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

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为一系列涉及大脑、神经和肌肉的疾病提供新的医疗干预模式。我们主要关注KCNQ （Kv7/ m型）K+通道家族，它们是几种神经元K+电流的基础，也关注CaV2.2 （n型）Ca2+通道。特别是，我们试图阐明Gq/11-介导的途径作用于这些离子通道的分子机制，以及这些途径对NT释放的功能影响。KCNQ和CaV2.2通道都被发现受到PIP2的调节，本项目研究PIP2介导的调节的分子机制。我们使用异种系统，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，啮齿动物交感颈上神经节（SCG）神经元的制备，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。在具体目标#1中，我们将使用内外大斑块、表面等离子体共振光谱和等温量热法研究PIP2、钙调蛋白及其在KCNQ通道上的结合域之间的生化和分子相互作用。我们假设PIP2和CaM作用于通道上的重叠区域，提供变构“串扰”。在特定目标#2中，我们将探讨受体介导的磷脂酰肌醇4-和5-激酶刺激机制，我们假设这是M通道调节中受体特异性的基础。我们还将探讨Ca2+i信号传导中受体特异性的潜在机制，重点关注Rho家族的IRBIT、DAG-激酶和小gtpase。在具体目标#3中，我们使用两种创新方法研究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，使用的细胞来自野生型或受体敲除小鼠。第二种方法使用分离的海马神经元，这些神经元形成autapses，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，因为这些通道在调节神经元的兴奋性中起主导作用，它们的调节可能是情绪状态、记忆和身体器官调节变化的基础。因此，我们的研究应该为开发针对各种神经疾病的新型治疗干预模式提供基础。

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.