MOLECULAR ANALYSIS OF NMDA RECEPTOR MODULATORY SITES

NMDA 受体调节位点的分子分析

• 批准号: 6349143
• 负责人: VINCENT CHEN
• 金额: $ 24.77万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349143
• 关键词: NMDA receptors; Xenopus oocyte; amantadine; cerebral ischemia /hypoxia; combinatorial chemistry; developmental neurobiology; disease /disorder model; drug screening /evaluation; electrophysiology; gene targeting; ion channel blocker; laboratory mouse; laboratory rat; molecular cloning; molecular site; neuroprotectants; nitric oxide; protein structure function; receptor binding; site directed mutagenesis

The dual role of NMDA receptors in the normal and abnormal functioning of the nervous system imposes important constraints on possible therapeutic strategies aimed at ameliorating ro abating developmental disorders and neurological disease. Based on our groups previous work, two groups of drugs are likely to show promise for safe but effective pharmacological intervention to curtail excessive activity of the NMDA receptor: (i) memantine, a use-dependent and uncompetitive antagonist that is an NMDA open-channel blocker, and (ii) nitric oxide (NO)-related species (provided by nitroglycerin) that interact with redox modulatory sites on the NMDA receptor. In our preliminary results, we have demonstrated the following: 1) the glutamine/arginine/asparagine (Q/R/N) sites in the second membrane spanning (M2) domains of NMDAR1 and NMDAR2 subunits strongly affect memantine binding; 2) there exist three kinetically-distinct components of redox modulation on NR1a/NR2A heteromeric channels that are affected by redox agents; 3) each kinetic NR2A component or redox modulation can be attributed to interaction with a pair of cysteine residues on the NR1 or NR2A subunits and these subunits also affect modulation by Zn; 4) one cysteine residue on NR2A is predominantly responsible for modulation of the NMDAR activity by NO-related species. Furthermore, we recently isolated a novel NMDAR subunit, NR3A (previously designated NMDAR-L or khi-1), from the rat CNS. Co-expression of NR1/nr2a OR b/nr3a IN Xenopus oocytes decreases unitary conductance in single-channel recordings. Conversely, NR3A-deficient mice have larger than normal NMDA-evoked currents. The new project will further study these knock-out mice. We also obtained a near full-length cDNA clone of a second novel NMDAR subunit, tentatively named NR3B since it shows high sequence identify to NR3A. We propose: [1] To study the structural determinants of memantine binding in the channel pore of the NMDAR, [2] To characterize the cysteine residues underlying redox, Zn, and No modulation of the NMDAR, [3] To elucidate the molecular mechanism of action of NR3A whereby it decreases NMDAR-activated current. [4] To clone and characterize a second novel NMDAR subunit, NR3B, in order to determine the function role that it subserves in the developing brain. Most importantly, drugs developed in Project will be used for neuroprotection from hypoxic-ischemic injury and AIDS-related neuronal damage.

NMDA受体在神经系统正常和异常功能中的双重作用对旨在减轻RO减轻发育障碍和神经系统疾病的可能治疗策略施加了重要限制。基于我们的小组以前的工作，两组药物可能会显示出对安全但有效的药理干预措施的希望，以减少NMDA受体的过度活性：（i）纪念碑，一种依赖于用途的且无竞争力的拮抗剂，是NMDA开放通道阻滞剂，以及与NITRIC氧化物（ii）构建的层压层（II）相互作用（II），该物种（ii）由NITRIC-ROOG ORGROOX CORDERS CORDINS CORNIN rog og og rog rog rog rog rog og rog rog rog rog rog rog rog rog rog sode consect NMDA受体。在我们的初步结果中，我们证明了以下内容：1）NMDAR1和NMDAR2亚基的第二个膜跨度（M2）结构域中的谷氨酰胺/精氨酸/天冬氨氨酸/天冬氨酸（q/r/n）位点强烈影响美联社的结合； 2）在NR1A/NR2A杂体通道上存在三个动力学的​​组件，这些组件受氧化还原剂影响； 3）每个动力学NR2A分量或氧化还原调制可以归因于与NR1或NR2A亚基上一对半胱氨酸残基的相互作用，这些亚基也会影响Zn的调制。 4）NR2A上的一个半胱氨酸残基主要负责无关物种对NMDAR活性的调节。此外，我们最近将一种新型的NMDAR亚基NR3A（以前称为NMDAR-L或KHI-1）与大鼠CNS分离出来。 Xenopus卵母细胞中NR1/NR2A或B/NR3A的共表达降低了单通道记录中的单一电导。相反，缺乏NR3A的小鼠具有比正常NMDA诱发的电流大。新项目将进一步研究这些淘汰小鼠。我们还获得了第二个小说NMDAR亚基的几乎全长cDNA克隆，暂定命名为NR3B，因为它显示出对NR3A的高序列。 We propose: [1] To study the structural determinants of memantine binding in the channel pore of the NMDAR, [2] To characterize the cysteine residues underlying redox, Zn, and No modulation of the NMDAR, [3] To elucidate the molecular mechanism of action of NR3A whereby it decreases NMDAR-activated current. [4]为了确定其在发育中的大脑中的函数作用，要克隆并表征第二个新型NMDAR亚基NR3B。最重要的是，在项目中开发的药物将用于缺氧 - 缺血性损伤和与AIDS相关的神经元损害的神经保护作用。