REARRANGMENTS AT THE NMDA RECEPTOR LIGAND BINDING DOMAIN INTERFACE

描述（由申请人提供）： 离子型谷氨酸受体（iGluRs）的配体结合域（LBD）二聚体界面的相互作用在受体激活和脱敏中起关键作用。目前的iGluR功能模型提出，激活反应开始于将激动剂对接在固定的LBD上叶（D1），然后下叶（D2）朝D1向上移动，以将激动剂锁定在LBD内。在3-D结构中鉴定了相邻亚基中D1叶之间的二聚体界面，并认为其有助于抑制D1并促进通道开放。相反，当激动剂被结合时，该界面的破裂被认为导致无活性的、脱敏的受体构象。虽然这个激活和脱敏的模型描述了宏观反应的第一近似值，但它们还有待在微观水平上进行测试。我建议利用对N-甲基-D-天冬氨酸受体（NMDAR）门控反应的更深入理解，通过记录LBD异二聚体界面内NMDAR的单通道和全细胞宏观电流，研究二聚体界面残基在通道激活中的作用。除了所有iGluR共有的相互作用外，NMDAR还含有独特的非共价相互作用和额外的位点II界面。因此，这些研究将研究所有iGluR共同的机制以及区分NMDAR的机制。二聚体界面的接触将通过二硫键交联和疏水相互作用进行修饰，以使两个D1-D1叶重叠，并可能产生非脱敏受体。相反，当接触将被丙氨酸取代废除时，界面将被削弱，并将产生具有更快和更深脱敏的受体。我还建议检查关键LBD异二聚体接口热点存在于NMDAR，但不是在其他iGluRs，包括网站II的联系和D1-D2的相互作用。在这个建议中概述的实验将允许前所未有的洞察力如何在LBD水平的亚基间接口的强度有助于NMDAR激活，失活和/或脱敏。他们还将确定NMDAR和非NMDAR激活机制之间的共性和本质区别。所提出的方法是唯一适合于解剖的贡献，不同的门控转换，并弥合目前的差距，在理解重排在特定位置内iGluR结构和整体功能的结果，通常与宏观电流记录进行评估。目前，由于对非NMDA iGluR微观动力学的理解仍然不足，本文提出的机制方法仅适用于NMDAR。这些研究的结果将阐明特定于NMDAR的机制，但也为所有iGluRs共同的机制提供了有用的有价值的见解。 公共卫生相关性： NMDAR在包括神经元发育、突触强度和通信的许多生理过程以及包括与中风、精神分裂症和神经变性（例如阿尔茨海默病和亨廷顿病）相关的兴奋性毒性细胞死亡的神经病理学中起重要作用。这项提议的结果将有助于我们进一步了解大脑中的兴奋性传递，以及如何纠正这种传递，以减轻神经系统疾病、成瘾和精神疾病的负担。

DESCRIPTION (provided by applicant): Interactions at the ligand binding domain (LBD) dimer interface of ionotropic glutamate receptors (iGluRs) play a pivotal role in receptor activation and desensitization. The current model of iGluR function proposes that the activation reaction starts with docking the agonist at a fixed LBD upper lobe (D1), followed by upward movement of the lower lobe (D2) towards D1 to lock the agonist within the LBD. A dimer interface between D1 lobes in adjacent subunits was identified in 3-D structures and is believed to help immobilize D1 and promote channel opening. In contrast, rupture of this interface, while agonist is bound, is thought to lead to an inactive, desensitized receptor conformation. While this proposed model for activation and desensitization describes macroscopic responses to a first approximation they have yet to be tested at the microscopic level. I propose to take advantage of the more advanced understanding of N-methyl-D-aspartate receptor (NMDAR) gating reaction to investigate the role in channel activation of residues at the dimer interface by recording both single- channel and whole-cell macroscopic currents from NMDARs with alterations within the LBD heterodimer interface. In addition to interactions common to all iGluRs, NMDARs also contain unique non-covalent interactions and an additional site II interface. Thus these studies will investigate mechanisms that are common to all iGluR as well as those that tell NMDARs apart. Contacts at the dimer interface will be modified by disulfide bond cross-linking and hydrophobic interactions, to immobilize the two D1-D1 lobes and presumably produce non-desensitizing receptors. Conversely, when contacts will be abolished by alanine substitution, the interface will be weakened and will produce receptors with faster and deeper desensitization. I also propose to examine key LBD heterodimer interface hotspots present in NMDARs but not in other iGluRs including site II contacts and D1-D2 interactions. The experiments outlined in this proposal will allow unprecedented insight into how the strength of intersubunit interface at the level of LBD contributes to NMDAR activation, deactivation and/or desensitization. They will also identify commonalities, as well as essential distinctions, between NMDAR and non- NMDAR activation mechanisms. The approach proposed is uniquely suited to dissect contributions to distinct gating transitions and to bridge the current gap in understanding between rearrangements at specific locations within iGluR structures and the overall functional result, usually evaluated with macroscopic current recordings. At present, the mechanistic approach proposed here is only possible for NMDARs, due to still inadequate understanding of non-NMDA iGluR microscopic kinetics. Results from these studies will illuminate mechanisms that are specific to NMDARs but also provide useful valuable insights into mechanisms that are common to all iGluRs. PUBLIC HEALTH RELEVANCE: NMDARs play a vital role in numerous physiological processes including neuronal development, synaptic strength and communication as well as neurological pathologies including excitotoxic cell death associated with stroke, schizophrenia, and neurodegeneration such as in Alzheimer's and Huntington's disease. The results of this proposal will help advance our understanding of excitatory transmission in the brain and of the ways in which this can be corrected to alleviate the burden of neurological diseases, addiction and mental illness.