PSD95 scaffolding of vascular K+ channels in hypertension

描述(由申请人提供)：振荡型电压门控K(KV1)通道是静息膜电位和小脑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变得迟钝，并被认为增加了脑循环中的生肌活性。然而，调节脑血管平滑肌细胞(CVSMCs)质膜上KV1通道表达的机制却鲜为人知。在这方面，我们最近在大鼠cVSMCs中发现了包括PSD95(突触后密度95)在内的支架蛋白，这些蛋白从未被描述过。PSD95是一种在神经元中具有良好特性的支架蛋白，具有50多个已知的结合伙伴，可以促进离子通道和受体之间的大分子信号转导。随后，我们确定在cVSMCs中KV1通道与PSD95支架相关，并且PSD95是KV1通道在小脑动脉中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通道介导的扩张器途径。因此，我们设想PSD95能够在cVSMCs中有效地将21AR信号通路与KV1通道偶联，我们设计了实验来表征这种新的PSD95复合体对脑血管反应性的影响。根据我们的早期发现，我们假设：在大鼠脑循环中，21AR和KV1通道在PSD95支架上形成了一个大分子血管扩张复合体。我们进一步认为，高血压期间脑血管KV1通道的下调破坏了PSD95支架，导致21AR-KV1信号通路的同步丢失和血管扩张剂缺陷。这些假说将用免疫共沉淀和共聚焦显微镜来检验，以辨别大脑小动脉中的蛋白质相互作用。通过膜片钳电生理学、微血管反应性分析和活体显微镜观察，将评估在体内外脑动脉中PSD95或KV1通道的siRNA敲除对生理的影响。该项目的发现将首次发现血管平滑肌中受支架蛋白调控的血管扩张剂复合体，并将为进一步研究了解离子通道如何与其在cVSMCs中的信号伙伴定位奠定基础。 公共卫生相关性：超过7000万美国人患有高血压，高血压是中风的主要风险因素。大脑循环中肌肉细胞中的一种称为钾通道的蛋白质调节着流向大脑的血液。该项目将在大脑小动脉的肌肉细胞中研究一种新的支架分子，该分子可能确保钾通道在大脑动脉的肌肉细胞中以足够的数量和正确的位置表达，以优化流向大脑的血液。

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.