Structure And Function Of Neurotransmitter Receptor Ion Channels

神经递质受体离子通道的结构和功能

基本信息

批准号：
7594147
负责人：
Mark L Mayer
金额：
$ 51.78万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7594147
关键词：
Affinity Agonist Allosteric Regulation Analgesics Anions Binding Binding Sites Biochemical Brain Bromine Calcium Cations Chloride Ion Chlorides Cleaved cell Closure Collaborations Complex Crystallization Crystallography Cycloleucine Development Electrostatics Excitatory Amino Acid Antagonists Excitatory Synapse Family Functional disorder Gated Ion Channel Gene Family Glues Glutamate Receptor Glutamates Glycine Halogens Human Individual Ion Channel Ion Channel Gating Ions Laboratories Ligand Binding Ligand Binding Domain Ligands Mammals Mediating Membrane Proteins Mental disorders Methionine Migraine Molecular Molecular Conformation Mutation N-Methyl-D-Aspartate Receptors N-Methylaspartate NR1 gene NR3A NMDA receptor Names Nervous system structure Neurologic Neurotransmitter Receptor Object Attachment Permeability Play Property Proteins Range Resolution Role Sampling Series Serine Side Signal Transduction Sodium Stroke Structure Testing Tryptophan Variant Water Work X ray diffraction analysis X-Ray Diffraction base carboxylate design dimer human NR1 protein kainate neurophysiology novel novel therapeutics patch clamp prevent receptor research study response transmission process willardiine

项目摘要

Ionotropic glutamate receptors (iGluRs) are membrane proteins which act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of GluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. This information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function. Crystallographic and functional analysis of glutamate receptor ligand complexes Alushin and Mayer in collaboration with Jane High resolution crystal structures were solved for four GluR5 subtype selective antagonist complexes; three are derivatives of willardiine based compounds for which we solved the 1st GluR5 antagonist complex in 2006, and one is for a decahydroisoquinoline which has potential as an analgesic and drug to reduce migraine. The structures reinforce the idea that even when bound with competitive antagonists glutamate receptors can sample a range of conformational space, similar in principle to the variation in domain closure observed for partial agonists, with the key difference that the difference in domain closure for individual antagonists does not cross the threshold necessary to trigger ion channel gating. One of the structures revealed a novel protein ligand interaction, named a halogen bond, formed by a contact between a carboxylate side chain and a ligand bromine atom. Crystallographic and functional analysis of allosteric ion binding sites Plested and Mayer in collaboration with Biggin Kainate subtype glutamate receptors are strongly modulated by monovalent anions and cations and in the absence of either chloride or sodium the receptors become non functional. A combined experimental approach using crystallography and patch clamp recording was used to identify the binding site for anions. The chloride ion binds in the dimer interface between two subunits and acts as electrostatic glue which helps to stabilize dimer assemblies in their active conformation. In the absence of chloride the dimers dissociate, and the receptor desensitizes. Mutations which disrupt chloride binding have the same effect in functional experiments. These results reveal that instead of acting in a modulatory, allosteric manner, anions are instead essential structural components of the receptor in its active conformation. In ongoing work using the same approach, we are working solve the structure of the cation binding site. Structural analysis of NR3 ligand binding selectivity Yao and Mayer The NMDA receptor NR3A subunit is expressed widely in the developing CNS of mammals. Co-assembly of NR3A with NR1 and NR2 modifies NMDA receptor-mediated responses, reducing calcium permeability. In previous work we characterized the ligand binding properties of NR3A using a highly purified water soluble NR3A ligand binding domain. High resolution crystal structures have now been solved for NR3A complexes with glycine, D-serine and ACPC, and for NR3B complexes with glycine and D-serine. These reveal that despite the substitution by methionine of a large tryptophan residue, which in the NR1 subunit fills up the binding pocket, preventing the binding of glutamate, the binding pocket of NR1, NR3A and NR3B is unusually small compared to other glutamate receptor subtypes which bind glutamate, indicating that steric occlusion is a common principle for achieving glycine selectivity. In ongoing work a series of mutations based on the crystal structures for NR1 and NR3 subunits are being tested to test the hypothesis that subunit specific differences in ligand affinity result in part from formation of closed cleft, i.e. active conformations

