PROJECT 1 - MOLECULAR ANALYSIS OF NMDA RECEPTOR MODULATORY SITES

项目I-张和陈项目主任/首席调查员(最后、第一、中间)：利普顿，斯图尔特·A。 项目总结(见说明)： 谷氨酸受体的N-甲基-D-天冬氨酸亚型(NMDAR)是中枢神经系统(CNS)正常功能所必需的。然而，NMDAR的过度激活，特别是突触外而不是突触受体的过度激活，至少在一定程度上介导了许多神经疾病的神经元或突触损伤，如最近提出的缺氧缺血性脑损伤和唐氏综合征。阻断过度的NMDAR活动必须在不干扰其正常大脑功能的情况下实现。我们采取了两种方法对NMDAR进行临床耐受的药理学和遗传学干预。一种方法是使用美金刚，但也没有物种通过S-亚硝化进一步下调NMDAR。 在这笔赠款的赞助下，将进一步描述美金刚和无类似物种作用的NMDARs的结构决定因素。另一种方法是利用由NRSA和NRSB组成的一族新的NMDAR亚单位的抑制作用，通过影响通道通透性来下调NMDAR，在某种意义上模仿了NMDAR拮抗剂的作用，这些药物也在这里正在开发中。我们将研究NRS亚单位的MS结构域在下调NMDAR活性中的作用，并设计基于NRS配体结合结构域(LBD)的筛选方法，以发现调节NRS受体的新化合物。这些试剂将有助于表征含有NRS的受体，并可能用于神经保护。 因此，这项建议的具体目的如下：1)从电生理角度研究S-亚硝化/氧化还原对NMDAR氨基末端结构域与LBD之间松散连接区域的调节作用；2)建立LBD衍生的筛选方法以筛选NMDAR NRS亚单位的选择性配体。这些配体将通过二次分析、化学修饰和共结晶进行进一步的表征和提纯；3)研究NRS亚基前庭外区多肽对NMDAR通透性的抑制作用。

Project I - Zhang & Chen Program Director/Principal Investigator (Last, First, Middle): Lipton, Stuart A. PROJECT SUMMARY (See instructions): The N-methyl-D-aspartate subtype of gluatmate receptor (NMDAR) is essential for normal function of the central nervous system (CNS). However, excessive activation of NMDARs, particulariy of extrasynaptic as opposed to synaptic receptors, mediates, at least in part, neuronal or synaptic damage in many neurological disorders, such as hypoxic-ischemic brain injury and, as recently suggested, in Down syndrome. Blockade of excessive NMDAR activity must be achieved without interference with its normal brain function. We have taken two approaches for clinically-tolerated pharmacological and genetic intervention on NMDARs. One approach is to use Memantine but also NO species to further down regulate the NMDAR by S-nitrosylation. The structural determinants on NMDARs for the action of Memantine and NO-like species will be characterized further under the auspices of this grant. Another approach is to utilize the inhibitory effect of a novel family of NMDAR subunits, composed of NRSA and NRSB, to downregulate NMDARs by affecting channel permeability, in a sense mimicking the effect of the NMDAR antagonist drugs that are also being developed here. We will study the role of the MS domain of NRS subunits that downregulate activity of NMDARs and also design NRS ligand-binding domain (LBD)-based screening assays to discover new compounds that modulate NRS-containing receptors. These agents will be useful for characterizing NRS-containing receptors, and possibly for neuroprotection. Accordingly, the Specific Aims of this proposal are as follows: 1) To study the effect of S-nitrosylation/redox modulation of the loose linker region between the amino-terminal domain (ATD) and the LBD of NMDARs by electrophysiology; 2) To develop LBD-derived screening assays to screen for ligands selective for the NRS subunit of the NMDAR. These ligands will be further characterized and refined by secondary assays, chemical modification, and co-crystallization; 3) To study the inhibitory effect of peptides derived from the out vestibule (MS) region of NRS subunits on NMDAR permeability.