Oxidation and Pharmacologic Activation of IK/SK Channels

中(IK)和小(SK)电导、钙激活的K+通道在血管平滑肌内皮依赖性松弛中的作用已被明确证明。在激动剂和血流诱导的血管扩张以及与几乎所有心血管疾病相关的活性氧簇(ROS)增加的情况下，IK和SK通道都被激活。这些通道在血管内皮细胞中的表达也被证明在球囊血管成形术后受到影响。最后，已知这些通道对EDHF介导的血管扩张至关重要，而EDHF介导的血管扩张在许多心血管疾病中受到损害。这些结果表明，内皮细胞IK和SK通道的药理学激活将在广泛的心血管疾病中发挥临床作用。我们的实验室首次鉴定出一系列结构相似的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 它们的功能以及它们如何被药理操控以获得临床益处，需要我们回答两个关键的未知问题。首先，ROS如何改变IK和SK通道功能，从而改变内皮功能？第二，已知的IK和SK通道开放剂的分子作用机制是什么？因此，我们提出了以下目标：(I)明确参与IK和SK通道活性氧类依赖调节的机制。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原代培养的内皮细胞上进行，(Ii)我们将确定药物作用的分子机制 IK和SK通道的激活剂增加了通道的活跃度。这些研究将利用膜片钳和突变技术的组合来进行。(Iii)我们将利用FRET来定义IK和SK通道中亚单位间和亚基内结构域的相互作用，以及通道功能的生理和药物调节如何改变这些相互作用。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。这些研究的结果将明确ROS激活内皮细胞IK和SK通道从而改变血管张力的机制，以及药物激活这些通道的分子机制；从而加深我们对这些通道如何被操纵以达到治疗效果的理解。

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.