Structure And Function Of Neurotransmitter Receptor Ion Channels

神经递质受体离子通道的结构和功能

• 批准号: 7968531
• 负责人: Mark L Mayer
• 金额: $ 128.12万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/7968531
• 关键词: AMPA Receptors; Acute Brain Injuries; Adopted; Affinity; Agonist; Allosteric Regulation; Amino Acids; Ammonium; Anions; Aspartate; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Brain; Calcium; Calcium Binding; Cations; Cells; Cesium; Chemicals; Chloride Ion; Chlorides; Cleaved cell; Complex; Coupled; Crystallization; Crystallography; Cycloleucine; DNA Sequence Rearrangement; Data; Data Set; Databases; Development; Divalent Cations; Epilepsy; Evolution; Excitatory Synapse; Family; Fluorescence; Free Energy; Functional disorder; Gated Ion Channel; Gene Family; Glutamate Receptor; Glutamates; Glycine; Goals; Human; Hydrogen Bonding; Ion Channel; Ion Channel Gating; Ions; Kainic Acid Receptors; Kinetics; Laboratories; Ligand Binding; Ligand Binding Domain; Ligands; Lithium; Lysine; Magnesium; Mammalian Cell; Measurement; Measures; Mediating; Mediator of activation protein; Membrane Proteins; Mental disorders; Methods; Modeling; Mole the mammal; Molecular; Molecular Conformation; Molecular Machines; Molecular Models; Molecular Profiling; Molecular Weight; Motion; Mutation; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NR1 gene; Names; Nervous system structure; Neurodegenerative Disorders; Neurologic; Neurotransmitter Receptor; Neurotransmitters; Oxygen; Phenotype; Physiology; Play; Post-Translational Protein Processing; Potassium; Preparation; Protein Sequence Analysis; Proteins; Publishing; Receptor Gene; Resolution; Robot; Role; Rubidium; Schizophrenia; Series; Serine; Signal Transduction; Site; Sodium; Solutions; Solvents; Stroke; Structure; Synaptic Transmission; Synaptic plasticity; System; Techniques; Thermodynamics; Titrations; Tryptophan; Vertebral column; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; analytical ultracentrifugation; base; carboxylate; chronic pain; depression; desensitization; design; dimer; electric field; kainate; member; molecular dynamics; molecular modeling; mutant; nanolitre; neurophysiology; novel; novel therapeutics; patch clamp; protein expression; protein folding; receptor; research study; response; sedimentation equilibrium; sedimentation velocity; simulation; structural biology; sugar; transmission process

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 iGluR 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 mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. 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 an allosteric binding site for sodium Kainate subtype glutamate receptors are strongly modulated by monovalent anions and cations. In the absence of either chloride or sodium the receptors become non functional. A combined experimental approach using crystallography, patch clamp recording, and all atom molecular dynamics simulations was used to identify the binding site for sodium, and the mechanism by which sodium modulates kainate receptor activity. Structures were solved for the GluR5 ligand binding domain dimer complex with lithium, sodium, potassium, rubidium, cesium and ammonium ions in the cation binding site. There are two sodium binding sites in a dimer assembly, one per subunit, and these flank the previously identified anion binding site which lies on the molecular two-fold axis of symmetry. Sodium acts by