Structure And Function Of Neurotransmitter Receptor Ion

神经递质受体离子的结构和功能

基本信息

项目摘要

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 GluR 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 allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. 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. The recent crystallization of the ligand binding cores of AMPA, kainate and NMDA receptor subunits, and a related bacterial receptor from the photosynthetic bacterium syncheocystis PCC 6803 which we named GluR0, has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists as well as providing insight into the mechanisms of activation and desensitization. During the past year experimental efforts in structural biology have been directed towards studies on the ligand binding cores of members of the kainate receptor gene family, and on the cytoplasmic domains of NMDA receptors. Although a large number of glutamate receptor agonist and partial agonist structures has been solved, relatively few antagonist complexes have been crystallized. This is likely the result of the different conformations of the closed cleft agonist bound structure and the open cleft antagonist bound structure and differences in mobility of the ligand protein complex. Agonists bound complexes are like rocks, crystallize easily, and have similar temperature factors in domain 1 and 2. Antagonist structures are hard to crystallize. High resolution structures of two novel GluR5 selective antagonists, UBP302 and UBP310, reveal a hyper extended conformation in which the ligand forces the domains to separate to a greater extent than observed in the GluR2 apo structure. Strikingly, electron density was excellent for domain 1, but less well defined for domain 2. Refinement of the final structure required TLS analysis and reveled both domain breathing motions and a higher overall mobility of domain 2. The structure of the UBP302 and 310 complexes is strikingly different from those previously solved for the GluR2 DNQX and NR1 5,7DCKA complexes and revealed a mode of ligand binding that could not be modeled from prior structural information. It is anticipated that this new information will facilitate the design of new subtype kainate receptor antagonists. Despite overall high amino acid sequence homology and similar gating kinetics, the exchange of single amino acids which differ between the AMPA and kainate receptor subtypes of iGluRs lead to identification of a non desensitizing AMPA receptor L483Y construct that during the past 5 years has been widely used as a tool to investigate numerous aspects of iGluR biology. The availability of similar constructs for kainate receptors would be of great interest, yet surprisingly paradoxical results have been obtained when mutations were introduced into the ligand binding core dimer interface of kainate receptors. To address this the GluR5 ligand binding core was crystallized in a dimeric assembly similar to that observed for AMPA receptors, and its structure determined at 2.1 ? resolution. Using this structure as a template a series of mutants was designed in the dimer interface with the goal of stabilizing the active conformation. These were also crystallized and their structures solved, and the sedimentation behavior of the purified ligand binding cores examined by analytical ultracentrifugation. We predicted that those mutations which stabilized kainate receptor dimer formation would attenuate desensitization. This was tested by rapid perfusion experiments using HEK293 cell patches which revealed complete block of GluR6 desensitization for constructs which stabilize the dimer assembly. Of interest for another construct which was designed to reproduce the local dimer structure of the GluR2 L483Y mutant, there was no block of desensitization or change in sedimentation behavior, even though when crystallized the resulting structure closely resembled the L483Y parent. Such results illustrate the importance of using a wide range of biophysical approaches to characterize receptor function, since although crystal structures capture in exquisite detail the molecular details of protein assembly, they cannot at present be used to reliably estimate the energetics of assembly of macromolecular complexes.
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GluR 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,以定义配体识别和离子通道活性门控的变构机制。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。 最近,AMPA、红藻氨酸和 NMDA 受体亚基的配体结合核心以及来自光合细菌合胞藻 PCC 6803(我们命名为 GluR0)的相关细菌受体的结晶,首次揭示了激动剂和拮抗剂结合的分子机制以及提供对激活和脱敏机制的深入了解。在过去的一年中,结构生物学的实验工作主要针对红藻氨酸受体基因家族成员的配体结合核心以及 NMDA 受体的细胞质结构域的研究。 尽管大量的谷氨酸受体激动剂和部分激动剂结构已被解析,但结晶的拮抗剂复合物相对较少。这可能是闭合裂隙激动剂结合结构和开放裂隙拮抗剂结合结构的不同构象以及配体蛋白复合物的迁移率差异的结果。激动剂结合的配合物就像岩石一样,容易结晶,并且在域1和域2中具有相似的温度因素。拮抗剂结构难以结晶。两种新型 GluR5 选择性拮抗剂 UBP302 和 UBP310 的高分辨率结构揭示了超延伸构象,其中配体迫使结构域分离到比 GluR2 apo 结构中观察到的更大程度。引人注目的是,域 1 的电子密度非常好,但域 2 的电子密度不太明确。最终结构的细化需要 TLS 分析,并揭示了域呼吸运动和域 2 更高的整体迁移率。UBP302 和 310 复合物的结构是与之前解决的 GluR2 DNQX 和 NR1 5,7DCKA 复合物的结果显着不同,并揭示了一种无法根据先前结构信息建模的配体结合模式。预计这一新信息将有助于新亚型红藻氨酸受体拮抗剂的设计。 尽管整体氨基酸序列同源性高且门控动力学相似,但 iGluR 的 AMPA 和红藻氨酸受体亚型之间不同的单个氨基酸的交换导致鉴定出非脱敏 AMPA 受体 L483Y 构建体,该构建体在过去 5 年中已被广泛使用作为研究 iGluR 生物学众多方面的工具。红藻氨酸受体的类似构建体的可用性将引起极大兴趣,但当将突变引入红藻氨酸受体的配体结合核心二聚体界面时,获得了令人惊讶的矛盾结果。为了解决这个问题,GluR5 配体结合核心在二聚体组装中结晶,类似于在 AMPA 受体中观察到的情况,其结构在 2.1 ?解决。使用该结构作为模板,在二聚体界面中设计了一系列突变体,目的是稳定活性构象。这些也被结晶并解析了它们的结构,并通过分析超速离心检查了纯化的配体结合核心的沉降行为。我们预测那些稳定红藻氨酸受体二聚体形成的突变会减弱脱敏作用。使用 HEK293 细胞贴片通过快速灌注实验对此进行了测试,结果显示完全阻断了稳定二聚体组装的构建体的 GluR6 脱敏作用。令人感兴趣的是另一种旨在复制 GluR2 L483Y 突变体的局部二聚体结构的构建体,没有脱敏的阻碍或沉降行为的变化,即使结晶时所得结构与 L483Y 亲本非常相似。这些结果说明了使用广泛的生物物理方法来表征受体功能的重要性,因为尽管晶体结构精确地捕获了蛋白质组装的分子细节,但它们目前不能用于可靠地估计大分子复合物组装的能量学。

项目成果

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Mark L Mayer其他文献

Mark L Mayer的其他文献

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{{ truncateString('Mark L Mayer', 18)}}的其他基金

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
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structure /Function Of Neurotransmitter Receptor Channel
神经递质受体通道的结构/功能
  • 批准号:
    6811642
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    9150065
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    8351116
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    7734704
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    8941443
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    7594147
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structure And Function Of Neurotransmitter Receptor Ion
神经递质受体离子的结构和功能
  • 批准号:
    6508745
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structure And Function Of Neurotransmitter Receptor Ion
神经递质受体离子的结构和功能
  • 批准号:
    6671852
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    8736824
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:

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Development of STEP Allosteric Inhibitors as Novel Therapeutics for Alzheimer's Disease
STEP 变构抑制剂的开发作为阿尔茨海默病的新疗法
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Development of STEP Allosteric Inhibitors as Novel Therapeutics for Alzheimer's Disease
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Drug Design: Glutamate Receptor Signaling
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    6466373
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