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：
8736824
负责人：
Mark L Mayer
金额：
$ 139.99万
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/8736824
关键词：
AMPA Receptors Acute Brain Injuries Adult Affinity Agonist Alanine Allosteric Regulation Anions Arginine Aspartate Binding Binding Sites Biochemical Biochemistry Brain Cations Chemicals Chemosensitization Chloride Ion Cleaved cell Complex Crystallization Cysteine DNA Sequence Rearrangement Development Distal Epilepsy Eukaryota Evolution Excision Excitatory Synapse Exhibits Family Fetus Free Energy Functional disorder Gated Ion Channel Gene Family Genes Genetic Crossing Over Glutamate Receptor Glutamates Glycine Goals Human Ion Channel Ion Channel Gating Ions Kainic Acid Receptors Kinetics Laboratories Learning Lectin Length Ligand Binding Ligand Binding Domain Ligands Link Maps Mediating Mediator of activation protein Membrane Proteins Memory Mental Depression Mental disorders Methionine Methods Modeling Molecular Molecular Conformation Molecular Machines Motion Mutation N-Methyl-D-Aspartate Receptors N-Methylaspartate Names Nervous system structure Neurodegenerative Disorders Neurotransmitter Receptor Phenylalanine Physiology Play Principal Component Analysis Prokaryotic Cells Property Proteins Regulation Reporting Resolution Rest Role Sampling Scheme Schizophrenia Sedimentation process Serine Signal Transduction Stroke Structural Models Structure Synapses Synaptic Transmission Thermodynamics Transmembrane Domain Variant Water Work X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction Zinc base carboxyl group chronic pain density desensitization design dimer insight kainate mutant nervous system disorder neural circuit neurophysiology novel novel therapeutics particle patch clamp receptor research study response tomography transmission process

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 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. NMDA RECEPTOR GLYCINE AND GLUTAMATE LIGAND-BINDING DOMAINS The NMDA receptor family of glutamate receptor ion channels are formed by obligate heteromeric assemblies of GluN1, GluN2 and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open and closed cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo state LBDs sample closed cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggests binding of glutamate via an induced fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels. STRUCTURAL STUDIES ON THE EVOLUTION OF iGLURS AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga , is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the gamma-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the gamma-carboxyl group. Removal of Cl_ lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. NOVEL ZINC BINDING SITES ON GLUK3 SUBTYPE KAINATE RECEPTORS Kainate receptors (KARs) play a key role in the regulation of synaptic networks. Here, we show that zinc, a cation released at a subset of glutamatergic synapses, potentiates glutamate currents mediated by homomeric and heteromeric KARs containing GluK3 at 10100 micro Molar concentrations, whereas it inhibits other KAR subtypes. Potentiation of GluK3 currents is mainly due to reduced desensitization, as shown by kinetic analysis and desensitization mutants. Crystallographic and mutation analyses revealed that a specific zinc binding site is formed at the base of the ligand binding domain (LBD) dimer interface by a GluK3-specific aspartate (Asp759), together with two conserved residues, His762 and Asp730, the latter located on the partner subunit. In addition, we propose that tetrameric GluK2/GluK3 receptors are likely assembled as pairs of heterodimeric LBDs. Therefore, zinc binding stabilizes the labile GluK3 dimer interface, slows desensitization, and potentiates currents, providing a mechanism for KAR potentiation at glutamatergic synapses. 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.Here,we report the use of single-particle cryoelectron tomography to determine the structures, at 21 Angstrom resolution, of full-lengthGluK2 kainate receptors trapped in antagonist-bound restingand agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2,withwell-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by 25 Angstroms. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.

