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: 8149250
• 负责人: Mark L Mayer
• 金额: $ 148.58万
• 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/8149250
• 关键词: Affinity; Allosteric Regulation; Biochemical; Biological; Crystallization; Cysteine; Development; Epilepsy; Excitatory Synapse; Family; Gene Family; Glutamates; Goals; Human; Ion Channel; Ion Channel Gating; Length; Ligand Binding; Mammalian Cell; Mediator of activation protein; Mental Depression; Molecular; Neurologic; Neurotransmitter Receptor; Resolution; Structure; Variant; X-Ray Crystallography; base; chronic pain; crosslink; kainate; protein expression; protein folding; 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. Expression and crystallization 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. The structure of the GluK2 (GluR6) ATD revealed dimer and tetramer assemblies.Using the crystal structure for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross linking experiments in full length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross links for the GluK2 LBD dimer of dimers assembly. The formation of cross linked tetramers in full length GluK2 by combinations of ATD and LBD mutants which individually produce only cross linked dimers, suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross linking either the ATD or LBD inhibits activation of GluK2 and that, in the LBD, cross links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture, and provide insight into the role of individual dimer assemblies in activation of ion channel gating. Kainate receptors are further classified into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptor families based on their affinity for the neurotoxin kainic acid. These two families share 42% sequence identity for the intact receptor but only 28% sequence identity at the level of ATD. We have determined for the first time high-resolution crystal structures for the GluK3 and GluK5 ATDs, both of which crystallize as dimers, albeit with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5 the R2 domain dimer assembly is similar to that reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-3. Our analysis also reveals that these non-NMDA receptor ATDs undergo only moderate variations in domain closure, of up to 10 in contrast to the 50 movement reported for the NMDA receptor GluN2A and GluN2B subunits. This restricted domain movement in non-NMDA receptor ATDs seems to result from both extensive intra-domain contacts, and from their assembly as dimers which interact at the R2 domains. Our results provide the first insights into the structure and function for GluK4-5, the least understood family of iGluRs. Expression studies are on going with the goal of obtaining a full length crystal structure of the ion channel assembly

离子型谷氨酸受体（iGlurs）是膜蛋白，在哺乳动物神经系统中大多数兴奋性突触上充当分子孔，并介导信号传递。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，在该框架中设计了电生理和生化实验，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。这些信息将允许使用新型治疗应用的亚型选择性拮抗剂和变构调节剂的发展，并揭示复杂蛋白机机器的内部起作用，该机器在大脑功能中起着关键作用。 iGlurs氨基末端结构域的表达和结晶研究 谷氨酸受体离子通道是多域膜蛋白，它们的分子量的四聚体约为440 kD。已经为配体结合结构域求解了许多晶体结构，这些结合结构域的分子量约为每个亚基约30 kd，约为完整受体的质量。细菌表达系统的广泛试验（除一种例外）已用于所有已发表的配体结合结构结构，未能为其他Iglur结构域生成单分散蛋白。氨基末端结构域（ATD）是一个重要的结构靶标，因为它控制着天然iGlurs中的亚型选择性组装，将组装限制在同一功能家族的成员身上。哺乳动物细胞中结构生物学的水平的蛋白质表达比在大肠杆菌中表达更加困难，但是具有多个检查点为正确折叠的蛋白质选择的优点，并添加糖和其他后翻译后的转化修饰。尽管需要多种细胞生物学和生化技术来修剪糖链，但为了获得结晶并衍射至高分辨率的蛋白质，并且产率低于原核表达的蛋白质，目前，这是唯一可能成功地研究ATD的方法。在正在进行的工作中，已经筛选了来自几个Iglur亚型的ATD，以在哺乳动物细胞中表达。已经使用纳米升移动机器人进行了结晶试验。 GLUK2（GLUR6）ATD的结构揭示了二聚体和四聚体组件。使用Gluk2 Kainate受体ATD的晶体结构作为指导，我们进行了全长四室GLUK2中的半胱氨酸突变体交叉链接实验，以建立如何在Dimers组件的Dimers组件中建立ATD包装。使用全长AMPA受体GLUA2晶体结构作为指导的类似方法被用于设计半胱氨酸突变体横链链接，以用于二聚体组件的GLUK2 LBD二聚体。 ATD和LBD突变体的组合单独产生交叉链接二聚体，在全长GLUK2中形成了交叉连接的四聚体，这表明ATD和LBD层中的亚基交换二聚体伙伴。功能研究表明，交叉连接ATD或LBD抑制GLUK2的激活，并且在LBD中，二聚体内部和二聚体之间的交叉连接具有不同的作用。这些结果表明，海藻酸盐和AMPA受体具有保守的细胞外结构，并洞悉了个体二聚体组件在激活离子通道门控的作用。 海藻酸盐受体进一步分为低亲和力（GLUK1-3）和高亲和力（Gluk4-5）受体家族，其基于它们对神经毒素海藻酸的亲和力。这两个家族的完整受体具有42％的序列身份，但在ATD水平上只有28％的序列身份。我们首次确定了Gluk3和Gluk5 ATD的高分辨率晶体结构，尽管在R1界面处的二聚体组件非常不同，但两者都将其结晶为二聚体。相比之下，对于Gluk3和Gluk5，R2域二聚体组件都类似于先前针对其他非NMDA iGlurs报道的。该观察结果与GLUK4-5无法形成功能同源离子通道的报道是一致的，并且需要与Gluk1-3结合起来。我们的分析还表明，与NMDA受体Glun2A和Glun2b亚基报道的50个运动相比，这些非NMDA受体ATD仅在域闭合中仅发生中等变化。非NMDA受体ATD中的这种限制域运动似乎是由广泛的内域接触以及它们作为在R2域相互作用的二聚体组装而造成的。我们的结果为Gluk4-5的结构和功能提供了第一个见解，Gluk4-5是最不知情的iGlurs家族。 表达研究的目的是获得离子通道组件的全长晶体结构