Regulation of GluN2B-NMDA Receptors by Interactions with the Actin Cytoskeleton

通过与肌动蛋白细胞骨架相互作用调节 GluN2B-NMDA 受体

基本信息

  • 批准号:
    10606121
  • 负责人:
  • 金额:
    $ 3.32万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-04-01 至 2025-12-31
  • 项目状态:
    未结题

项目摘要

Project Summary N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels composed of transmembrane GluN1, GluN2 (A-D), and GluN3 (A-B) subunits that mediate Ca2+ influx into the dendritic spine. Importantly, the unique intracellular tail of the GluN2B subunit is essential for learning and memory. Furthermore, autism spectrum disorder (ASD) and schizophrenia (SCZ) associated variants are found within the GluN2B intracellular tail. My overall goal is to elucidate a novel mechanism by which GluN2B tails contribute to the function of GluN2B- containing NMDARs (GluN2B-NMDARs). Previous experiments showed that actin-targeting drugs impact NMDAR activity, but the underlying mechanisms are unknown. My preliminary data strongly support the primary hypothesis of this project: a direct interaction between actin filaments and the GluN2B intracellular tail regulates NMDAR activity. The objective of my proposed project is to elucidate the biochemical basis for an interaction between the GluN2B tail and actin filaments and determining how this interaction regulates NMDAR function. I will achieve this objective by pursuing three highly complementary aims: Aim 1. Define the minimal fragment of GluN2B intracellular tail that mediates high affinity binding to actin filaments and the interfaces on GluN2B and actin that mediate binding. My preliminary data show a direct interaction between GluN2B tails and actin, but the key interfaces that mediate this interaction between the proteins is unknown. I will use recombinant proteins to perform quantitative binding assays and cross-linking assays to identify the minimal fragment of the GluN2B tail needed to mediate a high affinity interaction with actin. Aim 2. Characterize how Ca2+ and genetic variants impact GluN2B:actin binding. Ca2+ decreases NMDAR activity in cultured hippocampal neurons, but whether Ca2+ selectively impacts GluN2B-NMDAR activity is unknown. My preliminary data suggest Ca2+ strengthens the GluN2B tail:actin interaction. Genetic variants that lie within GluN2B tail regions that I found bind actin have been identified in patients with ASD and SCZ. Clusters of conserved and positively charged residues can contribute to actin binding of proteins. I will perform quantitative binding assays to identify how Ca2+ levels, disease-associated variants, and clusters of conserved and positive residues impact the GluN2B tail:actin interaction. Aim 3: Determine how manipulations of the actin cytoskeleton and GluN2B tail impact GluN2B-NMDAR activity. Changes in actin polymer state impact NMDAR activity in neurons. However, whether actin selectively impacts GluN2B-NMDARs or is mediated by GluN2B tail:actin interactions is unclear. I will monitor changes in GluN2B-NMDAR mediated currents in HEK293 cells to determine the impact of 1) actin-targeting drugs and 2) disrupting the GluN2B tail:actin interaction. After mastering these techniques, I will monitor how actin dynamics and GluN2B tail:actin interactions impact NMDAR activity in cultured hippocampal neurons.
项目摘要 N-甲基-D-天冬氨酸受体(NMDAR)是由跨膜组成的谷氨酸门控离子通道 介导钙离子流入树突棘的GluN1、GluN2(A-D)和GluN3(A-B)亚基。重要的是, GluN2B亚基独特的胞内尾巴对学习和记忆是必不可少的。此外,自闭症 在GluN2B细胞内发现了与谱系障碍(ASD)和精神分裂症(SCZ)相关的变体 尾巴。我的总体目标是阐明GluN2B尾巴参与GluN2B功能的一种新机制- 含有NMDAR(GluN2B-NMDAR)。先前的实验表明,肌动蛋白靶向药物影响 NMDAR活动,但潜在机制尚不清楚。我的初步数据有力地支持了 该项目的主要假设:肌动蛋白细丝和GluN2B之间的直接相互作用 细胞内Tail调节NMDAR活性。我提议的项目的目的是阐明 GluN2B尾部与肌动蛋白细丝相互作用的生化基础及其决定 相互作用调节NMDAR功能。我将通过追求三个高度相辅相成的目标来实现这一目标: 目的1.确定介导与肌动蛋白高亲和力结合的GluN2B胞内尾巴的最小片段 细丝以及GluN2B和肌动蛋白上介导结合的界面。我的初步数据显示 GluN2B尾巴和肌动蛋白之间的相互作用,但介导这种相互作用的关键接口是 蛋白质是未知的。我将使用重组蛋白进行定量结合分析和交联 鉴定与肌动蛋白高亲和力相互作用所需的GluN2B尾巴的最小片段。 目的2.研究钙离子和基因变异如何影响GluN2B:肌动蛋白结合。Ca2+降低NMDAR 培养的海马神经元的活性,但钙离子是否选择性地影响GluN2B-NMDAR的活性 未知。我的初步数据表明,钙离子加强了GluN2B尾巴:肌动蛋白的相互作用。基因变异 位于我发现在ASD和SCZ患者中发现的结合肌动蛋白的GluN2B尾部区域。集群 保守的和带正电的残基的结合有助于肌动蛋白与蛋白质的结合。我会表演量化的 结合分析以确定钙离子水平、疾病相关变异体和簇的保守和阳性 残基影响GluN2B尾部:肌动蛋白相互作用。 目的3:确定肌动蛋白细胞骨架和GluN2B尾部的操作如何影响GluN2B-NMDAR 活动。肌动蛋白聚合体状态的改变会影响神经元中NMDAR的活性。然而,肌动蛋白是否选择性地 影响GluN2B-NMDARs或由GluN2B Tail介导:肌动蛋白相互作用尚不清楚。我将监控以下方面的变化 GluN2B-NMDAR介导HEK293细胞电流以确定1)肌动蛋白靶向药物和2) 打乱GluN2B尾巴:肌动蛋白相互作用。在掌握了这些技术之后,我将监测肌动蛋白的动力学 以及GluN2B Tail:肌动蛋白相互作用影响培养的海马神经元的NMDAR活性。

项目成果

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