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。这些重要信息将使亚型选择性拮抗剂和变构调节剂具有新颖的治疗应用，并揭示复杂蛋白机的内部起作用，该机器在大脑功能中起着关键作用。 NMDA受体甘氨酸和谷氨酸配体结合结构域谷氨酸受体离子通道的NMDA受体家族是由Glun1，Glun2和Glun3亚基的专性异源组件形成的。 Glun1和Glun3结合甘氨酸，而Glun2结合谷氨酸。 Glun1和Glun3a配体结合结构域（LBD）的晶体结构出乎意料地揭示了水分子填充结合口袋，分别揭示了开放和封闭的裂缝构象。 GLUN1，GLUN2A和GLUN3A LBD的计算构象自由能景观表明，Apo状态LBDS样品封闭裂构构象，表明其激动剂通过构象选择机制结合。相比之下，AMPA受体GLUA2 LBD的自由能景观表明谷氨酸通过诱导的拟合机理结合。主成分分析揭示了丰富的铰链弯曲，摇动，扭曲和扫描运动，这些运动对于Glun1，Glun2a，Glun3a和Glua2 LBD都不同。这种变化突出了谷氨酸受体离子通道信号传导的结构复杂性。关于iGlurs进化的结构研究来自原始真核生物Adineta Vaga的谷氨酸受体离子通道AVGlUR1被丙氨酸，半胱氨酸，蛋氨酸和苯丙氨酸激活，它们产生凝集素敏感的脱敏反应，例如对谷氨酸，天冬氨酸和丝氨酸和丝氨酸的凝集素敏感性的反应。 AVGLUR1 LBD晶体结构揭示了一种不寻常的结合配体的方案，该方案可能被远距离相关的气味/化学感应受体使用。结构域中的精氨酸残基2坐标了谷氨酸的γ-羧基，而在丙氨酸，蛋氨酸和丝氨酸络合物中，氯离子的氯离子可以用作替代配体，以取代伽马 - 羧基。去除Cl_的降低对这些配体的亲和力，但对谷氨酸或天冬氨酸或苯丙氨酸的亲和力却不是对阴离子结合位点并具有低亲和力结合的。 AVGLUR1 LBD晶体结构和沉积分析还提供了有关原核生物和真核iGlurs之间进化联系的见解，并揭示了这两个类别所特有的特征，强调了对Iglur-ligand相互作用的其他基于结构的研究的需求。 GLUK3亚型海藻酸盐受体上的新型锌结合位点海藻酸盐受体（KARS）在突触网络的调节中起关键作用。在这里，我们表明锌是在谷氨酸能突触的子集中释放的阳离子，增强了由同源物和含Gluk3含有GLUK3的谷氨酸电流以10100微摩尔浓度介导的，而它抑制了其他KAR亚型。 Gluk3电流的增强主要是由于脱敏降低，如动力学分析和脱敏突变体所示。晶体学和突变分析表明，特定的锌结合位点是在配体结合结构域（LBD）二聚体界面的底部形成的，该二聚体是通过GLUK3特异性天冬氨酸（ASP759）以及两个保守的残基以及两个保守的残基，His762和Asp730，后者位于伙伴子unitiit上。此外，我们提出，四聚体Gluk2/gluk3受体可能被组装成成对的异二聚体LBD。因此，锌的结合稳定了不稳定的Gluk3二聚体界面，减慢脱敏并增强电流，从而为谷氨酸能突触提供了kar增强的机制。全长iglurs的冷冻结构 Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states.Here,we report the use of single-particle cryoelectron tomography to determine the structures, at 21 Angstrom resolution, of full-lengthGluK2 kainate receptors trapped in antagonist-bound restingand agonist-bound desensitized states.由竞争性拮抗剂LY466195稳定的静止状态与AMPA受体GluA2的晶体结构非常相似，并具有井分解的近端和远端亚基，在双重对称的氨基终端域和双重对称的氨基终端域和双重对称的对称性ligligand bitigand bitain bitain（lbyand Binding Dignain domerant）（lbbd）dimore（lbbd）dimerer（lbbd）dimore dimore dimerer dimerever（在脱敏状态下，LBD经历了重大重排，从而将四个亚基与25埃埃斯特罗姆分开。然而，受体的氨基末端结构域，跨膜和细胞质区域在静息和脱敏状态下具有相似的构象。在基于可溶性LBD二聚体组件的晶体结构的先前模型中，LBD重排是没有预期的，我们推测亚基分离可以更好地与四方对称的对称离子通道域匹配。从氨基末端结构域和LBD结构域的拟合到脱敏状态的密度图中，我们得出了一个结构模型，用于静止状态和脱敏状态之间的第四纪构象差异。