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：
9150065
负责人：
Mark L Mayer
金额：
$ 94.28万
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/9150065
关键词：
AMPA Receptors Acute Brain Injuries Adopted Adult Affinity Agonist Allosteric Regulation Amino Acid Sequence Back Binding Binding Sites Biochemical Biochemistry Biological Assay Biological Preservation Brain Chemicals Classification Complex Crystallization Ctenophora Data Development Disulfides Drosophila genus Electrons Epilepsy Eukaryota Excitatory Synapse Exhibits Family Fetus Functional disorder Gated Ion Channel Gene Family Genes Genome Glutamate Receptor Glutamates Glycine Goals Heterogeneity Human Hydrogen Bonding Image Ion Channel Ion Channel Gating Ions Kainic Acid Receptors Laboratories Learning Length Ligand Binding Ligand Binding Domain Ligands Maps Measures Mediating Mediator of activation protein Membrane Proteins Memory Mental Depression Mental disorders Methods Microscopic Mnemiopsis Modeling Molecular Molecular Conformation Molecular Machines Movement N-Methyl-D-Aspartate Receptors N-Methylaspartate Names Nervous system structure Neurodegenerative Disorders Neuromuscular Junction Neurotransmitter Receptor Neurotransmitters Phenotype Phylogenetic Analysis Physiology Play Polyamines Positioning Attribute Prokaryotic Cells Proline Property Proteins Reagent Reporting Resolution Rest Role Rotation Rupture Schizophrenia Sequence Alignment Side Signal Transduction Sodium Chloride Stroke Structure Synaptic Transmission System Testing Thermodynamics Vertebrates Water Work X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction arginylglutamate base chronic pain density desensitization design dimer electron density fly genetic analysis hydroxyl group insight kainate mutant nervous system disorder neural circuit neuromuscular neurophysiology novel novel therapeutics patch clamp prevent receptor receptor function reconstitution research study response synaptic function three dimensional structure trafficking transmission process voltage

项目摘要

Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. iGluRs are also found in primitive prokaryotes and eukaryotes suggesting an early evolutionary origin, followed by gene expansion to generate subtypes with diverse functional properties. 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 biochemical and electrophysiological experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This important information will allow the development of novel therapeutic reagents and reveal the inner workings of a complicated protein machine which plays a key role in brain function. CRYO EM STRUCTURES OF FULL LENGTH iGLURS Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. We completed a structural analysis of an iGluR gating cycle, progressing from resting, to activated and then desensitized states. Comparison of the closed and active state electron density maps reveals LBD clamshell closure that produces a 7 vertical contraction of the ATD-LBD assembly, measured as a downwards movement at the top of the ATD tetramer, as well as unanticipated movements in the LBD, in which the dimer pairs rotate about an axis offset from the local axis of 2-fold symmetry. The net result of these movements is a novel corkscrew-like rotation that drives the transition from the closed to the active conformation. Analysis of cryo-electron microscopic images for the GluA desensitized state revealed evidence of substantial conformational heterogeneity precluding determination of a single desensitized state 3D structure. Three-dimensional classification enabled separation of three dominant classes, with variable degrees of displacement between ATD dimers compared to the closed and active states, at contrast to the rigid assembly of the ATD in GluK2. A structure of the GluK2 desensitized state, determined at 7.6 resolution, revealed electron density for all α-helices in the ATD and LBD assemblies, and also for the M3 helix bundle, the upper segment of M1 and the pre-M1 cuff helix in the ion channel. The density map revealed preservation of 2-fold symmetry in the ATD layer while the LBD layer adopts a quasi 4-fold symmetric arrangement. The resolution of our map unambiguously shows that in the desensitized state the ion channel adopts a closed conformation in which the M3 helices form a crossed bundle assembly with the pre-M1 helices wrapped around the outside of the channel. The 4-fold symmetry in the LBD layer matches that of the ion channel in its non-conducting state, and thereby permits the channel to adopt a low energy conformation. It is notable that while both AMPA and kainate receptors adopt 4-fold symmetry in their desensitized LBD layers, in the AMPA receptor desensitization also causes a rupture in the ATD layer. This result can be understood by considering symmetry mismatch within the receptor, and changes in symmetry during the gating cycle. In the closed and open states, both the ATD and LBD layers have 2-fold symmetry. The strain resulting from agonist binding to the LBD is centered near the LBD-TM interface and is sufficient to open the channel. In the desensitization step, the LBD layer shifts from 2-fold to 4-fold symmetry, matching the 4-fold symmetry of the ion channel; the strain in the receptor now shifts to the 2-fold symmetric ATD. In GluK2 the ATD assembly appears to be able to withstand this strain, possibly relieving it by a drawbridge-like tilting at the ATD tetramer interface. However, in GluA2, this symmetry mismatch places sufficient strain on the ATD layer to rupture the tetramer interface. FUNCTIONAL RECONSTITUTION OF DROSOPHILA NMJ GLUTAMATE RECEPTORS The Drosophila larval neuromuscular junction, at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute neuromuscular glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. We performed electrophysiological studies to test if the auxiliary subunit Neto was required for the functional reconstitution of Drosophila NMJ iGluRs. This revealed that the major effect of Neto was to increase receptor activity, with only a small effect on receptor trafficking. We established that four different iGluR subunits are required for robust expression; that Drosophila NMJ iGluRs are Ca2+ permeable and exhibit voltage dependent channel block by cytoplasmic polyamines; and that they have a ligand binding profile different from that of vertebrate AMPA, kainate and NMDA receptors. To investigate the structural basis for this unique profile we identified GluRIIB as a promising candidate for crystallization. X-ray diffraction data for the GluRIIB S1S2 complex with glutamate, at a resolution of 2 , revealed the classical back to back LBD dimer assembly, as first reported for the GluA2 AMPA receptor with glutamate bound in a cavity of volume 208 3 together with three trapped water molecules. Within the binding site, the side chain of Asp509 forms a hydrogen bond with the hydroxyl group of Tyr481, a conserved aromatic residue that caps the entrance to the ligand binding cavity; this interaction locks these residues in place, producing steric clashes that prevents binding of AMPA and kainate. Amino acid sequence alignments reveal that Asp509 is conserved in all Drosophila NMJ iGluRs, while in all vertebrate AMPA and kainate receptor subunits there is a proline at this position. CTENOPHORE GLUTAMATE RECEPTORS Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, while ligand-binding assays show that glycine binds with nM affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species.

