Cryo-EM Studies of the Structure and Allosteric Mechanisms of Heteromeric Glycine Receptor

项目摘要与摘要：甘氨酸受体是突触的主要介体 脑干和脊髓的神经抑制。它们对于许多生理学来说是必不可少的 过程和耐人寻味但尚未开发的治疗靶点。这个项目的总体目标是 了解控制GlyR活性的过程，并为治疗设计提供模板。 GlyR是一种五聚体配基门控离子通道，以同聚体和异构体形式表达。 异构体GlyR对突触活动是必不可少的，因为它本身就可以结合突触锚定 蛋白质基因。这项提议使用低温电子显微镜(Cryo-EM)、电生理学 和电子顺磁共振(EPR)来实现对 异构体GlyR与基因表达谱结合。这将建立在过去对同性恋的冷冻-EM研究的基础上 GlyR.这项建议的具体目标是：第一，表征亚基特有的结构 已知的异型GlyR可以调节亚基组成、离子传导和细胞内调节。 第二，研究神经递质结合袋与通道之间的构象偶联 登机口。第三，探索结合在跨膜结构域中的配体的变构效应。 异型甘氨酸。申请者通过表达和纯化显示了显著的进步 异构体GlyR，并进行了初步的低温EM研究，得到了2.25-2.5ä低温EM 地图。在完成这个项目时，申请者将接受膜蛋白方面的培训。 生物化学、低温EM、电生理学和电子顺磁共振。该项目将在苏达进行 查克拉帕尼在凯斯西储大学的实验室。这个实验室最近取得了重大的进展 均聚体GlyR和其他配体门控离子的低温电子显微镜研究对该领域的贡献 频道。可用的资源包括一个带有泰坦-克里奥斯显微镜的冷冻-EM设施和实验室- 专用的电生理学仪器。该实验室的几名成员在低温方面拥有丰富的经验- EM、电生理学和/或EPR提供了良好的培训环境。

Project Summary and Abstract: Glycine receptors (GlyR) are the primary mediators of synaptic neural inhibition in the brainstem and spinal cord. They are essential to many physiological processes and intriguing, yet untapped, therapeutic targets. The overall goal of this project is to understand the processes that control GlyR activity and provide a template for therapeutic design. GlyR is a pentameric ligand-gated ion channel expressed in homomeric and heteromeric forms. Heteromeric GlyR is essential for synaptic activity as it alone can bind the synaptic anchoring protein gephryin. This proposal uses cryogenic electron microscopy (cryo-EM), electrophysiology and electron paramagnetic resonance (EPR) to achieve mechanistic understanding of heteromeric GlyR bound to gephryin. This will build upon past cryo-EM studies of homomeric GlyR. The specific aims of this proposal are: First, to characterize subunit-specific structures in hetero-GlyR known to regulate subunit composition, ion conduction and intracellular regulation. Second, study conformational coupling between the neurotransmitter binding pocket and channel gate. Third, explore the allosteric effects of ligands that bind within the transmembrane domain of hetero-GlyR. The applicant has shown significant progress by expressing and purifying heteromeric GlyR and performing preliminary cryo-EM studies resulting in a 2.25-2.5 Å cryo-EM map. In accomplishing this project, the applicant will receive training in membrane protein biochemistry, cryo-EM, electrophysiology and EPR. This project will take place in Sudha Chakrapani’s lab at Case Western Reserve University. This lab has made significant recent contributions to the field with cryo-EM studies of homomeric GlyR and other ligand-gated ion channels. Available resources include a cryo-EM facility with a Titan Krios microscope and lab- dedicated electrophysiology rigs. Several members of the lab have extensive experience in cryo- EM, electrophysiology and/or EPR providing an excellent training environment.