MOLECULAR ANALYSIS OF NMDA RECEPTOR MODULATORY SITES

NMDA 受体调节位点的分子分析

• 批准号: 7029203
• 负责人: DONGXIAN ZHANG
• 金额: $ 33.39万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029203
• 关键词: NMDA receptors; X ray crystallography; Xenopus; amantadine; antiparkinson drugs; binding sites; developmental neurobiology; drug screening /evaluation; hypoxia; ligands; membrane channels; neuroprotectants; neurotoxins; pharmacokinetics; receptor expression

N-methyl-D-aspartate receptors (NMDARs) are important for the normal function of the nervous system, but their excessive activation contributes to a number of developmental disorders and neurological diseases. This dual role imposes important constraints on possible therapeutic strategies aimed at ameliorating mental retardation and neurodegenerative disorders. Inhibition of NMDAR overactivity must be accomplished without interference of normal function. Our group was the first to show that the uncompetitive, open-channel blocker, Memantine, can curtail excessive activity of the NMDAR while leaving normal neurotransmission essentially unabated. Based in part on our work, Memantine was recently approved for clinical use in the USA. Here we develop dual-functional derivatives, the NO-Memantines, that use the Memantine moiety to target NO to S-nitrosylation site(s) on the NMDAR to further downregulate excessive activity better than Memantine alone. In our preliminary results, we have shown: (i) the glutamine/arginine/asparagine (Q/R/N) sites in the second transmembrane (M2) domains of NR1 and NR2 subunits form a specific binding site for Memantine; the binding of Memantine to this site can be used for targeting NO to the NMDAR nitrosylation site; (ii) hypoxia enhances downregulation of NMDAR activity via S-nitrosylation; (in) a novel family of NMDAR subunits, NR3A and NR3B, exist in the brain (we have cloned and characterized these subunits). Co-expression of NR1/NR2A/NR3A(3B) in recombinant systems decreases unitary conductance, Ca2+ permeability, and Mg2+ sensitivity in single-channel recordings of NMDA/glycine-activated currents. Decreased NMDAR activity engendered by NR3 may be protective during normal development; (iv) co-expression of NR3 and NR1 subunits (without NR2) in Xenopus oocytes and mammalian HEK 293 cells form excitatory lycine-activated cation channels (in the absence of glutamate); (v) S-nitrosylation of the NR1 structure in crystallography experiments. This finding allows us to begin to relate structure to function. We propose the following Specific Aims: [1] To prove the specificity of NO-Memantine targeting to the NMDAR, and to elucidate the mechanism of enhanced downregulation of NMDAR activity by S-nitrosylation under hypoxic conditions. Importantly, drugs studied here in Project I will be used for neuroprotection experiments in the other Projects of this P01 grant; [2] To characterize the channel pore region of NR3-containing channels; [3] To characterize the ligand-binding site of NR3-containing receptors, and use information from [2] and [3] to distinguish expression of NR1/NR3 "doublet" receptors from NR1/NR2/NR3 "triplet" receptors. [4] To further study the structure of NMDAR subunit ligand-binding and nitrosylation domains using crystallography.

N-甲基-D-天冬氨酸受体（NMDAR）对于神经系统的正常功能很重要，但其过度激活会导致许多发育障碍和神经系统疾病。这种双重作用对旨在改善精神发育迟滞和神经退行性疾病的可能治疗策略施加了重要限制。必须在不干扰正常功能的情况下抑制 NMDAR 过度活动。我们的团队是第一个证明无竞争力的开放通道阻断剂美金刚可以减少 NMDAR 的过度活性，而正常的神经传递基本不减弱。部分基于我们的工作，美金刚最近在美国被批准用于临床。在这里，我们开发了双功能衍生物 NO-Memantine，它使用 Memantine 部分将 NO 靶向 NMDAR 上的 S-亚硝基化位点，以比单独使用 Memantine 更好地进一步下调过度活性。在我们的初步结果中，我们表明：（i）NR1和NR2亚基的第二跨膜（M2）结构域中的谷氨酰胺/精氨酸/天冬酰胺（Q/R/N）位点形成美金刚的特异性结合位点；美金刚与该位点的结合可用于将NO靶向NMDAR亚硝基化位点； (ii) 缺氧通过 S-亚硝基化增强 NMDAR 活性的下调； （中）大脑中存在一个新的 NMDAR 亚基家族 NR3A 和 NR3B（我们已经克隆并表征了这些亚基）。 重组系统中 NR1/NR2A/NR3A(3B) 的共表达降低了 NMDA/甘氨酸激活电流单通道记录中的单一电导、Ca2+ 渗透性和 Mg2+ 敏感性。 NR3 引起的 NMDAR 活性降低可能在正常发育过程中具有保护作用； (iv) NR3 和 NR1 亚基的共表达 （不含 NR2）在非洲爪蟾卵母细胞和哺乳动物 HEK 293 细胞中形成兴奋性甘氨酸激活的阳离子通道（在没有谷氨酸的情况下）； (v) 晶体学实验中 NR1 结构的 S-亚硝基化。这一发现使我们能够开始将结构与功能联系起来。我们提出以下具体目标： [1] 证明NO-Memantine靶向NMDAR的特异性，并阐明缺氧条件下S-亚硝基化增强下调NMDAR活性的机制。重要的是，这里研究的药物 项目I将用于本次P01资助的其他项目中的神经保护实验； [2] 表征含NR3通道的通道孔区域； [3] 表征含 NR3 受体的配体结合位点，并使用 [2] 和 [3] 中的信息 区分 NR1/NR3“双联体”受体和 NR1/NR2/NR3“三联体”受体的表达。 [4] 利用晶体学进一步研究NMDAR亚基配体结合和亚硝基化结构域的结构。