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。 iGluR 也存在于原始原核生物和真核生物中，表明其具有早期进化起源，随后基因扩展产生具有不同功能特性的亚型。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生化和电生理学实验的框架，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机制。这一重要信息将有助于开发新型治疗试剂，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。全长 iGLURS 的冷冻电镜结构由于缺乏不同构象状态受体的结构信息，对谷氨酸受体门控分子机制的深入了解受到限制。我们完成了 iGluR 门控循环的结构分析，从静止状态到激活状态，再到脱敏状态。关闭状态和活动状态电子密度图的比较揭示了 LBD 蛤壳式闭合，它产生 ATD-LBD 组件的 7 垂直收缩，测量为 ATD 四聚体顶部的向下运动，以及 LBD 中的意外运动，其中二聚体对围绕偏离二次对称局部轴的轴旋转。这些运动的最终结果是一种新颖的螺旋状旋转，驱动从封闭构象到活跃构象的转变。对 GluA 脱敏状态的冷冻电子显微图像的分析揭示了显着构象异质性的证据，排除了单个脱敏状态 3D 结构的确定。三维分类能够分离三个主要类别，与闭合状态和活性状态相比，ATD 二聚体之间的位移程度不同，这与 GluK2 中 ATD 的刚性组装形成鲜明对比。以 7.6 分辨率测定的 GluK2 脱敏状态结构揭示了 ATD 和 LBD 组件中所有 α 螺旋的电子密度，以及 M3 螺旋束、M1 上段和前 M1 袖带螺旋的电子密度。离子通道。密度图显示 ATD 层保留了 2 重对称性，而 LBD 层则采用准 4 重对称排列。我们图的分辨率明确表明，在脱敏状态下，离子通道采用闭合构象，其中 M3 螺旋形成交叉束组件，前 M1 螺旋缠绕在通道外部。 LBD 层的四重对称性与非导电状态下离子通道的四重对称性相匹配，从而允许通道采用低能量构象。值得注意的是，虽然 AMPA 和红藻氨酸受体在其脱敏 LBD 层中均采用 4 重对称性，但在 AMPA 受体脱敏中也会导致 ATD 层破裂。这个结果可以通过考虑受体内的对称性失配以及门控循环期间对称性的变化来理解。在关闭和打开状态下，ATD 层和 LBD 层都具有 2 重对称性。激动剂与 LBD 结合产生的应变集中在 LBD-TM 界面附近，足以打开通道。在脱敏步骤中，LBD层从2重对称性转变为4重对称性，与离子通道的4重对称性相匹配；受体中的应变现在转变为 2 倍对称 ATD。在 GluK2 中，ATD 组件似乎能够承受这种应变，可能通过 ATD 四聚体界面处的吊桥状倾斜来缓解这种应变。然而，在 GluA2 中，这种对称性失配对 ATD 层施加了足够的应变，足以使四聚体界面破裂。果蝇 NMJ 谷氨酸受体的功能重建果蝇幼虫神经肌肉接头处的谷氨酸充当兴奋性神经递质，是一种广泛用于突触功能和发育遗传分析的模型。尽管经过数十年的研究，但无法使用异源表达系统重建神经肌肉谷氨酸受体功能，这使得受体功能的分析变得复杂，因此很难解析在突变果蝇中观察到的复合表型的分子基础。我们进行了电生理学研究，以测试果蝇 NMJ iGluR 的功能重建是否需要辅助亚基 Neto。这表明 Neto 的主要作用是增加受体活性，对受体运输的影响很小。