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

项目摘要 AMPA亚型离子型谷氨酸受体，介导神经元之间的快速信号传导， 高认知过程，也与许多神经系统疾病有关，包括阿尔茨海默氏症 疾病、肌萎缩侧索硬化、癫痫和缺血。调节AMPA受体的能力是 因此是一个重要的临床目标。然而，对于调节AMPA受体的药物存在未满足的需求 这表明我们对AMPA受体结构的认识存在深刻的差距 和功能我们的长期目标是了解AMPA受体分子机制如何在细胞中起作用。 原子水平。我们计划利用单粒子冷冻技术的进展来研究AMPA受体的结构和功能， 电子显微镜（cryo-EM）。现代冷冻电镜不仅可以达到与X射线相似或更好的分辨率， 晶体学，它也可以作为一个强大的工具来分析AMPA受体的异质群体 粒子和提取属于每个单独的功能构象的结构信息。因为 更经济的样品要求，cryo-EM也可以接近AMPA受体与 跨膜调节亚基，其难以大量产生，并且表现出高的 构象和化学计量异质性的程度，因此难以X射线晶体学。 我们的具体目标是：（1）阐明AMPA受体辅助亚基调控的结构基础;（2） 确定AMPA受体激活的分子基础;（3）建立AMPA的分子机制 受体脱敏为了达到我们的目的，我们将使用冷冻电镜获得大鼠GluA 2的结构， 在竞争性拮抗剂、激动剂和/或正变构剂存在下， 调制器。我们将使用双折射检测尺寸排阻色谱法（FSEC）和热稳定性 用于评估蛋白质表达、组装、均一性和稳定性的测定。我们还将聘请现场指导 诱变和电生理记录，以研究功能机制，并严格测试我们的 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，并将提供必要的分子水平的知识，以促进 设计治疗神经系统疾病的新药。

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.