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：
Melissa Carrizales
金额：
$ 3.32万
依托单位：
YALE UNIVERSITY
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

来源：
https://reporter.nih.gov/project-details/10606121
关键词：
Actin-Binding Protein Actins Affect Affinity Amino Acids Binding Biochemical Biological Assay Brain Diseases Cells Charge Complex Cytoplasmic Tail Cytoskeletal Proteins Cytoskeleton Data Dendritic Spines Development Disease Drug Targeting Epilepsy Excitatory Synapse Genes Glutamate Receptor Goals Hippocampus Intellectual functioning disability Ion Channel Gating Learning Maps Mass Spectrum Analysis Measures Mediating Membrane Memory Microfilaments Monitor N-Methyl-D-Aspartate Receptors NMDA receptor A1 Neurodevelopmental Disorder Neurons Patients Peptides Polymers Proteins Recombinant Proteins Recombinants Regulation Role Schizophrenia Sedimentation process Shapes Synapses Synaptic Receptors Tail Techniques Testing Therapeutic Therapeutic Intervention Variant Vertebral column Whole-Cell Recordings autism spectrum disorder crosslink density experimental study genetic variant mutant novel polymerization postsynaptic receptor function response schizophrenia risk small molecule stoichiometry synaptic function targeted treatment therapeutic development trafficking

Actin-Binding Protein Actins Affect Affinity Amino Acids Binding Biochemical Biological Assay Brain Diseases Cells Charge Complex Cytoplasmic Tail Cytoskeletal Proteins Cytoskeleton Data Dendritic Spines Development Disease Drug Targeting Epilepsy Excitatory Synapse Genes Glutamate Receptor Goals Hippocampus Intellectual functioning disability Ion Channel Gating Learning Maps Mass Spectrum Analysis Measures Mediating Membrane Memory Microfilaments Monitor N-Methyl-D-Aspartate Receptors NMDA receptor A1 Neurodevelopmental Disorder Neurons Patients Peptides Polymers Proteins Recombinant Proteins Recombinants Regulation Role Schizophrenia Sedimentation process Shapes Synapses Synaptic Receptors Tail Techniques Testing Therapeutic Therapeutic Intervention Variant Vertebral column Whole-Cell Recordings autism spectrum disorder crosslink density experimental study genetic variant mutant novel polymerization postsynaptic receptor function response schizophrenia risk small molecule stoichiometry synaptic function targeted treatment therapeutic development trafficking

展开

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（NMDARS）是由跨膜组成的谷氨酸门控通道 glun1，glun2（a-d）和glun3（a-b）亚基，将Ca2+涌入到树突状脊柱中。重要的是， Glun2b亚基的独特细胞内尾部对于学习和记忆至关重要。此外，自闭症频谱障碍（ASD）和精神分裂症（SCZ）相关的变体在Glun2b内细胞内发现尾巴。我的总体目标是阐明一种新型机制，通过该机制，Glun2b尾巴有助于Glun2b-的功能包含NMDAR（glun2b-nmdars）。先前的实验表明，靶向肌动蛋白的药物会影响 NMDAR活动，但潜在的机制尚不清楚。我的初步数据强烈支持该项目的主要假设：肌动蛋白丝与Glun2b之间的直接相互作用细胞内尾部调节NMDAR活性。我提议的项目的目的是阐明 Glun2b尾巴与肌动蛋白丝之间相互作用的生化基础，并确定如何相互作用调节NMDAR函数。我将通过追求三个高度互补的目标来实现这一目标：目标1。定义介导高亲和力结合到肌动蛋白的细胞内尾巴的最小片段细丝和glun2b和肌动蛋白上介导结合的界面。我的初步数据显示了一个直接的 Glun2b尾巴与肌动蛋白之间的相互作用，但是介导这种相互作用的关键接口蛋白质未知。我将使用重组蛋白进行定量结合测定和交联确定介导高亲和力与肌动蛋白相互作用所需的Glun2b尾巴的最小片段的测定。 AIM 2。表征Ca2+和遗传变体如何影响Glun2b：肌动蛋白结合。 Ca2+减少NMDAR 培养的海马神经元的活性，但是CA2+有选择地影响Glun2B-NMDAR活性是未知。我的初步数据表明Ca2+增强了Glun2b尾巴：肌动蛋白相互作用。遗传变异位于我发现的Glun2b尾部区域内，在ASD和SCZ患者中已经确定了结合肌动蛋白。集群保守和带正电的残基可以有助于蛋白质的肌动蛋白结合。我将执行定量结合测定，以确定Ca2+水平，疾病相关的变体以及保守和正的簇如何残基影响Glun2b尾巴：肌动蛋白相互作用。 AIM 3：确定肌动蛋白细胞骨架和Glun2b尾巴的操纵如何影响GLUN2B-NMDAR 活动。肌动蛋白聚合物状态影响神经元中NMDAR活性。但是，是否有选择地肌动蛋白影响glun2b-nMDAR或由glun2b尾巴介导：肌动蛋白相互作用尚不清楚。我将监视更改 HEK293细胞中Glun2b-NMDAR介导的电流确定1）靶向肌动蛋白的药物的影响和2）破坏glun2b尾巴：肌动蛋白相互作用。掌握了这些技术后，我将监视肌动蛋白的动态和Glun2b尾巴：肌动蛋白相互作用会影响培养的海马神经元的NMDAR活性。