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
• 负责人: Mark L Mayer
• 金额: --
• 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/6508745
• 关键词: AMPA receptors; X ray crystallography; biophysics; conformation; glutamate receptor; kainate; membrane channels; neuropharmacology; protein structure function; receptor expression; receptor sensitivity; tissue /cell culture

Ionotropic glutamate receptors (GluRs) are molecular pores which facilitate the passage of ions across cell membranes and mediate excitatory signal transmission in the mammalian nervous system. Because of their essential role in normal brain function and development and increasing evidence that dysfunction of GluR activity mediates multiple CNS diseases as well as damage during stroke a substantial effort has been directed towards analysis of GluR properties. The AMPA, kainate and NMDA subtypes of ionotropic glutamate receptors are encoded by at least 7 gene families. The recent crystallization of the ligand binding core of an AMPA receptor subunit in the Gouaux laboratory at Columbia University has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists. Surprisingly, despite their role in brain function, glutamate receptors appear to have evolved directly from bacterial ion channels. Direct evidence was obtained for this from the identification and analysis of the first GluR found in a prokaryote: GluR0 from the photosynthetic bacterium syncheocystis PCC 6803. GluR0 binds glutamate, forms potassium-selective channels, and is related in amino acid sequence to both eukaryotic GluRs and potassium channels. On the basis of the amino acid sequence and functional relationships between GluR0 and eukaryotic GluRs, it seems likely that a prokaryotic GluR was the precursor to eukaryotic GluRs. We have found that GluR0 can be gated not only by glutamate but also by external protons or by lowering the Ca2+ concentration of the external solution. The single-channel responses gated by either protons or by lowering Ca2+ exhibited a voltage-dependent block by external Na+, which was similar to that of desensitizing responses to glutamate. The single-channel activity gated by all three conditions typically displayed two types of gating behavior: long open times interspersed with fast flickering bursts. Proton gated currents, EC50 30 mM, exhibited a pH-dependent block of single channel conductance at -60 mV and were cross-desensitized by 1mM glutamate. Similarly, GluR0 responses activated by lowering extracellular calcium but not magnesium to 10 nM at pH 7.2, IC50 10 ?M, were desensitized by 1mM glutamate. The independence of these gating mechanisms was demonstrated by a binding site mutant, GluR0/R117K, which maintained normal pH and Ca2+ sensitivity while virtually abolishing activation by glutamate. Although the gating of many ion channels can be modulated by pH or by Ca2+ GluR activation by protons in the absence of ligand and spontaneous activity inhibited by Ca2+ has not been described before. The ligand binding core of GluR0 has been purified and over expressed. High resolution structures of the ligand binding core of GluR0, the glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction. The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate gamma-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating we solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 ?M. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. We propose that ligand induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface. The binding of agonists to glutamate receptors triggers ion channel gating which within milliseconds is followed by desensitization. The molecular mechanisms of desensitization are unknown. In collaboration with the Gouaux lab we have performed functional studies to address the molecular mechanisms of gating. Mutations have been identified which alter the extent and kinetics of desensitization. These map to a surface of the ligand binding core which crystallographic studies reveal to mediate interactions between GluR subunit dimers. Analytical ultracentrifugation was used to measure the Kd for dimerization of GluR2 ligand binding cores in solution. A piezo concentration jump apparatus was used to measure desensitization in outside out patches. Mutants which increase dimer stability reduce desensitization, while mutants which decrease dimer stability increase desensitization. Crystallographic analysis of the non desensitizing GluR2 mutant L748Y reveals that the Tyrosine side chain in one subunit interacts with a pocket in the opposite subunit of the dimer pair. Mutation to alanine of the side chains which form this pocket restores desensitization and increases the Kd for dimer formation. We propose that movement of GluR subunits about the dimer interface allow the ion channel to close even though the agonist binding domains remain in their closed-cleft -bound conformation which initially promotes ion channel gating. Our results reveal for the first time a molecular mechanism for GluR receptor desensitization.