stabilizing the dimer assembly in its active conformation required for ion channel gating, and in the absence of sodium the receptors desensitize much faster. Sodium selectivity is conferred by a high electric field strength in the cation binding site, but larger cations can bind with lower affinity. Functional studies show that the cation binding site is allosterically coupled to the anion binding site. All atom MD simulations and free energy calculations reveal that the binding of chloride is favored by 3-5 kcal per mole when the cation binding site is occupied by sodium. Mutational analysis and molecular modeling revealed that it is possible to convert the sodium binding site to one which has micromolar affinity for the divalent cations calcium and magnesium. This was achieved by substituting an aspartate residue for a hydrophobic amino acid which caps the sodium binding site in kainate receptors. AMPA receptors, which are insensitive to allosteric modulation by either sodium or calcium harbor a lysine at this site. Amino acid sequence analysis indicates that the divergence between iGluRs with and without allosteric binding sites for sodium arose early in evolution. We were unable to crystallize a kainate receptor with the aspartate mutation, and so made molecular models of the binding site. The model reveals a unique geometry, with three closely apposed carboxylate groups, together with two backbone carbonyl oxygen atoms, that provides ligands for binding calcium similar to those found in the protein databank for a diverse range of proteins with calcium binding sites. Direct measurements of the effects of allosteric ions on dimer assembly by kainate subtype iGluRs is not possible using electrophysiological techniques. In order to obtain proof that allosteric ions regulate dimer formation, a series of GluR6 dimer interface mutations, remote from the ion binding sites, was developed with the goal of developing a preparation amenable to analysis by analytical ultracentrifugation (AUC). The mutants were designed on the basis of crystal structures for wild type GluR5 dimers, using electrophysiological analysis to search for a phenotype with slowed kinetics of desensitization. The isolated ligand binding domains of the same mutants were prepared and their affinity for dimer formation measured both by sedimentation velocity analysis over a range of protein concentrations, and by sedimentation equilibrium. Structural studies on the amino terminal domain of iGluRs Glutamate receptor ion channels are multidomain membrane proteins which assemble of tetramers of molecular weight approximately 440 kD. Numerous crystal structures have been solved for the ligand binding domains which have a molecular weight of approximately 30 kD per subunit, approximately of the mass of an intact receptor. Extensive trials with bacterial expression systems, which with one exception, have been used for all published ligand binding domain structures, failed to produce monodisperse soluble protein for other iGluR domains. The amino terminal domain (ATD) is an important structural target because it controls subtype selective assembly in native iGluRs, limiting assembly to members of the same functional family. Protein expression at levels sufficient for structural biology in mammalian cells is much more difficult than expression in E.coli but has the advantages that multiple check points select for correctly folded proteins, and