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

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（CaN）、离子通道如M型（KCNQ，Kv 7）K+通道、L型Ca 2+通道和G蛋白偶联受体相互作用。活化T细胞核因子（NFAT）家族的转录因子已被证明是可兴奋细胞（包括中枢和外周神经元）中基因转录的活性依赖性调节的基础。结状神经节的感觉神经元负责将来自内脏器官的传入神经发送到脑干，在脑干中控制身体器官。在本项目中，我们将研究KCNQ 2 -3通道在上级颈神经节（SCG）交感神经元中通过NFATc 1-c4成员的转录调节，这些成员被细胞内Ca 2+（Ca 2 +i）信号激活，例如由活动依赖性去极化产生的信号。我们假设Ca 2 +i信号由CaN感知，由AKAP 79/150锚定到L型CaV 1.3 Ca 2+通道，随后是NFAT的去磷酸化，它们进入细胞核，以及M通道基因表达的上调，其在生理和病理生理状态下对兴奋性起负反馈作用。我们将使用各种尖端的方法来测试这一假设。我们进一步假设AKAP 79/150通过多种Gq/11相关受体类型（包括缓激肽、PAR-2、组胺、血管紧张素II和嘌呤能受体）协调NG神经元中M电流的调制，这些受体类型对疼痛和炎症至关重要。我们将确定哪些Gq/11偶联受体调节NG神经元，以及AKAP 79/150在将受体偶联至M型通道中的作用。待使用的技术包括天然SCG和NG神经元的膜片钳电生理学、单个活细胞成像、共聚焦显微镜、荧光素酶基因报告基因测定、基因改变小鼠和活体动物气道体积描记术。我们渴望发现AKAP 79/150在组织M型K+通道的转录调控中的机制，以及在感觉神经元中协调受体信号传导到这些通道中，这些通道在内脏感觉的控制中至关重要。

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 (CaN), ion channels such as M-type (KCNQ, Kv7) K+ channels, L-type Ca2+ channels and G protein-coupled receptors. The nuclear factor of activated T-cells (NFAT) family of transcription factors has been shown to underlie activity-dependent regulation of gene transcription in excitable cells, including central and peripheral neurons. Sensory neurons of the nodose ganglia are responsible for sending afferents from the visceral organs to the brainstem, where body organs are controlled. In this project, we will study the transcriptional regulation of KCNQ2-3 channels in superior cervical ganglion (SCG) sympathetic neurons via NFATc1-c4 members, which are activated by intracellular Ca2+ (Ca2+i) signals, such as those generated by activity-dependent depolarization. We hypothesize the Ca2+i signals are sensed by CaN, anchored by AKAP79/150 to L-type CaV1.3 Ca2+ channels, followed by dephosphorylation of NFATs, their import into the nucleus, and up-regulation of M-channel gene expression that acts as a negative feedback on excitability in physiological and pathophysiological states. We will test this hypothesis using a variety of cutting-edge approaches. We further hypothesize AKAP79/150 to orchestrate modulation of M current in NG neurons by a variety of Gq/11-linked receptor types that are critical to pain and inflammation, including bradykinin, PAR-2, histamine, angiotensin II and purinergic receptors. We will determine which Gq/11-coupled receptors modulate NG neurons, and the role of AKAP79/150 in coupling the receptors to the M-type channels. Techniques to be used include patch-clamp electrophysiology of native SCG and NG neurons, single live-cell imaging, confocal microscopy, luciferase gene-reporter assays, genetically-altered mice and live-animal airway plethysmography. We aspire to discover the mechanisms endowing AKAP79/150 in organizing the transcriptional regulation of M-type K+ channels, and in orchestrating the receptor signaling towards these channels in sensory neurons that are pivotal in control of visceral sensation.