离子型谷氨酸受体 (GluR) 是一种分子孔，可促进离子穿过细胞膜并介导哺乳动物神经系统中的兴奋信号传递。由于它们在正常脑功能和发育中的重要作用，并且越来越多的证据表明 GluR 活性功能障碍介导多种中枢神经系统疾病以及中风期间的损伤，因此大量努力致力于分析 GluR 特性。离子型谷氨酸受体的 AMPA、红藻氨酸和 NMDA 亚型由至少 7 个基因家族编码。哥伦比亚大学 Gouaux 实验室最近对 AMPA 受体亚基配体结合核心的结晶，首次揭示了激动剂和拮抗剂结合的分子机制。 令人惊讶的是，尽管谷氨酸受体在大脑功能中发挥着重要作用，但它似乎是直接从细菌离子通道进化而来的。从原核生物中发现的第一个 GluR 的鉴定和分析中获得了直接证据：来自光合细菌合胞藻 PCC 6803 的 GluR0。GluR0 结合谷氨酸，形成钾选择性通道，并且在氨基酸序列上与真核 GluR 相关。和钾通道。根据 GluR0 和真核 GluR 之间的氨基酸序列和功能关系，原核 GluR 似乎很可能是真核 GluR 的前体。 我们发现GluR0不仅可以被谷氨酸门控，还可以被外部质子或通过降低外部溶液的Ca2+浓度来门控。由质子或降低 Ca2+ 门控的单通道反应表现出外部 Na+ 的电压依赖性阻断，这类似于对谷氨酸的脱敏反应。由所有三种条件门控的单通道活动通常显示两种类型的门控行为：较长的开放时间和散布着快速闪烁的突发。质子门控电流 EC50 30 mM，在 -60 mV 时表现出 pH 依赖性单通道电导阻断，并通过 1 mM 谷氨酸交叉脱敏。类似地，通过在 pH 7.2 下将细胞外钙（而不是镁）降低至 10 nM（IC50 10 μM）来激活 GluR0 反应，并通过 1 mM 谷氨酸脱敏。这些门控机制的独立性由结合位点突变体 GluR0/R117K 证明，该突变体保持正常的 pH 值和 Ca2+ 敏感性，同时几乎消除了谷氨酸的激活。尽管许多离子通道的门控可以通过 pH 值或 Ca2+ GluR 来调节，但在没有配体的情况下，质子激活 GluR，并且以前没有描述过 Ca2+ 抑制自发活性。 GluR0 的配体结合核心已被纯化并过表达。 GluR0 的配体结合核心（集胞藻 PCC 6803 的谷氨酸受体离子通道）的高分辨率结构已通过 X 射线衍射解析。 GluR0 结构揭示了与细菌周质结合蛋白和大鼠 GluR2 AMPA 亚型神经递质受体的同源性。配体结合位点由两个球状 α/β 结构域之间的裂缝形成。 L-谷氨酸以扩展构象结合，类似于谷氨酰​​胺结合蛋白 (GlnBP) 观察到的构象。然而，L-谷氨酸 γ-羧基仅与结构域 1 中的 Asn51 相互作用，这与在 GluR2 和 GlnBP 中观察到的配体与结构域 2 残基的相互作用不同。为了解决中性氨基酸如何激活 GluR0 门控，我们解决了与 L-丝氨酸结合位点复合物的结构。这揭示了溶剂分子充当替代配体原子，使得丝氨酸 OH 基团与 Asn51 形成溶剂介导的氢键。无配体的闭裂构象的结构揭示了由溶剂分子介导的广泛的氢键网络。平衡离心分析揭示了 GluR0 配体结合核心的二聚化，解离常数为 0.8 µM。在晶体中，涉及结构域 1 中残基的对称二聚体沿着晶体学 2 倍轴出现，表明四聚体谷氨酸受体离子通道是由二聚体的二聚体组装而成的。我们认为，配体诱导的构象变化导致离子通道打开，这是由于域 2 相对于二聚体界面的分离增加的结果。 激动剂与谷氨酸受体的结合触发离子通道门控，随后在几毫秒内脱敏。脱敏的分子机制尚不清楚。我们与 Gouaux 实验室合作进行了功能研究，以解决门控的分子机制。已经确定了改变脱敏程度和动力学的突变。这些映射到配体结合核心的表面，晶体学研究表明其介导 GluR 亚基二聚体之间的相互作用。使用分析超速离心来测量溶液中 GluR2 配体结合核心二聚化的 Kd。使用压电浓度跳跃装置来测量外侧贴片中的脱敏。增加二聚体稳定性的突变体减少脱敏，而降低二聚体稳定性的突变体增加脱敏。非脱敏 GluR2 突变体 L748Y 的晶体学分析表明，一个亚基中的酪氨酸侧链与二聚体对的相对亚基中的口袋相互作用。形成该口袋的侧链丙氨酸突变可恢复脱敏并增加二聚体形成的 Kd。我们提出，GluR 亚基围绕二聚体界面的运动允许离子通道关闭，即使激动剂结合域仍保持其闭合裂隙结合构象，这最初促进离子通道门控。我们的结果首次揭示了 GluR 受体脱敏的分子机制。