Assembly and Trafficking of IK1 and SK3 in Endothelia

IK1 和 SK3 在内皮细胞中的组装和运输

• 批准号: 7730291
• 负责人: DANIEL C DEVOR
• 金额: $ 37.24万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730291
• 关键词: Address; Agonist; Atherosclerosis; Back; Balloon Angioplasty; Biochemical; Biotin; Blood Pressure; Blood Vessels; Bradykinin; Calcium-Activated Potassium Channel; Cardiovascular Diseases; Cell membrane; Cells; Cellular biology; Clinical; Disease; Elements; Endocytosis; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Environment; Enzymes; Event; Goals; Health; Hypertension; Imaging Techniques; Ion Channel; Ischemia; Knock-out; Knockout Mice; Knowledge; Laboratories; Life; Ligase; Mediating; Membrane; Molecular; Peptides; Pharmacology; Physiological; Physiology; Play; Probability; Process; Proteins; Qualifying; Recycling; Relaxation; Reperfusion Therapy; Research; Research Personnel; Role; Route; Second Messenger Systems; Stress; System; Therapeutic; Time; Transgenic Mice; Universities; Vascular Smooth Muscle; Vasoconstrictor Agents; Vasodilation; Work; base; blood pressure regulation; fluorescence imaging; innovation; insight; novel; protein complex; public health relevance; response; second messenger; trafficking; ubiquitin-protein ligase

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.

描述（由申请人提供）：中间（IK）和小（SK）电导的作用，Ca2+活化的K+通道在血管平滑肌的内皮依赖性弛豫中已无疑证明了。在激动剂诱导的血管舒张期间，IK和SK通道都被激活，这些通道的敲除与血压升高有关。同样，在气球血管成形术后，这些通道在内皮中的表达已被证明被妥协。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。要充分欣赏IK和SK频道在内皮功能中的作用以及如何为临床益处操纵它们，就需要我们回答以下问题：在质膜上维持IK1和SK3频道的数量如何，这是由血管activeive的调节激动剂，这些通道的内吞作用的途径是什么，这些通道会回收回膜，还是将其靶向溶酶体降解？迄今为止，这些关键生理问题的答案仍然是完全未知的，因此代表了我们对这些渠道如何调节健康和疾病中EDHF反应的重要差距。基于我们知识的这些差距，我们提出以下目的：（i）定义IK1被内吞和靶向内皮细胞中溶酶体降解的机制，以及这是通过血管活性化合物还是纯粹的应力来调节。 （ii）我们将定义SK3内吞回收的分子机制，以及在此过程中除外式化酶的作用。 （iii）。我们将定义参与IK1和SK3的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IK1和SK3内吞作用的机制，无论生理激动剂是否会改变，以及是否可以通过药理操作。鉴于质膜上的通道数量在细胞的生理反应中是确定性的，因此确定是否可以为治疗益处操纵这些运输事件是临床进口的。公共卫生相关性：中间（IK）和小（SK）电导，钙激活的钾通道在微血管内皮细胞中表达，在这些细胞中它们在维持血管张力和血压中起着至关重要的作用（1，2）。基于这些观察结果，很明显，调节IK和/或SK通道的活性或数量会改变血管张力，因此在治疗上有益。我们提出的研究将定义IK1和SK3通道在内皮细胞中溶酶体降解的内吞和/或靶向的机制，从而改变了通道数。我们的研究将提供有关如何操纵这些过程以获得临床收益的新见解。