Mechanism of functional modulation of glutamate receptors by their auxiliary subunits

项目总结 AMPA型嗜离子谷氨酸受体(AMPAR)是一种由神经细胞激活的配体门控离子通道。 谷氨酸轮状递质。它们调节大脑中的大部分兴奋性神经传递和信号 由这些复合体转导的信号对突触的可塑性、学习和记忆至关重要。安帕尔辅助分机 各单位对AMPAR的贩运和门控调制进行监管。在这项提案中，我们将研究这种机制 其辅助亚基对AMPAR的调节作用。核心AMPAR辅助亚单位是Tarp、GSG1L和 角鱼(CNIH)。人们对防水帆布进行了广泛的研究，并提出了缓解癫痫发作的治疗化合物 已可用于靶向强化海马区的TARP-8。GSG1L是AM的负调制子。 PAR，而TARP和CNIH则是正向调节因子。在人类体内，各种残留物位于 AMPAR和辅助亚基之间的作用界面对错义突变不耐受，表明它们 在大脑功能中的关键作用。我们假设不同的辅助亚基可以与改变共同组装- 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 有很高的生理相关性。在目标1中，我们假设AMPAR之间存在细微结构差异 组件是产生特性门控调制的基础，并建议揭示结构-- 在不同功能状态下含有多达两种辅助亚基的异四聚体AMPAR的结构 在使用冷冻EM的洗涤剂中。通过比较结构，新的机械模型可以解释AUX是如何- 辅助亚单位控制门控的时间进程和大小很可能出现，这将得到我们的验证- 电生理学。接下来，目前可用的冷冻-EM结构揭示了周围存在脂类 这个建筑群。我们假设这些脂质在AMPAR门控调制中发挥重要作用，这将 在AIM2中进行测试。最后，我们建议在近生理条件下制备AMPAR/辅助亚基复合体 必须研究不含洗涤剂的条件，以建立更精确的变构门控机理模型 调制。在目标3中，我们提出了解决AMPAR/辅助亚基的高分辨率低温电磁结构 嵌入在脂质双层模拟环境中的复合体，以解决已知的结构- 在洗涤剂和电生理学数据中获得的缝隙。辅助亚基在调节离子通道门控中的作用。 ING动力学被预测将对电路动力学产生重大影响。总而言之，这项研究的结果 以期促进我们对AMPAR功能的机理理解，并帮助开发新的Thera-TERA。 可以缓解神经和精神疾病中出现的AMPAR失调的传统化合物， 例如阿尔茨海默氏症、中风、自闭症、拉斯穆森和边缘脑炎以及癫痫。

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.