Clustering of individual and diverse ion channels together into complexes, and their functional coupling, mediated by A-kinase anchoring protein 79/150 in neurons

单个和不同的离子通道聚集成复合物，以及它们的功能耦合，由神经元中的 A-激酶锚定蛋白 79/150 介导

• 批准号: 9212929
• 负责人: MARK S SHAPIRO
• 金额: $ 2万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212929
• 关键词: A kinase anchoring protein; Affect; Afferent Neurons; Agonist; Binding; Biological Assay; Calcineurin; Calcineurin inhibitor; Calmodulin; Capsaicin; Cations; Cell Culture Techniques; Cells; Chinese Hamster Ovary Cell; Color; Complex; Confocal Microscopy; Coupled; Coupling; Dependence; Electrophysiology (science); Energy Transfer; Fluorescence; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Gene Expression Regulation; Grant; Health; Hydrolysis; Individual; Ion Channel; Knock-in Mouse; Knock-out; Knockout Mice; Life; Link; Macromolecular Complexes; Mammalian Cell; Mediating; Microscopy; Modality; Molecular; Mus; Muscarinic M1 Receptor; Muscle; Nanoscopy; Nerve; Neuronal Plasticity; Neurons; Nociception; Nodose Ganglion; Optics; Organism; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Physics; Physiology; Play; Potassium Channel; Primary Cell Cultures; Protein Kinase; Protein Kinase C; Proteins; Recruitment Activity; Regulation; Reporter; Resolution; Role; Scaffolding Protein; Sensory Ganglia; Signal Transduction; Signaling Protein; Specificity; Structure; Sympathetic Ganglia; Synaptic plasticity; System; TRPV1 gene; Technology; Testing; Time; Transgenic Organisms; Visible Radiation; channel blockers; desensitization; knockin animal; microscopic imaging; mutant; nanometer; neuronal excitability; optical sensor; patch clamp; peer; protein complex; receptor; receptor coupling; reconstitution; research study; scaffold; single molecule; spatiotemporal; tissue/cell culture

 DESCRIPTION (provided by applicant): Multi-protein complexes have emerged as a mechanism for spatiotemporal specificity and efficiency in the function and regulation of myriad cellular signals. In particular, many ion channels are clustered either with the receptors that modulate them, or with other ion channels whose activities are linked. Often, the clustering is mediated by scaffolding proteins, such as the AKAP79/150 protein that is a focus of this grant. We focus on three different channels critical to nervous function. One is the "M-type" (KCNQ, Kv7) K+ channel that plays fundamental roles in the regulation of excitability in nerve and muscle. It is thought to associate with Gq/11- coupled receptors, protein kinases, calcineurin (CaN), calmodulin (CaM) and phosphoinositides via AKAP79/150. Another channel of focus is TRPV1, a nociceptive channel in sensory neurons that is also thought to be regulated by signaling proteins recruited by AKAP79/150. The third are L-type Ca2+ (CaV1.2) channels that are critical to synaptic plasticity, gene regulation and neuronal firing. We will probe complexes containing AKAP79/150 and these three channels using "super-resolution" STORM imaging of primary sensory neurons and heterologously-expressed tissue-culture cells, in which individual complexes can be visualized at 10-20 nm resolution with visible light, breaking the diffraction barrier of physics. We hypothesize that AKAP79/150 brings several of these channels together to enable functional coupling, which we will examine by patch-clamp electrophysiology of the neurons. Förster resonance energy transfer (FRET) will also be performed under total internal reflection fluorescence (TIRF) or confocal microscopy, further testing for complexes containing KCNQ, TRPV1 and CaV1.2 channels. Since all three of these channels bind to AKAP79/150, we hypothesize that they co-assemble into complexes in neurons, together with certain G protein-coupled receptors. Furthermore, we hypothesize these complexes to not be static, but rather to be dynamically regulated by other cellular signals, which we will examine using rapid activation of kinases or phosphatases. Several types of mouse colonies of genetically altered AKAP150 knock-out or knock-in mice will be utilized. This project breaks new ground into the physiology of signaling in neurons, using several cutting-edge, high- powered approaches that have just recently been developed.

 描述（由适用提供）：多蛋白络合物已成为一种空间时间特异性和效率在众多细胞信号的功能和调节中的机制。特别是，许多离子通道都与调节它们的接收器或其他活动链接的离子通道聚集。通常，聚类是由脚手架蛋白（例如AKAP79/150蛋白）介导的，该蛋白是该赠款的重点。我们专注于至关重要的神经功能的三个不同渠道。一种是“ M型”（KCNQ，KV7）K+通道，它在调节神经和肌肉的兴奋性方面起着基本作用。人们认为它通过AKAP79/150与GQ/11-偶联受体，蛋白激酶，钙调蛋白（CAN），钙调蛋白（CAM）和磷酸肌醇相关联。另一个焦点是TRPV1，这是感觉神经元中的伤害感受通道，也被认为是通过AKAP79/150募集的信号蛋白来调节的。第三个是L型Ca2+（Cav1.2）通道，对合成可塑性，基因调节和神经元放电至关重要。我们将使用原代感觉神经元和异源表达的组织培养细胞的“超分辨率”风暴成像进行探测包含AKAP79/150的复合物，并使用这三个通道进行探测，其中可以在10-20 nm分辨率的情况下以可见光来可视化单个复合物，从而破坏了物理学的衍射屏障。我们假设AKAP79/150将这些通道中的几个汇总在一起以实现功能耦合，我们将通过神经元的贴片钳电生理学检查。 Förster共振能量转移（FRET）也将在总内反射荧光（TIRF）或共聚焦显微镜下进行，进一步测试含有KCNQ，TRPV1和CAV1.2通道的复合物。由于所有这三个通道都与AKAP79/150结合，因此我们假设它们与某些G蛋白偶联受体共同组装成神经元中的复合物。此外，我们假设这些复合物不是静态的，而是由其他细胞信号动态调节，我们将使用激酶或磷酸酶的快速激活进行检查。将使用几种类型的遗传改变AKAP150敲除或敲除小鼠的小鼠菌落。该项目使用最近开发的几种尖端，高功率的方法，将新的基础介绍为神经元中信号传导的生理。