PSD95 scaffolding of vascular K+ channels in hypertension

高血压血管 K 通道的 PSD95 支架

• 批准号: 8266034
• 负责人: Sung W Rhee
• 金额: $ 35.89万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8266034
• 关键词: Adrenergic Receptor; American; Binding; Biological Assay; Blood; Blood Vessels; Blood flow; Brain; Caliber; Cardiovascular system; Cell membrane; Cerebrovascular Circulation; Cerebrum; Co-Immunoprecipitations; Complex; Confocal Microscopy; Coupling; Defect; Dilator; Disease; Dominant-Negative Mutation; Down-Regulation; Electrophysiology (science); Ensure; Event; Genes; Goals; Hypertension; In Vitro; Ion Channel; Knowledge; Location; Mediating; Membrane; Membrane Potentials; Microcirculation; Modeling; Molecular; Muscle Cells; Neurons; Pathway interactions; Peptides; Physiological; Play; Potassium Channel; Protein Array; Proteins; Rattus; Receptor Signaling; Relaxation; Reporting; Rest; Risk; Risk Factors; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Staging; Stroke; Surface; Synapses; Testing; Time; Vascular Smooth Muscle; Vasodilator Agents; base; brain circulation; cerebral artery; cerebrovascular; design; in vivo; interest; intravital microscopy; novel; patch clamp; presynaptic density protein 95; public health relevance; receptor; research study; scaffold; vasoconstriction; voltage

DESCRIPTION (provided by applicant): Shaker-type, voltage-gated K+ (KV1) channels are an important determinant of the resting membrane potential and diameter of small cerebral arteries. During hypertension, KV1 channel-mediated dilation appears to be blunted and is postulated to increase myogenicity in the cerebral circulation. However, little is known about the mechanisms that regulate the expression of KV1 channels at the plasma membrane of cerebral vascular smooth muscle cells (cVSMCs). In this regard, we recently identified scaffolding proteins including PSD95 (postsynaptic density 95) in rat cVSMCs that have never been described. PSD95 is a well- characterized scaffolding protein in neurons with more than 50 known binding partners that can facilitate macromolecular signaling between ion channels and receptors. Subsequently we determined that KV1 channels associate with the PSD95 scaffold in cVSMCs, and that PSD95 is required for the normal expression and dilator function of KV1 channels in small cerebral arteries. Finally, we have evidence that the 21 adrenergic receptor (21AR) - another known binding partner of the PSD95 scaffold - activates a KV1 channel-mediated dilator pathway. Thus, we envision that PSD95 enables the efficient coupling of the 21AR signaling pathway to KV1 channels in cVSMCs and we have designed experiments to characterize the impact of this novel PSD95 complex on cerebrovascular reactivity. Based on our early findings, we hypothesize that: 21AR and the KV1 channels form a macromolecular vasodilator complex on a PSD95 scaffold in the rat cerebral circulation. We further propose that the down-regulation of cerebrovascular KV1 channels during hypertension disrupts the PSD95 scaffold resulting in a synchronized loss of the 21AR-KV1 signaling pathway and a vasodilator defect. These hypotheses will be tested using co-immunoprecipitation and confocal microscopy to discern protein interactions in small cerebral arteries. The physiological impact of siRNA knockdown of PSD95 or KV1 channels in cerebral arteries in vitro and in vivo will be evaluated using patch-clamp electrophysiology, microvessel reactivity assays, and intravital microscopy. The findings of this project will identify for the first time a vasodilator complex in vascular smooth muscle that is regulated by scaffolding proteins, and will set the stage for further studies to understand how ion channels are localized with their signaling partners in cVSMCs. PUBLIC HEALTH RELEVANCE: More than seventy million Americans are afflicted by high blood pressure that is the leading risk factor for stroke. Proteins called potassium channels in the muscle cells of the brain circulation regulate the blood flow to the brain. This project will investigate a novel scaffolding molecule in the muscle cells of small cerebral arteries that may ensure that potassium channels are expressed in adequate numbers and in the right location in the muscle cells of cerebral arteries to optimize blood flow to the brain.

描述（由申请人提供）：激振器型电压门控K+ (KV1)通道是静息膜电位和脑小动脉直径的重要决定因素。在高血压期间，KV1通道介导的扩张似乎被钝化，并被认为增加了脑循环中的肌原性。然而，对脑血管平滑肌细胞（cVSMCs）质膜上KV1通道表达的调控机制知之甚少。在这方面，我们最近在大鼠cVSMCs中发现了从未被描述过的包括PSD95（突触后密度95）在内的支架蛋白。PSD95是一种表征良好的支架蛋白，在神经元中有超过50种已知的结合伙伴，可以促进离子通道和受体之间的大分子信号传导。随后，我们确定了cVSMCs中KV1通道与PSD95支架相关，并且PSD95是脑小动脉中KV1通道正常表达和扩张功能所必需的。最后，我们有证据表明21肾上腺素能受体(21AR) - PSD95支架的另一个已知结合伙伴-激活KV1通道介导的扩张通路。因此，我们设想PSD95能够将21AR信号通路与cVSMCs中的KV1通道有效耦合，并且我们设计了实验来表征这种新型PSD95复合物对脑血管反应性的影响。基于我们的早期发现，我们假设：21AR和KV1通道在大鼠脑循环中的PSD95支架上形成大分子血管扩张复合物。我们进一步提出，高血压期间脑血管KV1通道的下调会破坏PSD95支架，导致21AR-KV1信号通路的同步丢失和血管扩张剂缺陷。这些假设将使用共免疫沉淀和共聚焦显微镜来检测大脑小动脉中的蛋白质相互作用。体外和体内脑动脉中PSD95或KV1通道siRNA敲低的生理影响将通过膜片钳电生理学、微血管反应性测定和活体显微镜进行评估。该项目的研究结果将首次确定血管平滑肌中受支架蛋白调节的血管扩张复合物，并为进一步研究离子通道如何与其信号伙伴在cVSMCs中定位奠定基础。