Mechanism and functional role of AKAP79/150 in M current control and excitability

描述（由申请人提供）：a激酶锚定蛋白（AKAPs）通过将其分子成分聚集到离散的亚细胞微域来组织许多细胞内信号通路。其中一个AKAP， AKAP79/150与蛋白激酶A、蛋白激酶C （PKC）、钙调蛋白（CaM）、钙调磷酸酶和磷脂酰肌醇4,5-二磷酸（PIP2）相互作用，并与m型（KCNQ、Kv7） K+通道和某些G蛋白偶联受体等效应物相互作用。在本项目中，我们将研究哪些KCNQ1-5亚基是AKAP79/150的靶点，以及交感神经节和结节神经节神经元的Gq/11偶联受体（毒毒碱M1、缓激肽B2、血管紧张素AT1和嘌呤能P2Y）利用AKAP79/150调节m型通道。我们还将研究CaM和PIP2分子与AKAP79/150之间的相互作用，这对M通道和AKAP79/150的功能都是至关重要的。AKAP79/150在Gq/11偶联受体控制神经元放电特性及其释放神经递质的作用将在单细胞水平上以及通过心肌细胞对交感神经元活动的变时反应的体外模型进行探索。我们将使用异源表达系统，其中m通道，受体和信号分子在中国仓鼠卵巢（CHO）细胞中表达，制备大鼠和小鼠颈上神经节（SCG）和结节神经节（NG）神经元，并将SCG神经元和心室心肌细胞共培养。将使用的技术包括荧光共振能量转移（FRET），全内反射荧光（TIRF），共聚焦显微镜，膜片钳电生理，碳纤维安培和视频成像。我们希望发现赋予AKAP79/150对受体和m型K+通道特异性的机制，以及它在塑造神经元的神经生理和神经递质释放特性中的功能作用。公共卫生相关性：构成生物信号中共同基序的信号通路是神经系统如何调节的基础，包括情绪状态和情绪、个性以及记忆的获取和使用的改变。我们将研究由a激酶锚定蛋白AKAP79/150介导的m型钾离子通道在神经细胞兴奋性调控中起主导作用。阐明AKAP79/150在m通道活性中的机制和功能作用，将有助于揭示神经系统在健康和疾病中的功能。

DESCRIPTION (provided by applicant): A-kinase anchoring proteins (AKAPs) organize numerous intracellular signaling pathways by bringing together their molecular components into discrete sub-cellular microdomains. One such AKAP, AKAP79/150 interacts with protein kinase A, protein kinase C (PKC), calmodulin (CaM), calcineurin, and phosphatidylinositol 4,5- bisphosphate (PIP2), along with effectors such as M-type (KCNQ, Kv7) K+ channels and certain G protein- coupled receptors. In this project, we will study which KCNQ1-5 subunits are targets of AKAP79/150, and which Gq/11-coupled receptors of sympathetic and nodose ganglia neurons (muscarinic M1, bradykinin B2, angiotensin AT1 and purinergic P2Y) use AKAP79/150 to modulate M-type channels. We will also investigate the interactions between the CaM and PIP2 molecules with AKAP79/150 that are critical to the function of both M channels and AKAP79/150. The role of AKAP79/150 in Gq/11-coupled receptor control of neuronal discharge properties of the neurons, and on their release of neurotransmitter will be explored, both at the single-cell level, and via an in vitro model of the chronotropic response of cardiomyocytes to sympathetic neuron activity. We will use a heterologous expression system in which M-channels, receptors and signaling molecules are expressed in Chinese hamster ovary (CHO) cells, preparations of rat and mouse superior cervical ganglia (SCG) and nodose ganglia (NG) neurons, and a co-culture of SCG neurons and ventricular cardiomyocytes. Techniques to be used include fluorescence resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), confocal microscopy, patch-clamp electrophysiology, carbon-fiber amperometry and video imaging. We aspire to discover the mechanisms endowing AKAP79/150 in specificity towards receptors and M-type K+ channels, and its functional role in shaping the neurophysiological and neurotransmitter release properties of neurons. PUBLIC HEALTH RELEVANCE: The signaling pathways that comprise a common motif in biological signaling underlie how the nervous system is regulated, including alterations in emotional state and mood, personality and the acquisition and use of memories. We will study the regulation of the M-type potassium ion channel, which plays dominant role in regulation of nerve-cell excitability, mediated by the A-kinase Anchoring Protein, AKAP79/150. The elucidation of the mechanisms and functional role of AKAP79/150 in M-channel activity will shed light on how the nervous system functions in health and disease.