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（AKAP）通过将其分子组分聚集到离散的亚细胞微区中来组织许多细胞内信号传导途径。一种这样的AKAP，AKAP 79/150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（PIP 2）相互作用，沿着效应物如M型（KCNQ，Kv 7）K+通道和某些G蛋白偶联受体。在本项目中，我们将研究哪些KCNQ 1 -5亚基是AKAP 79/150的靶点，以及哪些交感神经和结状神经节神经元的Gq/11偶联受体（毒蕈碱M1、缓激肽B2、血管紧张素AT 1和嘌呤能P2 Y）使用AKAP 79/150来调节M型通道。我们还将研究CaM和PIP 2分子与AKAP 79/150之间的相互作用，这对M通道和AKAP 79/150的功能至关重要。AKAP 79/150在Gq/11偶联受体控制神经元的神经元放电特性中的作用，以及它们释放神经递质的作用将在单细胞水平和通过心肌细胞对交感神经元活性的变时性反应的体外模型进行探索。我们将使用一种异源表达系统，其中M-通道，受体和信号分子在中国仓鼠卵巢（CHO）细胞，大鼠和小鼠颈上级神经节（SCG）和结状神经节（NG）神经元的制剂中表达，以及SCG神经元和心室心肌细胞的共培养物。所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，碳纤维安培和视频成像。我们希望发现AKAP 79/150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。公共卫生相关性：在生物信号传导中包含一个共同基序的信号传导途径是神经系统如何调节的基础，包括情绪状态和情绪的改变，个性以及记忆的获得和使用。我们将研究M型钾离子通道的调节，该通道在调节神经细胞兴奋性中起主导作用，由A激酶介导蛋白AKAP 79/150介导。AKAP 79/150在M通道活性中的机制和功能作用的阐明将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.