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.