Single-particle cryo-EM characterization of AMPA receptor functional states

AMPA 受体功能状态的单颗粒冷冻电镜表征

• 批准号: 9750158
• 负责人: Alexander Sobolevsky
• 金额: $ 37.41万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750158
• 关键词: AMPA Receptors; Agonist; Alzheimer' s Disease; Binding; Biochemistry; Biological Assay; Carnitine Palmitoyltransferase I; Cell membrane; Clinical; Communication; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Detection; Drug Design; Drug Targeting; Economics; Electrophysiology (science); Epilepsy; Fluorescence; Functional disorder; Future; Genes; Glutamate Receptor; Goals; Heterogeneity; Individual; Ion Channel; Ischemia; Knowledge; Length; Ligand Binding Domain; Light; Link; Mediating; Membrane; Mental disorders; Methodology; Modernization; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Mutagenesis; Neurons; Pathologic; Pharmaceutical Preparations; Physiological; Population Heterogeneity; Primary Lateral Sclerosis; Process; Property; Proteins; Rattus; Receptor Activation; Regulation; Research; Resolution; Sampling; Signal Transduction; Site-Directed Mutagenesis; Structural Models; Structure; Synapses; Testing; Work; X-Ray Crystallography; base; cognitive function; cognitive process; desensitization; design; genetic regulatory protein; improved; nervous system disorder; neurotransmission; novel therapeutics; particle; patch clamp; positive allosteric modulator; protein expression; receptor; receptor function; receptor structure function; response; stargazin; structural biology; thermostability; tool; trafficking

PROJECT SUMMARY AMPA-subtype ionotropic glutamate receptors, which mediate fast signaling between neurons and contribute to high cognitive processes, are also implicated in numerous neurological diseases, including Alzheimer's disease, amyotropic lateral sclerosis, epilepsy and ischemia. The ability to regulate AMPA receptors is therefore an important clinical goal. There is, however, an unmet need for drugs to regulate AMPA receptor activity in pathological conditions that indicates a profound gap in our knowledge of AMPA receptor structure and function. Our long-term goal is to understand how AMPA receptor molecular machinery works at the atomic level. We plan to study AMPA receptor structure and function using advances in single-particle cryo- electron microscopy (cryo-EM). Not only can modern cryo-EM reach similar or better resolution than X-ray crystallography, it can also be used as a powerful tool to analyze heterogeneous populations of AMPA receptor particles and extract structural information that belongs to each individual functional conformation. Because of much more economic sample requirements, cryo-EM can also approach complexes of AMPA receptor with transmembrane regulatory subunits, which are hard to produce in large quantities and demonstrate high degree of conformational and stoichiometric heterogeneity and are thus intractable to X-ray crystallography. Our Specific Aims are to (1) elucidate structural basis of AMPA receptor regulation by auxiliary subunits, (2) determine molecular bases of AMPA receptor activation, and (3) establish molecular mechanism of AMPA receptor desensitization. To reach our goals, we will use cryo-EM to obtain structures of rat GluA2 in complex with different auxiliary subunits in the presence of competitive antagonists, agonists and/or positive allosteric modulators. We will use Fluorescence-detection Size Exclusion Chromatography (FSEC) and thermostability assays to assess protein expression, assembly, homogeneity and stability. We will also employ site-directed mutagenesis and electrophysiological recordings to study functional mechanisms and to critically test our structural models. Reaching our research goals will have a significant impact on understanding the mechanisms of excitatory neurotransmission and will provide molecular-level knowledge essential to facilitate design of new drugs to treat neurological diseases.

项目摘要 AMPA-SUBTYPE离子型谷氨酸受体，介导神经元之间的快速信号传导并有助于 高度认知过程也涉及许多神经系统疾病，包括阿尔茨海默氏症 疾病，乳腺脊髓性硬化症，癫痫和缺血。调节AMPA受体的能力是 因此，一个重要的临床目标。但是，有未满足的药物调节AMPA受体的需求 病理条件下的活性表明我们对AMPA受体结构的了解有很大的差距 和功能。我们的长期目标是了解AMPA受体分子机械如何在 原子水平。我们计划使用单粒子冷冻的进步来研究AMPA受体结构和功能 电子显微镜（冷冻EM）。现代冷冻EM不仅可以达到比X射线相似或更好的分辨率 晶体学，它也可以用作分析AMPA受体异质种群的强大工具 粒子和提取属于每个单个功能构象的结构信息。由于 更高的经济样本要求，Cryo-EM还可以与AMPA受体的复合物与 跨膜调节亚基很难大量生产，并且表现出很高 构象和化学计量异质性的程度，因此与X射线晶体学很有棘手。 我们的具体目的是（1）通过辅助亚基阐明AMPA受体调节的结构基础，（2） 确定AMPA受体激活的分子碱基，（3）建立AMPA的分子机制 受体脱敏。为了实现我们的目标，我们将使用Cryo-Em在复杂的情况下获得大鼠Glua2的结构 在竞争性拮抗剂，激动剂和/或阳性变构的存在下，具有不同的辅助亚基 调节器。我们将使用荧光检测尺寸排除色谱法（FSEC）和热稳定性 评估蛋白质表达，组装，同质性和稳定性的测定。我们还将采用现场定向 诱变和电生理记录研究功能机制，并批判性测试我们 结构模型。实现我们的研究目标将对理解 兴奋性神经传递的机制，将提供分子级知识，以促进 设计新药以治疗神经疾病。