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

AKAP79/150 在 M 电流控制中的机制和功能作用

• 批准号: 8549448
• 负责人: MARK S SHAPIRO
• 金额: $ 46.5万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8549448
• 关键词: A kinase anchoring protein; Acetylcholine; Afferent Neurons; Agonist; Angiotensin II; Animals; Binding; Binding Sites; Biological; Biological Assay; Bradykinin; Brain; Brain Stem; Calcineurin; Calmodulin; Capsaicin; Cell Communication; Cell Nucleus; Cell physiology; Cells; Confocal Microscopy; Coughing; Cyclic AMP-Dependent Protein Kinases; Cyclosporine; Cytoplasm; DNA; Dependence; Depressed mood; Dimerization; Disease; Dose; Egtazic Acid; Electrophysiology (science); Elements; Emotional; Enhancers; Esthesia; Family; Feedback; Fura-2; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Gene Expression; Genes; Genetic Transcription; Genomics; Health; Hippocampus (Brain); Histamine; Image; Inflammation; Ion Channel; Irritants; Life; Light; Link; Luc Gene; Luciferases; Measures; Mediating; Membrane; Memory; Messenger RNA; Methods; Modeling; Molecular; Moods; Mus; Mutation; Nervous System Physiology; Nervous system structure; Neurons; Nodose Ganglion; Organ; PC12 Cells; Pain; Pain Measurement; Peripheral; Personality; Pharmaceutical Preparations; Physiological; Play; Plethysmography; Potassium Channel; Protein Dephosphorylation; Protein Isoforms; Protein Kinase C; Purinoceptor; Rattus; Receptor Signaling; Recruitment Activity; Regulation; Regulatory Element; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Seizures; Sensory; Shapes; Signal Pathway; Signal Transduction; Site; Structure of superior cervical ganglion; T-Cell Activation; Techniques; Testing; Time; Transcriptional Regulation; Up-Regulation; VIVIT peptide; Visceral; Visceral Afferents; cellular imaging; channel blockers; chromatin immunoprecipitation; constriction; ganglion cell; knock-down; member; mouse model; mutant; neuronal excitability; nuclear factors of activated T-cells; patch clamp; promoter; receptor; receptor coupling; research study; response; sensor; small hairpin RNA; transcription factor; voltage

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.

描述（由申请人提供）：a激酶锚定蛋白（AKAPs）通过将其分子成分聚集到离散的亚细胞微域来组织许多细胞内信号通路。其中一个AKAP， AKAP79/150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、Kv7） K+通道、l型Ca2+通道和G蛋白偶联受体相互作用。活化t细胞核因子（NFAT）转录因子家族已被证明是可兴奋细胞（包括中枢和外周神经元）中基因转录活性依赖调节的基础。结节神经节的感觉神经元负责将内脏器官的传入信号发送到控制身体器官的脑干。在本项目中，我们将通过NFATc1-c4成员研究KCNQ2-3通道在上颈神经节（SCG）交感神经元中的转录调控，这些成员被细胞内Ca2+ (Ca2+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Ca2+i信号由CaN感知，由AKAP79/150锚定到l型CaV1.3 Ca2+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，这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方法来检验这一假设。我们进一步假设AKAP79/150通过多种Gq/11相关受体（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电流的调节，这些受体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AKAP79/150在将受体偶联到m型通道中的作用。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，荧光素酶基因报告分析，转基因小鼠和活体气道容积描记术。我们渴望发现赋予AKAP79/150组织m型K+通道转录调控的机制，以及在控制内脏感觉的关键感觉神经元中向这些通道协调受体信号的机制。