离子型谷氨酸受体 (iGluR) 是膜蛋白，充当分子孔并介导哺乳动物神经系统中大多数兴奋性突触的信号传递。人类离子型谷氨酸受体 (iGluR) 的 7 个基因家族编码 18 个亚基，这些亚基组装形成 3 个主要功能家族，以 20 世纪 70 年代末首次用于识别 iGluR 亚型的配体命名：AMPA、红藻氨酸和 NMDA。由于它们在正常大脑功能和发育中发挥重要作用，并且越来越多的证据表明 iGluR 活性功能障碍会介导多种神经和精神疾病以及中风期间的损伤，细胞和分子神经生理学实验室的大量工作致力于分析GluR 在分子水平上发挥作用。通过蛋白质结晶和 X 射线衍射解析的原子分辨率结构提供了一个设计电生理和生化实验的框架，以定义配体识别和离子通道活性门控的变构机制。这些信息将有助于开发具有新颖治疗应用的亚型选择性拮抗剂和变构调节剂，并揭示在大脑功能中发挥关键作用的复杂蛋白质机器的内部运作。谷氨酸受体配体复合物的晶体学和功能分析阿卢辛和梅耶尔与简合作解析了四种 GluR5 亚型选择性拮抗剂复合物的高分辨率晶体结构；其中三种是基于 Willardiine 的化合物的衍生物，我们于 2006 年解决了第一个 GluR5 拮抗剂复合物的问题，另一种是十氢异喹啉，它具有作为镇痛剂和减少偏头痛的药物的潜力。这些结构强化了这样的想法，即即使与竞争性拮抗剂结合，谷氨酸受体也可以采样一系列构象空间，原则上类似于部分激动剂观察到的结构域闭合变化，其关键区别在于各个拮抗剂的结构域闭合差异不超过触发离子通道门控所需的阈值。其中一个结构揭示了一种新的蛋白质配体相互作用，称为卤素键，由羧酸侧链和配体溴原子之间的接触形成。别构离子结合位点的晶体学和功能分析 Plested 和 Mayer 与 Biggin 合作红藻氨酸亚型谷氨酸受体受到单价阴离子和阳离子的强烈调节，并且在缺乏氯或钠的情况下，受体变得无功能。使用晶体学和膜片钳记录的组合实验方法来识别阴离子的结合位点。氯离子结合在两个亚基之间的二聚体界面中，并充当静电胶，有助于稳定二聚体组件的活性构象。在没有氯化物的情况下，二聚体解离，受体脱敏。破坏氯离子结合的突变在功能实验中具有相同的效果。这些结果表明，阴离子不是以调节、变构的方式起作用，而是活性构象中受体的重要结构成分。在使用相同方法的持续工作中，我们正在努力解决阳离子结合位点的结构。 NR3配体结合选择性的结构分析姚和迈耶 NMDA 受体 NR3A 亚基在哺乳动物发育中的 CNS 中广泛表达。 NR3A 与 NR1 和 NR2 的共组装可改变 NMDA 受体介导的反应，降低钙渗透性。在之前的工作中，我们使用高度纯化的水溶性 NR3A 配体结合域来表征 NR3A 的配体结合特性。 NR3A 与甘氨酸、D-丝氨酸和 ACPC 的复合物以及 NR3B 与甘氨酸和 D-丝氨酸的复合物现已解析出高分辨率晶体结构。这些表明，尽管用蛋氨酸取代了一个大的色氨酸残基，该残基在 NR1 亚基中填充了结合袋，阻止了谷氨酸的结合，但与其他谷氨酸受体亚型相比，NR1、NR3A 和 NR3B 的结合袋异常小。结合谷氨酸，表明空间封闭是实现甘氨酸选择性的常见原理。在正在进行的工作中，正在测试一系列基于 NR1 和 NR3 亚基晶体结构的突变，以检验以下假设：配体亲和力的亚基特异性差异部分是由于闭合裂口（即活性构象）的形成造成的。