Mechanism of functional modulation of glutamate receptors by their auxiliary subunits

项目摘要 AMPA型离子型谷氨酸受体（AMPAR）是由神经元激活的配体门控离子通道， 谷氨酸递质。它们介导大脑中的大部分兴奋性神经传递， 由这些复合物转导的信号对突触可塑性、学习和记忆至关重要。AMPAR辅助分泵 监管AMPAR的贩运和门控调制的单位。在本提案中，我们将研究 通过其辅助亚基进行AMPAR调节。核心AMPAR辅助亚基是TARP、GSG 1 L和 cornichons（CNIH）。广泛地研究了TARP，并且研究了缓解癫痫发作的治疗性化合物。 已经可以用于靶向富含TARP γ-8的海马体。GSG 1 L是AM的负调节剂。 PAR，而TARP和CNIH作为正调节剂。在人类中，位于中间的各种残基- AMPAR和辅助亚基之间的作用界面对错义突变不耐受，表明其 在大脑功能中的重要作用。我们假设不同的辅助亚基可以与改变的共组装， nel并产生丰富多样的门控调制，这是调节突触传递的基础 和可塑性。为了建立结构和机械基础，我们将研究复杂的AMPAR组件 具有高度的生理相关性。在目标1中，我们假设AMPAR之间的精细结构差异 组件是产生特征选通调制的基础，并提出揭示建筑学， 含有两种不同功能状态的辅助亚基的异源四聚体AMPAR的结构 在洗涤剂中使用冷冻EM。通过比较结构，新的机制模型，可以解释如何辅助- 纤毛亚单位控制门控的时间进程和幅度可能会出现，这将被我们验证。 电生理学接下来，目前可用的冷冻电镜结构揭示了脂质周围的存在， 复杂的。我们假设这些脂质在AMPAR门控调节中起重要作用， 测试目标2最后，我们认为AMPAR/辅助亚基复合物可以在近生理条件下制备， 必须研究无去污剂的条件，以建立更精确的变构门控机制模型 调变在目标3中，我们提出解决AMPAR/辅助亚基的高分辨率cryo-EM结构 包埋在脂质双层模拟环境中的复合物，以解决结构之间的已知差异， 在洗涤剂和电生理学数据中获得的结果。辅助亚基在调节离子通道门控中的作用 预测振动动力学对电路动力学具有显著影响。总之，本研究的结果 有望促进我们对AMPAR功能的机制理解，并有助于开发新的治疗方法， 本发明涉及能够减轻神经和精神疾病中AMPAR调节异常的药物化合物， 如阿尔茨海默病、中风、自闭症、拉斯穆森和边缘系统脑炎以及癫痫。

PROJECT SUMMARY The AMPA type ionotropic glutamate receptors (AMPARs) are ligand gated ion channels activated by the neu- rotransmitter glutamate. They mediate the majority of excitatory neurotransmission in the brain and the signals transduced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary sub- units regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The core AMPAR auxiliary subunits are TARPs, GSG1L, and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target hippocampus enriched TARP gamma-8. GSG1L is a negative modulator of AM- PARs, while TARPs and CNIHs serve as positive modulators. In humans, various residues located at the inter- action interface between AMPAR and auxiliary subunits are intolerant to missense mutations, indicating their critical roles in brain function. We hypothesize that different auxiliary subunits can co-assemble with the chan- nel and produce a rich variety of gating modulations, which are fundamental in regulating synaptic transmission and plasticity. To establish the structural and mechanistic basis, we will study complex AMPAR assemblies that have high physiological relevance. In Aim 1 we hypothesize that fine structural differences among AMPAR assemblies are fundamental for producing characteristic gating modulation and propose to reveal the architec- tures of heterotetrameric AMPARs containing up to two types of auxiliary subunits at different functional states in detergent using cryo-EM. By comparing the structures, new mechanistic models that could explain how aux- iliary subunits control the time course and magnitude of gating are likely to emerge, which will be validated us- ing electrophysiology. Next, currently available cryo-EM structures revealed the presence of lipids surrounding the complex. We hypothesize that these lipids play important function in AMPAR gating modulation, which will be tested in Aim2. Finally, we suggest that AMPAR/auxiliary subunit complex prepared in near physiological conditions void of detergent must be studied to build more precise mechanistic models of its allosteric gating modulation. In Aim 3, we propose to solve high resolution cryo-EM structures of AMPAR/auxiliary subunit complex embedded in a lipid bilayer mimetic environment to resolve the known discrepancies between struc- tures obtained in detergent and electrophysiology data. The role of auxiliary subunits in tuning ion channel gat- ing kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new thera- peutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer’s disease, stroke, autism, Rasmussen’s and limbic encephalitis, and seizure.