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，但是它们的过度激活有助于多种发育障碍和神经系统疾病。这种双重作用对旨在减轻智力低下和神经退行性疾病的可能的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完成NMDAR过度活动的抑制。我们的小组是第一个表明无竞争，开放通道阻滞剂美体的人，可以减少 NMDAR的活动过多，而离开正常的神经递送基本上没有减弱。纪念碑最近在美国批准了美国的临床用途。在这里，我们开发了双功能衍生物，即无象征的衍生物，这些衍生物使用美容部分来靶向NMDAR上的s-硝基化位点，以进一步下调过度活性，而不是单独的纪念碑。在我们的初步结果中，我们显示了：（i）NR1和NR2亚基的第二个跨膜（M2）域中的谷氨酰胺/精氨酸/天冬氨氨酸/天冬氨酸（q/r/n）位点形成了纪念碑的特定结合位点；美金刚与该部位的结合可用于靶向NMDAR硝基化位点。 （ii）缺氧通过S-硝基化增强了NMDAR活性的下调； （在）大脑中存在一个新型的NMDAR亚基NR3A和NR3B家族（我们已经克隆并表征了这些亚基）。 重组系统中NR1/NR2A/NR3A（3B）的共表达可降低NMDA/甘氨酸激活水流的单渠道记录中单位电导，Ca2+渗透性和MG2+敏感性。 NR3产生的NMDAR活性降低在正常发育过程中可能具有保护性； （iv）NR3和NR1亚基的共表达 （无NR2）在异爪蟾卵母细胞和哺乳动物HEK 293细胞中形成兴奋性lycine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联系起来。我们提出以下具体目的：[1]证明非甲虫靶向NMDAR的特异性，并阐明在低氧条件下通过S-硝基化增强NMDAR活性下调的机制。重要的是，这里研究的药物 我将在该P01赠款的其他项目中用于神经保护实验； [2]表征含NR3的通道的通道孔区； [3]表征含NR3受体的配体结合位点，并使用[2]和[3]的信息 区分NR1/NR3“ Doublet”受体的表达与NR1/NR2/NR3“ Triplet”受体的表达。 [4]进一步研究了使用晶体学的NMDAR亚基配体结合和亚硝基化结构域的结构。