ZINC-DEPENDENT APPARENT DESENSITIZATION OF NMDA RECEPTOR

描述(申请人提供)：这项研究探索了N-甲基-D-天冬氨酸(NMDA)受体NR2A亚单位的高亲和力锌位点在谷氨酸诱导的神经细胞死亡(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。本实验室已经证明，NR2A的氨基末端结构域(ATD)中的高亲和力锌位点与谷氨酸结合域之间的变构相互作用可导致快速的锌依赖脱敏。锌依赖脱敏的作用现在将通过“敲入”小鼠模型来研究，在该模型中，锌位点被一个点突变(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。为了探索结构决定因素，ATD和跨膜结构域的连接区将被系统地突变。具体目标如下：(1)检验锌结合部位和谷氨酸结合部位之间的变构相互作用在不显著改变正常突触NMDA反应的情况下降低兴奋性毒性的假说；(2)检验NR2的ATD强烈影响NMDA受体亚型特异性门控的假说；(3)确定NR2AATD中强烈影响门控和锌依赖脱敏的关键结构元件；(4)验证跨膜区及其与配体结合区的连接物在ATD和S1/S2区之间的pH敏感门控和变构相互作用中起关键作用的假说。相关性：这项研究将确定锌如何调节一种名为NMDA受体的分子。谷氨酸是一种从濒临死亡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受体在细胞死亡的恶性循环中扮演着关键角色。锌可以通过引起一种称为“脱敏”的过程来降低NMDA受体的活性。关于NMDA受体“锌依赖脱敏”的知识可能会导致新的药物，可以用来减少或阻止中风、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.