PSD95 scaffolding of vascular K+ channels in hypertension

描述（由申请人提供）：振动器型电压门控K+（KV 1）通道是脑小动脉静息膜电位和直径的重要决定因素。在高血压期间，KV 1通道介导的扩张似乎变钝，并被假定为增加脑循环中的肌生成。然而，关于脑血管平滑肌细胞（cVSMCs）质膜上KV 1通道表达的调控机制知之甚少。在这方面，我们最近确定了支架蛋白，包括PSD 95（突触后密度95）在大鼠cVSMCs从未被描述。PSD 95是神经元中充分表征的支架蛋白，具有超过50种已知的结合配偶体，其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KV 1通道与cVSMC中的PSD 95支架相关，并且PSD 95是小脑动脉中KV 1通道的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（21 AR）-另一个已知的结合伙伴的PSD 95支架-激活KV 1通道介导的扩张剂途径。因此，我们设想PSD 95能够有效地将21 AR信号通路与cVSMC中的KV 1通道偶联，并且我们设计了实验来表征这种新型PSD 95复合物对脑血管反应性的影响。基于我们的早期研究结果，我们假设：21 AR和KV 1通道在大鼠脑循环中在PSD 95支架上形成大分子血管扩张剂复合物。我们进一步提出，高血压期间脑血管KV 1通道的下调会破坏PSD 95支架，导致21 AR-KV 1信号通路同步丧失和血管扩张剂缺陷。这些假设将使用免疫共沉淀和共聚焦显微镜来检测小脑动脉中的蛋白质相互作用。将使用膜片钳电生理学、微血管反应性测定和活体显微镜检查来评价体外和体内脑动脉中PSD 95或KV 1通道的siRNA敲低的生理影响。该项目的研究结果将首次确定血管平滑肌中由支架蛋白调节的血管扩张剂复合物，并将为进一步研究奠定基础，以了解离子通道如何与cVSMC中的信号伙伴一起定位。

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.