Oxidation and Pharmacologic Activation of IK/SK Channels

钙激活的K+通道在中(IK)和小(SK)电导中的作用是明确的 表现在血管平滑肌的内皮依赖性松弛。IK和SK通道都是 在激动剂和血流诱导的血管扩张过程中被激活，以及在活性氧物种增加的情况下激活 (ROS)，它们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 在球囊血管成形术后也被证明是安全的。最后，众所周知，这些渠道是至关重要的 到EDHF介导的血管扩张，这在许多心血管疾病中受到影响。这些结果导致了 药物激活内皮细胞的IK和SK通道将在临床上有益于 心血管疾病种类繁多。我们的实验室是第一个发现一系列结构相似的 IK和SK通道的药理开放剂。进一步明确IK和SK通道在内皮细胞中的作用 它们的功能以及如何从药理上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？第二，什么是 已知的IK和SK通道开放剂的分子作用机制是什么？因此，我们提出如下建议 目的：(I)明确参与对IK和SK通道的活性氧依赖调节的机制。 我们将利用膜片钳和突变技术的组合来确定氧化的机制 代理激活IK和SK通道。这些研究将在两个异源表达的渠道上进行，如 以及在原代培养的内皮细胞上，(Ii)我们将定义药理学上 IK和SK通道的激活剂增加了通道的活跃度。这些研究将利用以下组合进行 膜片钳和突变技术，(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物调节因子如何改变 这些互动。定义这些相互作用是如何改变的，对于我们理解这些渠道是如何改变至关重要的 在炎症过程中被调控，以及它们可能如何被药理学操纵。这些研究的结果 研究将清楚地确定ROS激活内皮细胞IK和SK通道从而改变 血管张力，以及确定这些通道药物激活的分子机制； 从而加深了我们对如何操纵这些通道以获得治疗益处的理解。

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.