add sugars and other post translational modifications required for normal function. Although a variety of cell biological and biochemical techniques are required to subsequently trim the sugar chains, in order to obtain proteins which crystallize and diffract to high resolution, and the yields are lower than for prokaryotic expression, currently this is the only approach likely to succeed for studies of the ATD. In ongoing work the ATDs from several iGluR subtypes have been screened for expression in mammalian cells. Crystallization trials have been performed using a nano liter pipetting robot, and for one diffraction data to a resolution of 2.65 for a complete data set was obtained at APS. Structure solution and refinement is complete. Structural analysis of NR3 ligand binding selectivity NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well characterized members of the NMDA receptor gene family which play key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations we investigated mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. Replica exchange MD simulations of 650 ns duration revealed numerous interdomain contacts which stabilize the agonist bound closed cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits. Mutation of these sites destabilized ligand binding measured by titration assays using quenching of endogenous tryptophan fluorescence.

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iGluR 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、离子通道活性的门控和变构调节剂的作用的潜在机制。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。 钠变构结合位点的晶体学和功能分析 红藻氨酸亚型谷氨酸受体受单价阴离子和阳离子强烈调节。在没有氯或钠的情况下，受体变得不起作用。使用晶体学、膜片钳记录和所有原子分子动力学模拟的组合实验方法来确定钠的结合位点以及钠调节红藻氨酸受体活性的机制。 解析了在阳离子结合位点中具有锂、钠、钾、铷、铯和铵离子的 GluR5 配体结合域二聚体复合物的结构。二聚体组装体中有两个钠结合位点，每个亚基一个，这些结合位点位于先前确定的位于分子双重对称轴上的阴离子结合位点的侧面。钠的作用是使二聚体组件稳定在离子通道门控所需的活性构象中，并且在没有钠的情况下，受体的脱敏速度更快。钠选择性是由阳离子结合位点的高电场强度赋予的，但较大的阳离子可以以较低的亲和力结合。功能研究表明，阳离子结合位点与阴离子结合位点变构偶联。所有原子 MD 模拟和自由能计算表明，当阳离子结合位点被钠占据时，每摩尔 3-5 kcal 有利于氯离子的结合。突变分析和分子模型表明，可以将钠结合位点转化为对二价阳离子钙和镁具有微摩尔亲和力的结合位点。这是通过用天冬氨酸残基取代覆盖红藻氨酸受体中钠结合位点的疏水氨基酸来实现的。 AMPA 受体对钠或钙的变构调节不敏感，在此位点含有赖氨酸。氨基酸序列分析表明，有或没有钠变构结合位点的 iGluR 之间的差异在进化早期就出现了。我们无法结晶具有天冬氨酸突变的红藻氨酸受体，因此制作了结合位点的分子模型。该模型揭示了一种独特的几何结构，具有三个紧密并列的羧酸基团以及两个主链羰基氧原子，提供了用于结合钙的配体，类似于蛋白质数据库中针对具有钙结合位点的各种蛋白质的配体。 使用电生理学技术不可能直接测量变构离子对红藻氨酸亚型 iGluR 的二聚体组装的影响。为了获得变构离子调节二聚体形成的证据，开发了一系列远离离子结合位点的 GluR6 二聚体界面突变，目的是开发一种适合分析超速离心 (AUC) 分析的制剂。突变体是根据野生型 GluR5 二聚体的晶体结构设计的，使用电生理分析来寻找脱敏动力学减慢的表型。制备相同突变体的分离配体结合结构域，并通过一系列蛋白质浓度的沉降速度分析和沉降平衡来测量它们对二聚体形成的亲和力。 iGluRs 氨基末端结构域的结构研究 谷氨酸受体离子通道是多域膜蛋白，由分子量约为 440 kD 的四聚体组装而成。已经解决了配体结合域的许多晶体结构，其每个亚基的分子量约为 30 kD，大约是完整受体的质量。对细菌表达系统的广泛试验（除了一个例外）已用于所有已发表的配体结合结构域结构，但未能产生其他 iGluR 结构域的单分散可溶性蛋白。氨基末端结构域 (ATD) 是一个重要的结构靶标，因为它控制天然 iGluR 中的亚型选择性组装，限制组装到同一功能家族的成员。在哺乳动物细胞中以足以进行结构生物学的水平表达蛋白质比在大肠杆菌中表达要困难得多，但其优点是多个检查点选择正确折叠的蛋白质，并添加糖和正常功能所需的其他翻译后修饰。虽然随后需要多种细胞生物学和生化技术来修剪糖链，以获得高分辨率结晶和衍射的蛋白质，并且产量低于原核表达，但目前这是唯一可能成功的方法用于 ATD 的研究。在正在进行的工作中，已经筛选了几种 iGluR 亚型的 ATD 在哺乳动物细胞中的表达情况。结晶试验已使用纳升移液机器人进行，并且在 APS 上获得了分辨率为 2.65 的完整数据集的一个衍射数据。结构解算和细化完成。 NR3配体结合选择性的结构分析 NR3 亚型谷氨酸受体具有独特的发育表达谱，但却是 NMDA 受体基因家族中特征最不明确的成员，该家族在突触可塑性和大脑发育中发挥着关键作用。使用配体结合测定、晶体学分析和全原子 MD 模拟，我们研究了甘氨酸和 D-丝氨酸的 NR3A 和 NR3B 结合的机制，甘氨酸和 D-丝氨酸是包含 NR3 亚基的 NMDA 受体的候选神经递质。两个 NR3 亚基的配体结合结构域采用与相应 NR1 复合物中相似的结构域闭合程度，但具有与所有其他谷氨酸受体离子通道不同的独特环 1 结构。在其配体结合口袋内，NR3A 和 NR3B 具有与 NR1 中发现的显着不同的氢键网络和溶剂结构，并且在结合部分激动剂 ACPC 时未能经历 NR1 中观察到的构象重排。 650 ns 持续时间的复制品交换 MD 模拟揭示了许多稳定激动剂结合的闭合裂隙构象的域间接触，以及 NR3 亚基中独特的环 1 螺旋的新颖扭转运动。这些位点的突变使配体结合不稳定，通过使用内源色氨酸荧光淬灭的滴定测定来测量。