有关。在球囊血管成形术后，这些通道在内皮中的表达也已被妥协。 最后，已知这些通道对EDHF介导的血管舒张至关重要，后者在许多心血管疾病中受到损害。这些结果导致了这样的提议，即在多种心血管疾病中，内皮IK和SK通道的药理激活将具有临床益处。我们的实验室是第一个识别IK和SK通道的一系列结构相似的药理学开瓶器的实验室。进一步定义IK和SK频道在内皮中的作用 功能以及如何在药理学上操纵它们以获得临床益处，这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，IK和SK通道开瓶器的作用分子机制是什么？因此，我们提出以下目的：（i）定义与IK和SK通道的活性氧依赖性调节所涉及的机制。 我们将利用斑块夹和诱变技术的组合来定义氧化剂激活IK和SK通道的机制。这些研究将在异源表达的通道以及内皮细胞的原发性培养物上进行，（ii）我们将定义分子机制 IK和SK通道的激活剂增加了通道活动。这些研究将利用斑块夹和诱变技术的组合进行，（iii）我们将利用FRET来定义IK和SK通道中的 - 基因内和基因内的域间相互作用，以及通道功能的生理和药理调节剂如何修改这些依据互动。定义这些相互作用的改变是我们对这些渠道的理解至关重要的 在炎症过程中受到调节，以及如何通过药理操作。这些研究的结果将清楚地定义了ROS激活内皮IK和SK通道的机制，从而改变了血管张力，并定义了这些通道的药理激活的分子机制；从而进一步了解如何为治疗益处操纵这些渠道。