ZINC-DEPENDENT APPARENT DESENSITIZATION OF NMDA RECEPTOR

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状况有关，包括中风、癫痫和阿尔茨海默病。对其中一些疾病的有效治疗仍然缺乏。这项研究的长期目标是确定开发新疗法的新靶点。本实验室已经证明，NR2A的氨基末端结构域（ATD）的高亲和力锌位点与谷氨酸结合结构域之间的变构相互作用导致了快速的锌依赖性脱敏。锌依赖性脱敏的作用现在将通过“敲入”小鼠模型进行研究，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进行评估。为了探究结构决定因素，ATD和连接到跨膜结构域的连接区域将被系统地突变。以下具体目标将被解决：(1)验证锌结合位点和谷氨酸结合位点之间的变构相互作用在不显著改变正常突触NMDA反应的情况下减少兴奋毒性的假设；(2)验证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中强烈影响门控和锌依赖性脱敏的关键结构元件；(4)验证跨膜结构域及其与配体结合结构域的连接体在ATD与S1/S2结构域的ph敏感门控和变构相互作用中起关键作用的假设。相关性：这项研究将确定锌如何调节一种叫做NMDA受体的分子。谷氨酸是一种从死亡的神经元中泄漏出来的分子，它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起着关键作用。锌可以通过引起一种叫做“脱敏”的过程来降低NMDA受体的活性。关于NMDA受体的“锌依赖性脱敏”的知识可能会导致新的药物，可用于减少或阻止中风，癫痫发作中的细胞死亡。

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.