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（胶质）是分子孔，促进离子跨细胞膜的传递并介导哺乳动物神经系统中的兴奋性信号传递。由于它们在正常的大脑功能和发育中的重要作用，并且越来越多的证据表明，Glur活动功能障碍介导多种中枢神经系统疾病以及中风期间损害的损害已大量努力用于分析Glur性质。离子谷氨酸受体的AMPA，海藻酸盐和NMDA亚型由至少7个基因家族编码。哥伦比亚大学Gouaux实验室中AMPA受体亚基的配体结合核心的最新结晶是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，尽管它们在脑功能中作用，但谷氨酸受体似乎还是直接从细菌离子通道中演变而成。从光合细菌同步细菌PCC 6803中发现的第一个GLUR：GLUR0中发现的第一个GLUR：GLUR0结合谷氨酸，形成钾选择性通道，与氨基酸序列相关，从光合细菌同步性细胞PCC 6803中获得了直接证据。基于Glur0和真核胶的氨基酸序列和功能关系，核酸glur似乎是真核粘着的前体。 我们发现，GLUR0不仅可以通过谷氨酸，而且可以通过外部质子或降低外部溶液的Ca2+浓度来门控。由质子或通过降低Ca2+门控的单通道响应表现出外部Na+的电压依赖性块，这与对谷氨酸的脱敏反应相似。由所有三个条件进行门控的单渠道活动通常显示出两种类型的门控行为：漫长的开放时间散布着快速闪烁的爆发。质子门控电流EC50 30毫米，在-60 mV时表现出单通道电导的pH依赖性块，并通过1mm谷氨酸透明敏感。同样，通过将1mm谷氨酸脱敏在pH 7.2，IC50 10？m时，通过降低细胞外钙而不是镁降低到10 nm的GLUR0反应。这些门控机制的独立性通过结合位点突变体GLUR0/R117K证明，该突变体保持正常的pH和Ca2+敏感性，同时实际上消除了谷氨酸的激活。尽管在没有配体的情况下，质子可以通过pH或Ca2+ Glur激活来调节许多离子通道的门控，并且以前尚未描述Ca2+抑制的自发活性。 GLUR0的配体结合核已被纯化并过度表达。 X射线衍射已解决了GLUR0的配体结合核的高分辨率结构，Sychocystis PCC 6803的谷氨酸受体离子通道。 GLUR0结构揭示了细菌性周质结合蛋白和大鼠GlUR2 AMPA亚型神经递质受体的同源性。配体结合位点是由两个球状α/β域之间的裂缝形成的。 L-谷氨酸在扩展构象中结合，类似于谷氨酰​​胺结合蛋白（GLNBP）的构象。然而，L-谷氨酸γ-羧基与域1中的ASN51仅相互作用，与配体与GLUR2和GLNBP观察到的域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-丝氨酸的结合位点复合物的结构。这揭示了作用为替代配体原子的溶剂分子，因此丝氨酸OH组使溶剂介导的氢键与ASN51。不含配体的，闭合闭合构象的结构表明，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配体结合核的二聚化，离解常数为0.8？m。在晶体中，涉及域1中残基的对称二聚体沿着2倍轴的晶体学发生，并表明四聚谷氨酸受体离子通道是从二聚体二聚体组装的。我们建议配体诱导的构象变化导致离子通道相对于二聚体界面的增加域2分离而打开。 激动剂与谷氨酸受体的结合触发离子通道门控，后者在毫秒内进行脱敏。脱敏的分子机制未知。与Gouaux实验室合作，我们进行了功能研究，以解决门控机制。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的表面，晶体学研究表明，介导Glur亚基二聚体之间的相互作用。分析性超速离心用于测量溶液中GLUR2配体结合核的KD二聚化。使用压电浓度跳跃设备来测量外部斑块中的脱敏。提高二聚体稳定性的突变体会减少脱敏，而降低二聚体稳定性的突变体会增加脱敏。对非脱敏的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相反亚基的口袋相互作用。侧链的丙氨酸突变会恢复脱敏并增加二聚体形成的KD。我们提出，Glur亚基在二聚体界面上的运动允许离子通道即使动力学结合结构域仍在其闭合结合构象中，该构型最初促进了离子通道门控。我们的结果首次揭示了Glur受体脱敏的分子机制。