MOLECULAR ANALYSIS OF NMDA RECEPTOR MODULATORY SITES

N-甲基-D-天冬氨酸受体(NMDAR)对神经系统的正常功能非常重要，但它们的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的限制。抑制NMDAR过度活性必须在不干扰正常功能的情况下完成。我们小组是第一个证明缺乏竞争力的开放通道阻滞剂美金刚可以抑制 NMDAR的过度活动，而正常的神经传递基本上没有减弱。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-Memantines，它利用Memangine部分靶向NMDAR上的S-亚硝酸化位点(S)，进一步下调过度活性，比单独使用Memangine更好。在我们的初步结果中，我们发现：(I)NR1和NR2亚基的第二跨膜结构域(M2)上的谷氨酰胺/精氨酸/天冬酰胺(Q/R/N)区域形成了美金刚的特异性结合部位；美金刚与该部位的结合可用于将NO靶向NMDAR亚硝化部位；(Ii)低氧通过S亚硝化增强NMDAR活性的下调；(In)脑中存在一个新的NMDAR亚基家族，NR3A和NR3B(我们已克隆并鉴定了这些亚基)。 NR1/NR2A/NR3A(3B)在重组系统中的共表达降低了NMDA/甘氨酸激活电流单通道记录的单位电导、钙通透性和镁敏感性。NR3引起的NMDAR活性降低可能在正常发育过程中起到保护作用；(Iv)NR3和NR1亚单位的共同表达 在非洲爪哇卵母细胞和哺乳动物HEK293细胞中(不含NR2)形成兴奋性的赖氨酸激活的阳离子通道(在没有谷氨酸的情况下)；(V)结晶学实验中Nr1结构的S-亚硝化。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]证明非美金刚靶向N-甲基-D-天冬氨酸受体的特异性，并阐明缺氧条件下S-亚硝酸酯增强下调N-甲基-D-天冬氨酸受体活性的机制。重要的是，在这里研究的药物 项目I将用于本P01资助的其他项目的神经保护实验； [2]表征含NR3通道的通道孔区；[3]表征含NR3受体的配体结合部位，并利用[2]和[3]中的信息 区分NR1/NR3“双联”受体和NR1/NR2/NR3“三联”受体的表达。 [4]用结晶学方法进一步研究NMDAR亚单位配体结合域和亚硝化结构域的结构。

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.