Mechanisms and functional role of lipid-mediated modulation of neuronal channels

脂质介导的神经通道调节的机制和功能作用

• 批准号: 8462002
• 负责人: MARK S SHAPIRO
• 金额: $ 42.7万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462002
• 关键词: 1,2-diacylglycerol; 1-Phosphatidylinositol 4-Kinase; Action Potentials; Adrenergic Agents; Affinity; Affinity Chromatography; Agonist; Astrocytes; Bathing; Binding; Binding Proteins; Biochemical; Biological Assay; Biosensor; Brain; CDC42 gene; Calcified; Calmodulin; Calorimetry; Cardiac Myocytes; Cell membrane; Cells; Characteristics; Chimeric Proteins; Chinese Hamster Ovary Cell; Coculture Techniques; Consumption; Dependence; Development; Diacylglycerol Kinase; Diglycerides; Disease; Emotional; Family; Fluorescence Resonance Energy Transfer; Generations; Giant Cells; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hippocampus (Brain); Human; Hydrolysis; Individual; Intervention; Ion Channel; Island; Knock-out; Knockout Mice; Life; Link; Lipids; Measurement; Measures; Mediating; Medical; Memory; Methods; Modeling; Molecular; Molecular Biology; Monitor; Monomeric GTP-Binding Proteins; Mus; Muscle; Nerve; Nervous system structure; Neurons; Oocytes; Optics; Organ; Organism; Output; Pathway interactions; Peripheral Nervous System; Phosphatidic Acid; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phosphotransferases; Play; Polylysine; Potassium Channel; Preparation; Production; Productivity; Protein Isoforms; Protein Kinase C; Proteins; Reading; Receptor Signaling; Regulation; Reporter; Research; Rho-associated kinase; Rodent; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Specificity; Spectrum Analysis; Structure of superior cervical ganglion; Surface Plasmon Resonance; Synapses; System; Techniques; Testing; Therapeutic Intervention; Time; Ventricular; Wild Type Mouse; Work; adrenergic; base; innovation; mutant; neuronal excitability; neurotransmitter release; novel; patch clamp; public health relevance; receptor; receptor coupling; reconstitution; research study; rho; tissue/cell culture; voltage

DESCRIPTION (provided by applicant): Ion channel currents are the fundamental units of electrical activity in most organisms. In our nervous system, K+ and Ca2+ channels are critical, and their regulation provides a way to directly control neuronal excitability and release of neurotransmitter (NT) at synapses. The modulations are crucial to basic nervous function and their understanding should contribute to novel modes of medical interventions for a range of disorders involving the brain, nerves and muscles. We focus primarily on the family of KCNQ (Kv7/M-type) K+ channels that underlie several neuronal K+ currents, and also on CaV2.2 (N-type) Ca2+ channels. In particular, we seek to elucidate the molecular mechanisms of Gq/11- mediated pathways that act on these ion channels, and the functional effect of these pathways on release of NT. Both KCNQ and CaV2.2 channels have emerged as being regulated by PIP2, and this project studies the molecular mechanisms of the PIP2-mediated regulation. We use heterologous systems in which the channels, receptors and signaling molecules are expressed in mammalian tissue- culture cells or oocytes, preparations of rodent sympathetic superior cervical ganglion (SCG) neurons, hippocampal neurons grown on astrocyte "micro-islands" and a co-culture of SCG neurons and mouse cardiomyocytes. In specific aim #1, we will study the biochemical and molecular interactions between PIP2, calmodulin and their binding domains on KCNQ channels using inside-out macropatches, surface plasmon resonance spectroscopy and isothermal calorimetry. We hypothesize PIP2 and CaM to act on overlapping domains on the channels, providing for allosteric "cross-talk." In specific aim #2, we will probe the mechanism of receptor-mediated stimulation of phosphatidylinositol 4- and 5-kinases that we hypothesize to underlie the receptor specificity in modulation of M channels. We will also probe the underlying mechanism of receptor-specificity in Ca2+i signaling, focusing on the proteins IRBIT, DAG- kinase, and small GTPases of the Rho family. In specific aim #3, we investigate the functional role of M-channel regulation in control over NT release, using two innovative approaches. The first is a co- culture in which SCG neurons make adrenergic synapses on spontaneously-beating cardiomyocytes cultured in a dish, using cells taken from wild-type or receptor knock-out mice. The second uses isolated hippocampal neurons which form autapses, allowing the input/output relation between action potential and NT release to be directly determined. The molecules and signaling pathways that we study have broad relevance to human health and disease, as these channels play a dominant role in regulating excitability of neurons, and their regulation likely underlies changes in emotional state, memory and regulation of body organs. Thus, our research should provide the basis for the development of novel modes of therapeutic intervention for a variety of nervous diseases.

描述（由申请人提供）：离子通道电流是大多数生物体中电活动的基本单位。在我们的神经系统中，K+和Ca 2+通道是至关重要的，它们的调节提供了一种直接控制神经元兴奋性和突触处神经递质（NT）释放的方法。这些调节对基本神经功能至关重要，对它们的理解应该有助于对涉及大脑，神经和肌肉的一系列疾病进行新的医疗干预模式。我们主要关注KCNQ（Kv 7/M型）K+通道家族，这些通道是几种神经元K+电流的基础，也关注CaV2.2（N型）Ca 2+通道。特别是，我们试图阐明Gq/11介导的途径，这些离子通道的作用的分子机制，以及这些途径对NT释放的功能性影响。KCNQ和CaV2.2通道都是由PIP 2调节的，本项目研究PIP 2介导的调节的分子机制。我们使用其中通道、受体和信号分子在哺乳动物组织培养细胞或卵母细胞中表达的异源系统、啮齿动物交感上级颈神经节（SCG）神经元的制备物、在星形胶质细胞“微岛”上生长的海马神经元以及SCG神经元和小鼠心肌细胞的共培养物。在具体目标#1中，我们将使用由内而外的macropatches，表面等离子体共振光谱和等温量热法研究PIP 2，钙调素及其在KCNQ通道上的结合结构域之间的生物化学和分子相互作用。我们假设PIP 2和CaM作用于通道上的重叠结构域，提供变构“串扰”。“在具体目标#2中，我们将探索受体介导的磷脂酰肌醇4-和5-激酶刺激机制，我们假设这些机制是M通道调节中受体特异性的基础。我们还将探索Ca 2 +i信号传导中受体特异性的潜在机制，重点关注Rho家族的蛋白质伊尔比特、DAG激酶和小GTP酶。在具体目标#3中，我们使用两种创新方法研究了M通道调节在控制NT释放中的功能作用。第一种是共培养，其中SCG神经元在培养皿中培养的自发跳动的心肌细胞上产生肾上腺素能突触，使用取自野生型或受体敲除小鼠的细胞。第二种方法使用形成autapses的分离的海马神经元，允许直接确定动作电位和NT释放之间的输入/输出关系。我们研究的分子和信号通路与人类健康和疾病具有广泛的相关性，因为这些通道在调节神经元的兴奋性方面发挥着主导作用，它们的调节可能是情绪状态，记忆和身体器官调节的基础。因此，我们的研究应该为各种神经疾病的治疗干预的新模式的发展提供基础。