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: 8217085
• 负责人: MARK S SHAPIRO
• 金额: $ 38.7万
• 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/8217085
• 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. PUBLIC HEALTH RELEVANCE: K+ and Ca2+ ion channels underlie critical activities in the brain and peripheral nervous system, and their modulation is involved in many diseases, and in many therapeutic interventions. We study regulation of these channels by lipid signaling molecules, and by Ca2+-binding proteins, using primary nerve cells, molecular biology, and biochemical/biophysical techniques allowing study of signaling pathways at the single-cell and molecular levels.

描述（由申请人提供）：离子通道电流是大多数生物体电活动的基本单位。在我们的神经系统中，K+和Ca2+通道至关重要，它们的调节提供了一种直接控制神经元兴奋性和神经递质（NT）在突触下的方法。该调制对于基本神经功能至关重要，它们的理解应为涉及大脑，神经和肌肉的一系列疾病的新型医疗干预方式做出贡献。我们主要关注KCNQ（KV7/M型）K+通道的家族，该家族是几个神经元K+电流的基础，也是CAV2.2（N型）Ca2+通道。特别是，我们试图阐明作用于这些离子通道的GQ/11-介导途径的分子机制，以及这些途径对NT释放的功能效应。 KCNQ和CAV2.2通道都已被PIP2调节，该项目研究了PIP2介导的调节的分子机制。 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.在特定的目标＃1中，我们将使用内而外的宏观捕获，表面等离子体的谐振光谱和等温量热法研究PIP2，钙调蛋白及其结合结构域之间的生化和分子相互作用。我们假设PIP2和CAM在通道上的重叠域作用，提供了变构的“串扰”。在特定的目标＃2中，我们将探测受体介导的磷脂酰肌醇4和5-激酶的刺激的机制，我们假设这些磷脂醇的受体特异性是M通道调节中受体特异性的基础。我们还将探测Ca2+I信号传导中受体特异性的潜在机制，重点是Rho家族的蛋白质IRIT，DAG-KINASE和小GTPase。在特定的目标＃3中，我们使用两种创新方法研究了M通道调控在NT释放中的功能作用。第一个是一种共同培养，其中SCG神经元在自发性地抑制的心肌细胞上，使用野生型或受体敲除小鼠培养的细胞，使肾上腺素能突触在菜肴中培养。第二种使用形成自动化的孤立海马神经元，从而可以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，因为这些渠道在调节神经元的兴奋性方面起主要作用，并且它们的调节可能是情绪状态，身体器官的记忆和调节的变化。因此，我们的研究应为各种神经疾病的新型治疗干预方式开发的基础。 公共卫生相关性：K+和Ca2+离子渠道是大脑和周围神经系统中关键活动的基础，并且它们的调节涉及许多疾病以及许多治疗性干预措施。我们通过脂质信号分子和Ca2+结合蛋白来研究这些通道的调节，使用原代神经细胞，分子生物学和生化/生物物理技术，从而可以研究单细胞和分子水平的信号通路。