Assembly and Trafficking of IK1 and SK3 in Endothelia

描述（由申请方提供）：已明确证明了中等（IK）和小（SK）电导、Ca 2+激活的K+通道在血管平滑肌内皮依赖性舒张中的作用。IK和SK通道在激动剂诱导的血管舒张期间被激活，并且这些通道的敲除与血压升高相关。此外，这些通道在内皮细胞中的表达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。为了充分理解IK和SK通道在内皮功能中的作用以及如何操纵它们以获得临床益处，我们需要回答以下问题：IK 1和SK 3通道的数量如何维持在质膜上，是否受血管活性激动剂的调节，这些通道的内吞途径是什么？这些通道是循环回膜还是被溶酶体降解？迄今为止，这些关键生理问题的答案仍然完全未知，因此代表了我们对这些通道如何调节健康和疾病中的EDHF反应的理解中的重要差距。基于我们知识中的这些空白，我们提出以下目标：（i）定义IK 1被内吞并靶向内皮细胞中溶酶体降解的机制，以及这是否由血管活性化合物或剪切应力调节。(ii)我们将定义SK 3的内吞再循环的分子机制以及去泛素化酶在这一过程中的作用。（三）.我们将定义参与IK 1和SK 3泛素化和降解的泛素蛋白连接酶（E3）。我们将利用分子，蛋白质生化和活细胞荧光成像技术的组合来进行这些研究。这些研究的结果将清楚地定义IK 1和SK 3内吞作用的机制，这是否被生理激动剂改变，以及这是否可以被操纵。鉴于质膜上通道的数量在细胞的生理反应中是确定性的，确定这些运输事件是否可以被操纵以获得治疗益处具有临床意义。公共卫生相关性：中等（IK）和小（SK）电导，钙激活钾通道在微血管内皮细胞中表达，在维持血管张力和血压方面起着至关重要的作用（1，2）。基于这些观察，很明显，调节IK和/或SK通道的活性或数量将改变血管张力，因此在治疗上是有益的。我们提出的研究将定义IK 1和SK 3通道被内皮细胞内吞、回收和/或靶向溶酶体降解的机制，从而改变通道数量。我们的研究将为如何操纵这些过程以获得临床收益提供新的见解。

DESCRIPTION (provided by applicant): The role of intermediate (IK) and small (SK) conductance, Ca2+-activated K+ channels has been unequivocally demonstrated in the endothelial-dependent relaxation of vascular smooth muscle. Both IK and SK channels are activated during agonist-induced vasodilation, and knockout of these channels is associated with increased blood pressure. Also, the expression of these channels in endothelia has 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. To fully appreciate the role of IK and SK channels in endothelial function and how they may be manipulated for clinical benefit requires us to answer the following questions: How are the number of IK1 and SK3 channels maintained at the plasma membrane, is this regulated by vasoactive agonists, what is the route of endocytosis for these channels and do these channels recycle back to the membrane or are they targeted for lysosomal degradation? To date, the answers to these critical physiological questions remain completely unknown and thus represent an important gap in our understanding of how these channels modulate the EDHF response in both health and disease. Based on these gaps in our knowledge, we propose the following aims: (i) Define the mechanism by which IK1 is endocytosed and targeted for lysosomal degradation in endothelial cells and whether this is modulated by vasoactive compounds or sheer stress. (ii) We will define the molecular mechanisms involved in the endocytic recycling of SK3 as well as the role of deubiquitylating enzymes in this process. (iii). We will define the ubiquitin-protein ligases (E3) involved in the ubiquitylation and degradation of IK1 and SK3. We will utilize a combination of molecular, protein biochemical and live-cell fluorescence imaging techniques to carry out these studies. The results of these studies will clearly define the mechanism of IK1 and SK3 endocytosis, whether this is altered by physiologic agonists and whether this can be manipulated pharmacologically. Given that the number of channels at the plasma membrane is deterministic in the physiological response of the cell, determining whether these trafficking events can be manipulated for therapeutic benefit is of clinical import. PUBLIC HEALTH RELEVANCE: Both intermediate (IK) and small (SK) conductance, calcium-activated potassium channels are expressed in microvascular endothelial cells where they play a crucial role in maintaining vascular tone and hence blood pressure (1, 2). Based upon these observations, it is clear that modulating the activity or number of IK and/or SK channels would alter vascular tone and therefore be therapeutically beneficial. Our proposed research will define the mechanisms by which IK1 and SK3 channels are endocytosed, recycled and/or targeted for lysosomal degradation in endothelial cells, thereby altering channel number. Our studies will provide novel insight into how these processes can be manipulated for clinical gain.