Oxidation and Pharmacologic Activation of IK/SK Channels

中（IK）和小（SK）电导，钙激活的K+通道的作用已明确证明在血管平滑肌的内皮依赖性舒张。IK和SK通道在激动剂和流量诱导的血管舒张期间以及在活性氧（ROS）增加的情况下被激活，这与几乎所有的心血管疾病相关。这些通道在内皮中的表达也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EDHF介导的血管舒张至关重要，而血管舒张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，即内皮IK和SK通道的药理学激活将在广泛的心血管疾病中具有临床益处。我们的实验室是第一个确定一系列结构相似的IK和SK通道的药理学开放剂。为了进一步明确IK和SK通道在内皮细胞中的作用， 功能以及如何为临床获益而对它们进行重复操作，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？第二，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）定义参与IK和SK通道的活性氧依赖性调节的机制。 我们将利用膜片钳和诱变技术相结合，以确定氧化剂激活IK和SK通道的机制。这些研究将在异源表达的通道以及内皮细胞的原代培养物上进行。（ii）我们将确定药理学作用于内皮细胞的分子机制。 IK和SK通道的激活剂增加通道活性。这些研究将利用膜片钳和诱变技术的组合进行。（iii）我们将利用FRET来定义IK和SK通道中的亚基结构域间和亚基结构域内的相互作用，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，对于我们理解这些通道是如何 在炎症过程中的调节，以及它们如何被操纵。这些研究的结果将清楚地定义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，从而进一步了解这些通道如何被操纵以获得治疗益处。

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.