Targeting specific interactions between A-kinase Anchoring Proteins (AKAPs) and ion channels with cell-permeant peptides as a novel mode of therapeutic intervention against pain disorders

针对 A 激酶锚定蛋白 (AKAP) 和离子通道与细胞渗透肽之间的特异性相互作用，作为针对疼痛疾病的治疗干预的新模式

• 批准号: 9815836
• 负责人: MARK S SHAPIRO
• 金额: $ 37.36万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-02 至 2020-01-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9815836
• 关键词: A kinase anchoring protein; Affect; Afferent Neurons; Agonist; Binding; Biological Assay; Calcineurin; Calcineurin inhibitor; Calmodulin; Capsaicin; Cations; Cells; Chinese Hamster Ovary Cell; Color; Complex; Confocal Microscopy; Coupled; Coupling; Dependence; Electrophysiology (science); Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Gene Expression Regulation; Grant; Hydrolysis; Individual; Ion Channel; Knock-in Mouse; Knock-out; Knockout Mice; Link; Macromolecular Complexes; Mammalian Cell; Mediating; Microscopy; Modality; Molecular; Multiprotein Complexes; Mus; Muscarinic M1 Receptor; Muscle; Nanoscopy; Nerve; Neuronal Plasticity; Neurons; Nociception; Nodose Ganglion; Optics; Organism; Pain Disorder; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Physics; Physiology; Play; Potassium Channel; Protein Kinase; Protein Kinase C; Proteins; Regulation; Reporter; Resolution; Role; Scaffolding Protein; Sensory Ganglia; Signal Transduction; Signaling Protein; Specificity; Structure; Sympathetic Ganglia; Synaptic plasticity; System; TRPV1 gene; Technology; Testing; Therapeutic Intervention; Time; Transgenic Organisms; Visible Radiation; channel blockers; desensitization; experimental study; knockin animal; microscopic imaging; mutant; nanometer resolution; neuronal excitability; novel; patch clamp; peer; public health relevance; receptor; reconstitution; reconstruction; recruit; scaffold; sensor; 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（由申请人提供）：多蛋白复合物已经成为无数细胞信号的功能和调节中的时空特异性和效率的机制。特别是，许多离子通道与调节它们的受体或与活性相关的其他离子通道聚集在一起。通常，这种聚集是由支架蛋白介导的，例如AKAP 79/150蛋白，这是这项资助的重点。我们专注于对神经功能至关重要的三种不同通道。一种是“M型”（KCNQ，Kv 7）K+通道，其在神经和肌肉的兴奋性调节中起基本作用。它被认为通过AKAP 79/150与Gq/11偶联受体、蛋白激酶、钙调磷酸酶（CaN）、钙调蛋白（CaM）和磷酸肌醇相关。另一个焦点通道是TRPV 1，这是感觉神经元中的伤害性通道，也被认为是由AKAP 79/150募集的信号蛋白调节的。第三种是L型Ca 2+（CaV1.2）通道，对突触可塑性，基因调控和神经元放电至关重要。我们将使用初级感觉神经元和异源表达的组织培养细胞的“超分辨率”STORM成像来探测含有AKAP 79/150和这三个通道的复合物，其中单个复合物可以用可见光以10-20 nm的分辨率可视化，打破了物理学的衍射障碍。我们假设AKAP 79/150将这些通道中的几个通道聚集在一起以实现功能耦合，我们将通过神经元的膜片钳电生理学来检查。Förster共振能量转移（FRET）也将在全内反射荧光（TIRF）或共聚焦显微镜下进行，进一步测试含有KCNQ，TRPV 1和CaV 1.2通道的复合物。由于这三种通道都与AKAP 79/150结合，我们假设它们与某些G蛋白偶联受体一起在神经元中共同组装成复合物。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的，我们将使用激酶或磷酸酶的快速激活来检查。将使用遗传改变的AKAP 150敲除或敲入小鼠的几种类型的小鼠集落。这个项目使用几种最近才开发出来的尖端、高性能的方法，在神经元信号生理学方面开辟了新的天地。