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: 8065878
• 负责人: DANIEL C DEVOR
• 金额: $ 37.2万
• 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/8065878
• 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; 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（IK）和小（SK）电导、Ca2+激活的K+通道的作用已在血管平滑肌的内皮依赖性松弛中得到明确证明。 IK 和 SK 通道在激动剂诱导的血管舒张过程中都会被激活，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，已知这些通道对于 EDHF 介导的血管舒张至关重要，而血管舒张会在许多心血管疾病中受到损害。这些结果表明，内皮 IK 和 SK 通道的药理学激活对于多种心血管疾病具有临床益处。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，需要我们回答以下问题： 质膜上的 IK1 和 SK3 通道的数量是如何维持的，这是否受到血管活性物质的调节激动剂，这些通道的内吞作用途径是什么？这些通道是否循环回到膜上，还是以溶酶体降解为目标？迄今为止，这些关键生理问题的答案仍然完全未知，因此在我们理解这些通道如何调节健康和疾病中的 EDHF 反应方面存在着重要差距。基于我们知识上的这些差距，我们提出以下目标：（i）定义 IK1 被内吞并靶向内皮细胞中溶酶体降解的机制，以及这是否受到血管活性化合物或纯粹应激的调节。 (ii) 我们将定义 SK3 内吞再循环所涉及的分子机制以及去泛素化酶在此过程中的作用。 （三）。我们将定义参与 IK1 和 SK3 泛素化和降解的泛素蛋白连接酶 (E3)。我们将结合分子、蛋白质生化和活细胞荧光成像技术来开展这些研究。这些研究的结果将清楚地定义 IK1 和 SK3 内吞作用的机制，是否会被生理激动剂改变以及是否可以通过药理学来操纵。鉴于质膜上的通道数量在细胞的生理反应中是确定性的，确定是否可以操纵这些运输事件以获得治疗益处具有临床意义。公共健康相关性：中电导 (IK) 和小电导 (SK) 钙激活钾通道均在微血管内皮细胞中表达，在维持血管张力和血压方面发挥着至关重要的作用 (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 IK1 和 SK3 通道被内吞、回收和/或靶向内皮细胞溶酶体降解的机制，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。