Single-Particle Cryo-EM Characterization of AMPA Receptor Functional States

项目总结 AMPA亚型离子型谷氨酸受体介导神经元之间的快速信号传递并参与高密度脂蛋白 认知过程。由于AMPA受体也与许多神经疾病有关，包括 阿尔茨海默病、肌萎缩侧索硬化症、癫痫和缺血，调节它们的能力 代表了一个重要的临床目标。然而，对调节AMPA受体的药物的需求尚未得到满足 病理条件下的活性，这突显了我们对AMPA受体认识的深刻差距 结构和功能。我们的长期目标是了解ampa受体分子机制是如何运作的。 在原子水平上。我们计划利用单粒子冷冻电子来研究AMPA受体的结构和功能 显微镜(低温电子显微镜)。以“分辨率革命”为标志的低温电磁技术的进步为我们带来了 不同门控构象的AMPA受体结构的数量以及具有几个小分子的复合体 分子和调节蛋白。然而，只有一小部分AMPA受体构象状态 并对结合伙伴进行了结构表征，所报道的结构的分辨率仍然存在 相对较低，限制了我们查看原子细节的能力。因此，我们计划使用低温电磁先进技术来填补 弥合这一认识差距，并专注于以下具体目标：(1)解开AMPA的结构原则 辅助亚基对受体的调节，(2)揭示AMPA受体激活的分子决定因素和 (3)确定AMPA受体脱敏的分子机制。要到达我们的 研究目的：我们将利用冷冻-EM技术获得AMPA受体的单独结构或与不同受体的复合体结构 辅助蛋白，在激动剂、竞争性拮抗剂存在的情况下，正负变构 调制器，以及在有利于各种构象状态的条件下。我们将使用荧光检测大小 排除层析(FSEC)和热稳定性分析评估蛋白质的表达、组装、 同质性和稳定性。我们还将采用定点突变结合单通道和 全细胞膜片钳电生理记录以研究其作用机制并对OUR 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，并将提供分子水平的必要知识，以促进 治疗神经疾病的新药的设计。

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.