Structural and functional investigation of negative allosteric NMDA receptor modulation

NMDA受体（NMDARs）是介导兴奋性神经传递的谷氨酸受体离子通道。 大多数NMDAR由两个GluN 1和两个GluN 2亚基组成。一个GluN 1亚基已经被 克隆，但有四个GluN 2亚基（GluN 2 A-D）赋予NMDAR不同的功能特性 以及不同的发展和区域表达。编码GluN 2A亚基的基因突变， 与儿童癫痫/失语症综合征相关，这些突变的一个子集导致 在含GluN 2A的受体中起作用，导致严重的神经系统并发症。的高频率 与神经系统疾病相关的GluN 2A突变为神经系统疾病的发展提供了令人信服的理由。 GluN 2A选择性治疗剂。GluN 2A-选择性调节剂也可以是有用的和非常需要的 神经生理学研究的药理学工具。我们发现了新的GluN 2A选择性阴性 通过减少甘氨酸与GluN 1亚基的结合来抑制NMDAR的变构调节剂（NAM）。这些 NAMs可以改变我们评估GluN 2A对正常脑功能和疾病的贡献的能力。在 此外，我们还测定了GluN 1和GluN 2A激动剂形成的异源二聚体的晶体结构 结合结构域（ABD）与在亚基界面处结合的NAM。这些晶体结构代表了 发现了一个新的，未开发的调节位点，并为亚基的发展提供了新的机会， 选择性配体我们将揭示这个调节位点的结构和机制特征，并提供 使用GluN 2A选择性NAM作为药理学工具所需的重要理解。目标1） GluN 2A选择性NAMs的选择性、效力和功效的结构基础是什么？我们将使用 晶体学、诱变和电生理学记录NMDA受体功能，以确定 NAM抑制的结构决定因素。目的2）变构相互作用的机理基础是什么 有什么联系吗晶体学、药理学和突变分析将 剖析了NAM与H2O之间的构象变化及其引起变构相互作用的机制。 甘氨酸结合位点。目的3）GluN 2A选择性NAMs是否能抑制三异聚体和神经元NMDA 受体？我们将定义在与突触传递相关的条件下NAM对NMDAR的抑制，以及 NAM抑制癫痫患者中鉴定的含有功能获得性GluN 2A突变的NMDAR 神经元NMDAR是由两个GluN 1和GluN 2组成的二异聚体受体的异质群体。 两个相同的GluN 2亚基（例如GluN 1/GluN 2A）以及含有两个GluN 1的三异聚体受体 和两个不同的GluN 2亚基（例如GluN 1/GluN 2A/GluN 2B）。GluN 2A-选择性调节剂对人胰岛素抵抗的评估 二异聚体和三异聚体受体都需要用作药理学工具。此外，本发明还提供了一种方法， 我们将在表达不同的神经元的自发性神经元的原代培养物中抑制NMDAR介导的突触电流， GluN 2A和GluN 2B的比例以及表达具有功能获得性突变的GluN 2A的自适应神经元。

NMDA receptors (NMDARs) are glutamate receptor ion channels that mediate excitatory neurotransmission. The majority of NMDARs are composed of two GluN1 and two GluN2 subunits. One GluN1 subunit has been cloned, but there are four GluN2 subunits (GluN2A-D) that endow NMDARs with distinct functional properties and different developmental and regional expression. Mutations in the gene encoding the GluN2A subunit have been associated with childhood epilepsy/aphasia syndromes, and a subset of these mutations cause gain-of- function in GluN2A-containing receptors that lead to severe neurologic complications. The high frequency of GluN2A mutations linked to neurologic conditions provides a compelling rationale for the development of GluN2A-selective therapeutic agents. GluN2A-selective modulators can also be useful and much needed pharmacological tools for neurophysiological studies. We have identified novel GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDARs by reducing glycine binding to the GluN1 subunit. These NAMs can transform our ability to evaluate the contribution of GluN2A to normal brain function and disease. In addition, we have determined crystal structures of the heterodimer formed by GluN1 and GluN2A agonist binding domains (ABDs) with NAMs bound at the subunit interface. These crystal structures represent the discovery of a novel, unexplored modulatory site and provide new opportunities for the development of subunit- selective ligands. We will uncover structural and mechanistic features of this modulatory site and provide important understanding required for the use of GluN2A-selective NAMs as pharmacological tools. Aim 1) What are the structural bases for selectivity, potency, and efficacy of GluN2A-selective NAMs? We will use crystallography, mutagenesis, and electrophysiological recordings of NMDA receptor function to define the structural determinants for NAM inhibition. Aim 2) What is the mechanistic basis for allosteric interaction between NAM and glycine binding sites? Crystallographic, pharmacological, and mutational analyses will dissect the conformational changes and mechanism that causes the allosteric interaction between NAM and glycine binding sites. Aim 3) Can GluN2A-selective NAMs inhibit triheteromeric and neuronal NMDA receptors? We will define NAM inhibition of NMDARs in conditions relevant to synaptic transmission as well as NAM inhibition of NMDARs that contain gain-of-function GluN2A mutations identified in epilepsy patients. Neuronal NMDARs are heterogeneous populations of diheteromeric receptors comprised of two GluN1 and two identical GluN2 subunits (e.g. GluN1/GluN2A) as well as triheteromeric receptors containing two GluN1 and two different GluN2 subunits (e.g. GluN1/GluN2A/GluN2B). Evaluation of GluN2A-selective modulators on both diheteromeric and triheteromeric receptors is required for their use as pharmacological tools. In addition, we will inhibit NMDAR-mediated synaptic currents in primary cultures of autaptic neurons that express different ratios of GluN2A and GluN2B as well as autaptic neurons that express GluN2A with gain-of-function mutations.