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，并促进高水平的谷氨酸受体表达。 认知过程由于AMPA受体也涉及许多神经系统疾病，包括 阿尔茨海默氏病、肌萎缩侧索硬化症、癫痫和缺血，调节它们的能力 这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。 在病理条件下的活性，这突出了我们对AMPA受体知识的深刻差距 结构和功能。我们的长期目标是了解AMPA受体分子机制的运作方式 在原子水平上。我们计划利用单粒子低温电子显微镜研究AMPA受体的结构和功能 显微镜（cryo-EM）。以“分辨率革命”为标志的冷冻电镜技术的进步为我们带来了一个 在不同的门控构象和复杂的几个小的AMPA受体结构的数量 分子和调节蛋白。然而，只有一小部分AMPA受体的构象状态， 和结合伴侣的结构特征和决议的报告结构仍然 相对较低，限制了我们看到原子细节的能力。因此，我们计划利用冷冻EM技术的进步， 填补这一知识空白，并侧重于以下具体目标：（1）阐明AMPA的结构原则 通过辅助亚基调节AMPA受体，（2）揭示AMPA受体活化的分子决定因素， 电导，和（3）确定AMPA受体脱敏的分子机制。向着我们既定的 研究目标，我们将使用冷冻电镜获得AMPA受体单独或与不同的复合物的结构， 辅助蛋白，在激动剂、竞争性拮抗剂、正或负变构剂、 调节剂，并在有利于各种构象状态的条件下。我们将使用重复检测大小 排阻色谱法（FSEC）和热稳定性测定，以评估蛋白质表达、组装， 均匀性和稳定性。我们还将采用定点诱变结合单通道， 全细胞膜片钳电生理记录研究功能机制，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 mediate fast signaling between neurons and contribute to high cognitive processes. Since AMPA receptors are also implicated in numerous neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, epilepsy, and ischemia, the ability to regulate them represents an important clinical goal. However, there is an unmet need for drugs to regulate AMPA receptor activity in pathological conditions, which highlights a profound gap in our knowledge of AMPA receptor structure and function. Our long-term goal is to understand how AMPA receptor molecular machinery operates at the atomic level. We plan to study AMPA receptor structure and function using single-particle cryo-electron microscopy (cryo-EM). Advances in cryo-EM that were signified by the “resolution revolution” brought us a number of AMPA receptor structures in different gating conformations and in complex with several small molecules and regulatory proteins. Nevertheless, only a little fraction of AMPA receptor conformational states and binding partners have been characterized structurally and the resolution of the reported structures remains relatively low, limiting our ability to see the atomic details. We, therefore, plan to use cryo-EM advances to fill up this knowledge gap and to focus on the following Specific Aims: (1) unravel structural principles of AMPA receptor regulation by auxiliary subunits, (2) reveal molecular determinants of AMPA receptor activation and conductance, and (3) determine molecular mechanisms of AMPA receptor desensitization. To reach our research goals, we will use cryo-EM to obtain structures of AMPA receptors alone or in complex with different auxiliary proteins, in the presence of agonists, competitive antagonists, positive or negative allosteric modulators, and in conditions favoring various conformational states. 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 combined with single-channel and whole-cell patch-clamp 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 the design of new drugs for the treatment of neurological disorders.