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

描述(申请人提供)：A-激酶锚定蛋白(AKAP)通过将它们的分子组成聚集到离散的亚细胞微域中来组织许多细胞内信号通路。AKAP79/150与蛋白激酶A、蛋白激酶C(PKC)、钙调蛋白(CaM)、钙调神经磷酸酶(Calcineurin)、磷脂酰肌醇4，5-二磷酸(PIP2)以及M型(KCNQ、KV7)K通道和某些G蛋白偶联受体等效应分子相互作用。在本项目中，我们将研究哪些KCNQ1-5亚基是AKAP79/150的靶点，以及交感和结节神经元的GQ/11偶联受体(毒鼠碱M1、缓激肽B2、血管紧张素AT1和嘌呤能P2Y)使用AKAP79/150来调节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神经递质释放中的作用将在单细胞水平和通过心肌细胞对交感神经元活动的变时性反应的体外模型来探索。我们将使用在中国仓鼠卵巢(CHO)细胞中表达M通道、受体和信号分子的异源表达系统，大鼠和小鼠颈上神经节(SCG)和结状神经节(NG)神经元的制备，以及SCG神经元和心室肌细胞的共同培养。可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、膜片钳电生理学、碳纤维安培法和视频成像。我们渴望发现AKAP79/150对受体和M型K通道具有特异性的机制，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。与公共健康相关：构成生物信号中的共同主题的信号通路是神经系统调节的基础，包括情绪状态和情绪、个性以及记忆的获得和使用的变化。我们将研究A-激酶锚定蛋白AKAP79/150对M型钾离子通道的调节，该通道在神经细胞兴奋性的调节中起主导作用。阐明AKAP79/150在M通道活动中的机制和功能作用将有助于阐明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.