Mechanism of action of novel GluN2C NMDA receptor potentiators

新型 GluN2C NMDA 受体增强剂的作用机制

• 批准号: 8554784
• 负责人: Alpa Khatri
• 金额: $ 5.39万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-28 至 2014-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8554784
• 关键词: Affect; Age; Agonist; Autistic Disorder; Binding; Brain; Cell Line; Cells; Cerebellar Mossy Fibers; Cerebellum; Chemosensitization; Chimera organism; Complex; Data; Disease; Epilepsy; Family; Funding; Glutamate Receptor; Glutamates; Glycine; Goals; Ion Channel; Knockout Mice; Lead; Learning; Long-Term Potentiation; Mammalian Cell; Measures; Mediating; Memory; Molecular Probes; Motor; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; National Institute of Mental Health; Neuraxis; Oocytes; Pain; Paper; Parkinson Disease; Perfusion; Play; Point Mutation; Preclinical Drug Evaluation; Probability; Property; Psychotic Disorders; Psychotropic Drugs; Pump; Receptor Signaling; Research; Role; Schizophrenia; Site; Slice; Stimulus; Synapses; Synaptic Transmission; Synaptic plasticity; System; Techniques; Testing; Time; Xenopus laevis; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; delta opioid receptor; design; gain of function; granule cell; in vivo; inhibitor/antagonist; insight; kainate; loss of function; member; nervous system disorder; neuron development; novel; patch clamp; prevent; programs; public health relevance; receptor; receptor function; research study; tool; voltage clamp

DESCRIPTION (provided by applicant): The ionotropic glutamate receptors family includes the N-methyl D-aspartate (NMDA), alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and delta receptors, mediate the excitatory synaptic transmission in the central nervous system. The NMDA receptor is a heteromeric receptor formed by two glycine binding GluN1 and two glutamate binding GluN2 subunits. The GluN2 subunit, of which there are four (GluN2A, B, C, and D) mediate the ion channel properties of the NMDA receptors. The GluN1/GluN2C NMDA receptor is highly expressed in the cerebellum, specifically on the cerebellar granule cell at the mossy fiber-cerebellar granule cell synapse. Less than 2% of the papers on NMDA receptors are on the GluN1/GluN2C receptor, probably because there has been a lack of pharmacological tools that can specifically identify the GluN1/GluN2C receptors in vivo. This application presents the first known compound class (ketopyrrolines) that selectively potentiates the GluN2C containing receptors. This application aims to identify the structural determinates and mechanism of action of ketopyrrolines on the GluN2C receptors, as well as the role of GluN2C in the induction of LTP in the cerebellum. These experiments will determine ketopyrrolines usefulness as a pharmacological tool to understand the role of GluN2C in the CNS. Aim 1: What are the possible targets and structural determinants of ketopyrrolines? Ketopyrrolines will be tested for GluN2C selectivity within the lab and at the NIMH funded Psychoactive Drug Screening Program at UNC. Gain of function and loss of function chimeras will be constructed between the GluN2A and Glun2C subunits expressed in Xenopus laevis oocytes. This technique was successfully used in the lab to identify the structural determinates for a GluN2C/D potentiator (CIQ) and inhibitor (QNZ46). Aim 2: What is the mechanism of action for ketopyrrolines? Whole-cell and single channel recording will be performed by expressing the GluN1/GluN2C receptor in a mammalian cell line. Ketopyrrolines will be applied by a fast perfusion system to measure its association, disassociation, open probability, and channel open and shut times. Aim 3. How do the GluN2A and GluN2C subunits affect the threshold for plasticity in the cerebellum? The threshold for plasticity will be determined in the presence and absence of ketopyrrolines for the wild type, GluN2A and GluN2C knockout mice by varying the input stimulus. Patch Clamp recording will be carried out in cerebellar slices at an appropriate age for GluN2C expression. The successful completion of this application will lead to an understanding of the structural determinants, mechanism of potentiation, the role of GluN2C within the threshold of plasticity and in vivo effects of ketopyrrolines, a novel compound class that selectively potentiates the GluN1/GluN2C NMDA receptor.

描述（由申请人提供）：离子型谷氨酸受体家族包括N-甲基D-天冬氨酸（NMDA）、α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）、红藻氨酸和δ受体，介导中枢神经系统中的兴奋性突触传递。NMDA受体是由两个甘氨酸结合GluN 1和两个谷氨酸结合GluN 2亚基形成的异聚体受体。GluN 2亚基有四个（GluN 2 A、B、C和D），介导NMDA受体的离子通道特性。GluN 1/GluN 2C NMDA受体在小脑中高度表达，特别是在苔藓纤维-小脑颗粒细胞突触的小脑颗粒细胞上。不到2%的关于NMDA受体的论文是关于GluN 1/GluN 2C受体的，这可能是因为缺乏可以在体内特异性识别GluN 1/GluN 2C受体的药理学工具。本申请提出了第一个已知的化合物类别（酮吡咯啉），其选择性地增强含有GluN 2C的受体。本申请旨在确定酮吡咯啉对GluN 2C受体的结构决定因素和作用机制，以及GluN 2C在小脑LTP诱导中的作用。这些实验将确定酮吡咯啉作为药理学工具的有用性，以了解GluN 2C在CNS中的作用。目的1：吡咯酮的可能作用靶点和结构决定因素是什么？酮吡咯啉将在实验室内和NIMH资助的精神活性药物筛选计划中进行GluN 2C选择性测试。将在非洲爪蟾卵母细胞中表达的GluN 2A和Glun 2C亚基之间构建功能获得和功能丧失嵌合体。该技术在实验室中成功地用于鉴定GluN 2C/D增效剂（CIQ）和抑制剂（QNZ 46）的结构决定簇。目的2：酮吡咯啉的作用机制是什么？将通过在哺乳动物细胞系中表达GluN 1/GluN 2C受体进行全细胞和单通道记录。将通过快速灌注系统应用酮吡咯啉，以测量其缔合、解离、开放概率和通道开放和关闭时间。目标3. GluN 2A和GluN 2C亚基如何影响小脑可塑性的阈值？对于野生型、GluN 2A和GluN 2C敲除小鼠，在存在和不存在酮吡咯啉的情况下，通过改变输入刺激来确定可塑性的阈值。将在适当年龄的小脑切片中进行膜片钳记录，用于GluN 2C表达。本申请的成功完成将导致了解的结构决定因素，增强的机制，GluN 2C的可塑性和酮吡咯啉，一种新的化合物类，选择性地增强GluN 1/GluN 2C NMDA受体的体内效应的阈值内的作用。