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)：多蛋白质复合体已经作为一种机制出现，在无数细胞信号的功能和调节中具有时空特异性和效率。特别是，许多离子通道要么与调节它们的受体聚集，要么与其他活动相连的离子通道聚集在一起。通常，聚集是由支架蛋白介导的，例如AKAP79/150蛋白，这是这项拨款的重点。我们关注对神经功能至关重要的三个不同的渠道。一种是“M型”(KCNQ，KV7)K+通道，它在神经和肌肉的兴奋性调节中发挥基础作用。它被认为通过AKAP79/150与GQ/11偶联受体、蛋白激酶、钙调神经磷酸酶(CaN)、钙调蛋白(CaM)和磷脂酰肌醇结合。另一个焦点通道是TRPV1，这是感觉神经元中的一种伤害性通道，也被认为受到AKAP79/150招募的信号蛋白的调节。第三类是L钙通道，它对突触可塑性、基因调控和神经元放电起关键作用。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”风暴成像来探测含有AKAP79/150和这三个通道的复合体，其中单个复合体可以用可见光以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AKAP79/150将这些通道中的几个结合在一起，以实现功能耦合，我们将通过神经元的膜片钳电生理学来研究这一点。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，进一步测试包含KCNQ、TRPV1和CaV1.2通道的复合体。由于所有这三个通道都与AKAP79/150结合，我们假设它们在神经元中共同组装成复合体，与某些G蛋白偶联受体一起。此外，我们假设这些复合体不是静态的，而是受到其他细胞信号的动态调节，我们将使用激酶或磷酸酶的快速激活来研究这些信号。将利用几种类型的转基因AKAP150基因敲除或敲入小鼠的小鼠群体。这个项目使用了最近开发的几种尖端的、高功率的方法，为神经元信号的生理学开辟了新的天地。