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（iGlurs）是膜蛋白，在哺乳动物神经系统中的大多数兴奋性突触上充当分子孔，并介导信号传递。在原始原核生物和真核生物中也发现了伊吉尔斯，提示早期进化起源，其次是基因扩展，以产生具有不同功能特性的亚型。人类中离子谷氨酸受体（iGlurs）的7个基因家族编码18个亚基，它们组装，形成了3个主要功能家族，该家族以配体命名，该家族最初用于鉴定1970年代后期的Iglur亚型：AMPA，Kainate和NMDA。由于它们在正常的大脑功能和发育中具有重要作用，并且越来越多的证据表明，iGlur活性功能障碍介导了多种神经系统和精神疾病，以及中风期间的损害，因此在细胞和分子神经生理学实验室的实验努力旨在针对分析分子水平的iGlur功能。通过蛋白质结晶和X射线衍射解决的原子分辨率结构提供了一个框架，在该框架中设计生化和电生理实验，以定义配体识别的机制，离子通道活性的门控和变构调节剂的作用。这些重要信息将允许开发新型的治疗试剂，并揭示复杂蛋白机的内部工作，该机器在大脑功能中起着关键作用。全长iglurs的冷冻结构对谷氨酸受体门控的分子机制的洞察力受到陷入不同构象状态中的受体缺乏结构信息的限制。我们完成了一个从静止，激活，然后脱敏状态的igur门控循环的结构分析。封闭状态电子密度图的比较显示LBD盖壳闭合会产生ATD-LBD组件的7个垂直收缩，以ATD Tetramer顶部的向下移动测量，以及LBD的意外运动，在LBD中，Dimer对轴心旋转的位置旋转了局部轴心，该轴是由局部轴心旋转的。这些运动的最终结果是一种新型的开瓶器样旋转，可以驱动从闭合到活动构象的过渡。分析GLUA脱敏状态的冷冻电子显微镜图像揭示了实质构象异质性的证据，排除了单个脱敏状态3D结构的确定。与闭合状态和活动状态相比，三维分类能够分离三个主要类别，与封闭状态和活动状态相比，ATD二聚体之间的位移程度变化，与GLUK2中ATD的刚性组装相反。 GLUK2脱敏状态的结构，以7.6分辨率确定，揭示了ATD和LBD组件中所有α-螺旋的电子密度，也显示了M3螺旋束，M1的上段和离子通道中的M3螺旋束，M1的上段和前M1袖带螺旋。密度图显示在ATD层中保留了2倍对称性，而LBD层采用了4倍对称排列的准层。我们的地图的分辨率明确表明，在脱敏状态下，离子通道采用封闭构型，其中M3螺旋形成一个交叉束组件，并带有前M1螺旋，并包裹在通道外部。 LBD层中的4倍对称性与离子通道的非传导状态匹配，因此允许该通道采用低能构象。值得注意的是，尽管AMPA和海谷酸酯受体在其脱敏的LBD层中都采用4倍对称性，但在AMPA受体脱敏的LBD层中也会导致ATD层破裂。可以通过考虑受体内的对称性不匹配以及在门控循环中的对称性变化来理解该结果。在封闭和开放状态下，ATD和LBD层都有2倍对称性。激动剂结合与LBD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倍向对称性移动，与离子通道的4倍对称性匹配。现在，受体中的应变转移到2倍对称ATD。在Gluk2中，ATD组件似乎能够承受这种菌株，可能通过在ATD四聚体界面处的吊桥状倾斜来缓解它。但是，在GLUA2中，这种对称不匹配在ATD层上施加了足够的应变，以破裂四聚体界面。果蝇NMJ谷氨酸受体的功能重构果蝇幼虫神经肌肉结，谷氨酸在该连接处充当兴奋性神经递质，是一种广泛使用的模型，用于突触功能和发育的遗传分析。尽管进行了数十年的研究，但无法使用异源表达系统重新构建神经肌肉谷氨酸受体功能使受体功能的分析变得复杂，因此很难解决在突变蝇中观察到的复合表型的分子基础。我们进行了电生理研究，以测试果蝇NMJ iglurs功能重构是否需要辅助亚基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个不同的iglur亚基。果蝇NMJ iglurs是Ca2+可渗透的，并且通过细胞质多胺表现出依赖电压的通道阻滞。并且它们的配体结合谱与脊椎动物AMPA，海藻酸盐和NMDA受体不同。为了研究这种独特概况的结构基础，我们将格鲁里布确定为结晶的有前途的候选者。 Gluriib S1S2复合物与谷氨酸的X射线衍射数据以2分辨率揭示了经典的背对面LBD二聚体组件，正如GluA2 AMPA受体与谷氨酸结合的GluA2 AMPA受体在体积208 3的腔中结合的，这首先报道了3个型号的谷氨酸。在结合位点中，ASP509的侧链与Tyr481的羟基形成氢键，Tyr481是一种保守的芳族残基，限制了配体结合腔的入口；这种相互作用将这些残基锁定在适当的位置，从而产生阻止AMPA和海藻酸盐结合的空间冲突。氨基酸序列比对表明，ASP509在所有果蝇NMJ iglurs中都是保守的，而在所有脊椎动物AMPA和Kainate受体亚基中，该位置都有一个脯氨酸。 c型谷氨酸受体最近针对c型的基因组项目揭示了我们最早的后代祖先之一，在mnemiopsis leidyi和pleurobrachia bachei中存在许多离子型谷氨酸受体（iglurs）。序列比对和系统发育分析表明，这些形成了与特征良好的AMPA，Kainate和NMDA Iglur亚型的明显进化枝。尽管被注释为谷氨酸受体，但ML0322222A和PBIGLUR3配体结合结构域（LBD）的晶体结构揭示了结合口袋中的内源性甘氨酸，而配体结合测定法显示甘氨酸具有NM亲和力；生化测定和结构分析表明，谷氨酸是从结合腔中阻塞的。进一步的分析揭示了Cenophore特异性的特征，例如仅在结合甘氨酸的亚基中存在的域间Arg-Glu盐桥，但在脊椎动物NMDA中发现的LBD的LOOP 1中也是保守的二硫化物，但在脊椎动物NMDA中发现的二硫化物1，但没有AMPA或Kainate受体。我们假设Cenophore iglurs与NMDA受体的早期祖先有关，这表明c脑和双脑物种的常见进化途径。

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.