我们确定稳健表达需要四种不同的 iGluR 亚基；果蝇 NMJ iGluR 具有 Ca2+ 渗透性，并且通过细胞质多胺表现出电压依赖性通道阻断作用；并且它们具有与脊椎动物 AMPA、红藻氨酸和 NMDA 受体不同的配体结合特征。为了研究这种独特特征的结构基础，我们确定 GluRIIB 是一种有前途的结晶候选物。分辨率为 2 的 GluRIIB S1S2 与谷氨酸复合物的 X 射线衍射数据揭示了经典的背对背 LBD 二聚体组装，正如首次报道的 GluA2 AMPA 受体与谷氨酸结合在体积为 208 3 的空腔中以及三个被困住的水分子。在结合位点内，Asp509 的侧链与 Tyr481 的羟基形成氢键，Tyr481 是一个保守的芳香族残基，覆盖配体结合腔的入口；这种相互作用将这些残基锁定在适当的位置，产生空间冲突，阻止 AMPA 和红藻氨酸的结合。氨基酸序列比对表明，Asp509 在所有果蝇 NMJ iGluR 中都是保守的，而在所有脊椎动物 AMPA 和红藻氨酸受体亚基中，该位置都有一个脯氨酸。栉水母谷氨酸受体最近的栉水母基因组计划揭示了我们最早的后生动物祖先中的 Mnemiopsis leidyi 和 Pleurobrachia bachei 中存在大量离子型谷氨酸受体 (iGluR)。序列比对和系统发育分析表明，它们与脊椎动物中发现的充分表征的 AMPA、红藻氨酸和 NMDA iGluR 亚型形成了一个独特的进化枝。尽管注释为谷氨酸受体，ML032222a 和 PbiGluR3 配体结合域 (LBD) 的晶体结构揭示了结合口袋中的内源性甘氨酸，而配体结合分析表明甘氨酸以 nM 亲和力结合；生化测定和结构分析证实谷氨酸被封闭在结合腔中。进一步的分析揭示了栉水母特有的特征，例如仅存在于结合甘氨酸的亚基中的域间 Arg-Glu 盐桥，而且 LBD 环 1 中也存在保守的二硫键，这种二硫键存在于脊椎动物 NMDA 中，但不存在于 AMPA 或红藻氨酸受体中。我们假设栉水母 iGluR 与 NMDA 受体的早期祖先有关，这表明栉水母和两侧对称动物有共同的进化路径。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Emerging models of glutamate receptor ion channel structure and function.

DOI：
10.1016/j.str.2011.08.009
发表时间：
2011-10-12
期刊：
STRUCTURE
影响因子：
5.7
作者：
Mayer, Mark L.
通讯作者：
Mayer, Mark L.

Zinc potentiates GluK3 glutamate receptor function by stabilizing the ligand binding domain dimer interface.

锌通过稳定配体结合域二聚体界面来增强 GluK3 谷氨酸受体功能。

DOI：
10.1016/j.neuron.2012.08.027
发表时间：
2012
期刊：
Neuron
影响因子：
16.2
作者：
Veran,Julien;Kumar,Janesh;Pinheiro,PauloS;Athané,Axel;Mayer,MarkL;Perrais,David;Mulle,Christophe
通讯作者：
Mulle,Christophe

Structure and mechanism of glutamate receptor ion channel assembly, activation and modulation.