ZINC-DEPENDENT APPARENT DESENSITIZATION OF NMDA RECEPTOR

NMDA 受体锌依赖性表观脱敏

• 批准号: 7405411
• 负责人: FANG ZHENG
• 金额: $ 28.4万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-09 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405411
• 关键词: Address; Adverse effects; Affinity; Alzheimer' s Disease; Amino Acids; Binding; Binding Sites; Brain; Cell Death; Cells; Chromosome Pairing; Classification; Clinical; Condition; Disease; Drug Interactions; Elements; Glucose; Glutamates; Hippocampal Mossy Fibers; Hippocampus (Brain); Indium; Intraperitoneal Injections; Kainic Acid; Knock-in Mouse; Knowledge; Laboratories; Lead; Ligand Binding Domain; Location; Membrane; Mus; Mutate; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Nature; Neurons; Numbers; Oxygen; Pathogenesis; Patients; Pharmaceutical Preparations; Play; Point Mutation; Potassium Channel; Process; Property; Protons; Psychotic Disorders; Rate; Research; Research Personnel; Role; Seizures; Series; Site; Slice; Stroke; Synapses; System; Testing; Time; Transmembrane Domain; Zinc; base; deprivation; desensitization; excitotoxicity; improved; in vivo; mouse genome; mouse model; mutant; nervous system disorder; neuron loss; novel; outcome forecast; patch clamp; programs; receptor; receptor function; response

DESCRIPTION (provided by applicant): This study explores the role of the high affinity zinc site in the NR2A subunit of N-methyl-D-aspartate (NMDA) receptors in glutamate-induced neuronal cell death (i.e. excitotoxicity). Excitotoxicity is implicated in a variety of neuropathological conditions, including stroke, seizures and Alzheimer's disease. Effective treatment for some of these diseases is still lacking. A long-term objective of this study is to identify novel targets for developing new therapy. This laboratory has shown that an allosteric interaction between the high affinity zinc site in the amino-terminal domain (ATD) and the glutamate-binding domain of NR2A causes fast zinc-dependent desensitization. The role of zinc-dependent desensitization will now be studied with a "knock-in" mouse model in which the zinc site is abolished by a point mutation (NR2AH128A). The mechanism and structural determinants of this allosteric interaction will be studied in an artificial expression system (HEK293 cells). The receptor function will be assessed by whole cell patch-clamp recording and single channel recording. To probe the structural determinants, the ATD and the linker regions to the transmembrane domains will be mutated systematically. The following specific aims will be addressed: (1) To test the hypothesis that the allosteric interaction between the zinc binding site and the glutamate binding site reduces excitotoxicity without significant alteration of normal synaptic NMDA responses; (2) To test the hypothesis that the ATD of NR2 strongly influences the subtype-specific gating of NMDA receptors; (3) To identify the critical structural element in the ATD of NR2A that strongly influences the gating and zinc- dependent desensitization; (4) To test the hypothesis that transmembrane domains and their linkers to the ligand-binding domain play a critical role in the pH-sensitive gating and the allosteric interaction between the ATD and the S1/S2 domain. Relevance: This study will determine how zinc modulates a molecule called NMDA receptors. Glutamate is a molecule leaked from dying neurons that casuses more neurons to die and NMDA receptors play a critical role in this vicious circle of cell death. Zinc can reduce the activity of NMDA receptors by causing a process called "desensitization". Knowledge about "zinc-dependent desensitization" of NMDA receptors may lead to new drugs that can be used to reduce or block the cell death in stroke, seizures.

描述（由申请人提供）：本研究探讨了 N-甲基-D-天冬氨酸 (NMDA) 受体 NR2A 亚基中的高亲和力锌位点在谷氨酸诱导的神经元细胞死亡（即兴奋性毒性）中的作用。兴奋性毒性与多种神经病理学病症有关，包括中风、癫痫发作和阿尔茨海默病。其中一些疾病仍然缺乏有效的治疗方法。这项研究的长期目标是确定开发新疗法的新靶点。该实验室表明，氨基末端结构域 (ATD) 中的高亲和力锌位点与 NR2A 的谷氨酸结合结构域之间的变构相互作用会导致快速的锌依赖性脱敏。现在将通过“敲入”小鼠模型来研究锌依赖性脱敏的作用，其中锌位点被点突变（NR2AH128A）消除。这种变构相互作用的机制和结构决定因素将在人工表达系统（HEK293 细胞）中进行研究。受体功能将通过全细胞膜片钳记录和单通道记录来评估。为了探测结构决定因素，ATD 和跨膜结构域的接头区域将被系统地突变。将解决以下具体目标：（1）检验锌结合位点和谷氨酸结合位点之间的变构相互作用降低兴奋性毒性而不显着改变正常突触 NMDA 反应的假设； (2) 检验NR2的ATD强烈影响NMDA受体亚型特异性门控的假设； (3) 确定NR2A ATD中对门控和锌依​​赖性脱敏有强烈影响的关键结构元件； (4) 检验跨膜结构域及其与配体结合结构域的接头在 pH 敏感门控以及 ATD 和 S1/S2 结构域之间的变构相互作用中发挥关键作用的假设。相关性：这项研究将确定锌如何调节 NMDA 受体分子。谷氨酸是一种从垂死的神经元中泄漏出来的分子，会导致更多的神经元死亡，而 NMDA 受体在这种细胞死亡的恶性循环中发挥着关键作用。锌可以通过引起“脱敏”过程来降低 NMDA 受体的活性。关于 NMDA 受体“锌依赖性脱敏”的知识可能会带来新药，可用于减少或阻止中风、癫痫发作时的细胞死亡。