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

甘氨酸受体（GlyR）是突触的主要介质， 脑干和脊髓的神经抑制。它们对许多生理功能至关重要， 过程和有趣但尚未开发的治疗靶点。该项目的总体目标是 了解控制GlyR活性的过程，并为治疗设计提供模板。 GlyR是以同聚体和异聚体形式表达的五聚体配体门控离子通道。 异聚体GlyR对于突触活动是必不可少的，因为它可以单独结合突触锚定。 gephryin蛋白。该提案使用低温电子显微镜（cryo-EM）、电生理学 和电子顺磁共振（EPR），以实现机械的理解 异聚GlyR与杰弗灵结合。这将建立在过去的同聚体的冷冻电镜研究的基础上。 GlyR.本提案的具体目标是：第一，确定 已知hetero-GlyR调节亚基组成、离子传导和细胞内调节。 第二，研究神经递质结合口袋与通道之间的构象耦合 门第三，探索结合在细胞膜跨膜结构域内的配体的变构效应。 hetero-GlyR.申请人通过表达和纯化， 异聚体GlyR，并进行初步的冷冻-EM研究，得到2.25-2.5 μ l的冷冻-EM 地图在完成这个项目时，申请人将接受膜蛋白的培训 生物化学、冷冻电镜、电生理学和电子顺磁共振。该项目将在Sudha进行 凯斯西储大学的查克拉帕尼实验室。这个实验室最近在 对同聚体GlyR和其他配体门控离子的冷冻EM研究领域的贡献 渠道可用的资源包括一个冷冻EM设施与泰坦Krios显微镜和实验室- 专用的电生理设备实验室的几位成员在低温方面有着丰富的经验， 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.