REARRANGMENTS AT THE NMDA RECEPTOR LIGAND BINDING DOMAIN INTERFACE

项目摘要 离子型谷氨酸受体(IGluRs)配体结合域(LBD)二聚体界面的相互作用在受体激活和脱敏过程中起着关键作用。目前的iGluR功能模型认为，激活反应始于将激动剂停靠在固定的LBD上叶(D1)，然后将下叶(D2)向上移动到d1，将激动剂锁定在LBD内。在三维结构中，相邻亚基中的D1叶之间存在一个二聚体界面，这被认为有助于固定D1区并促进通道的开放。相反，当激动剂结合时，这种界面的破裂被认为会导致不活跃、不敏感的受体构象。虽然这个提出的激活和脱敏模型描述了对一级近似的宏观反应，但它们还有待于在微观层面上进行测试。我建议利用对N-甲基-D-天冬氨酸受体(NMDAR)门控反应的更高级理解，通过记录LBD异二聚体界面内发生变化的NMDAR的单通道和全细胞宏观电流，来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还包含独特的非共价相互作用和额外的Site II界面。因此，这些研究将调查所有iGluR共有的机制以及区分NMDAR的机制。二聚体界面上的接触将被二硫键交联和疏水相互作用所修饰，以固定两个D1-D1叶，并可能产生非脱敏受体。相反，当接触被丙氨酸取代而被消除时，界面将被削弱，并将产生具有更快和更深脱敏作用的受体。我还建议研究NMDAR中存在的关键LBD异二聚体界面热点，而不是其他iGluR中的关键LBD异源二聚体界面热点，包括Site II接触和D1-D2相互作用。这项提案中概述的实验将使人们能够前所未有地深入了解LBD水平上亚单位间界面的强度如何有助于NMDAR的激活、失活和/或脱敏。它们还将确定NMDAR和非NMDAR激活机制之间的共性和本质区别。所提出的方法特别适合于剖析对不同门控转变的贡献，并弥合目前在理解iGluR结构内特定位置的重排与总体功能结果之间的差距，通常使用宏观电流记录进行评估。目前，由于对非NMDAiGluR微观动力学的了解仍不充分，这里提出的机制方法仅适用于NMDARs。这些研究的结果将阐明NMDAR特有的机制，但也将为所有iGluR共同的机制提供有用的有价值的见解。

Project Summary 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.