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

异聚甘氨酸受体结构和变构机制的冷冻电镜研究

基本信息

批准号：
10388533
负责人：
Eric D. Gibbs
金额：
$ 6.98万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10388533
关键词：
Address Affect Agonist Alcohol abuse Alcohols Anesthetics Antiparasitic Agents Binding Binding Proteins Binding Sites Brain Brain Stem Cannabinoids Complex Coupling Cryoelectron Microscopy Data Disease Electron Microscopy Electron Spin Resonance Spectroscopy Electrophysiology (science)Environment Glycine Glycine Receptor Binding Glycine Receptors Goals Health Homo Ion Channel Gating Ions Ivermectin Kinetics Knowledge Ligand Binding Ligands Lipid Binding Maps Mediating Mediator of activation protein Membrane Proteins Microscope Molecular Conformation Movement Muscle Mutation Neural Inhibition Neurotransmitter Receptor Neurotransmitters Outcome Study Pain Pathology Perception Physiologic pulse Physiological Physiological Processes Preparation Process Protein Biochemistry Proteins Protocols documentation Receptor Signaling Regulation Resolution Resources Review Literature Rewards Role Sampling Sequence Analysis Site Spinal Cord Stimulus Structure Synapses Techniques Tetrahydrocannabinol Therapeutic Tinnitus Titan Training Transmembrane Domain Universities Visual Visual Perception antagonist chronic pain cryogenics design experience experimental study extracellular gephyrin insight member particle receptor binding receptor function spasticity therapeutic target

项目摘要

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.

项目总结和摘要：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于许多生理学至关重要。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 是一种五聚体配体门控离子通道，以同聚和异聚形式表达。异聚 GlyR 对于突触活动至关重要，因为它单独可以结合突触锚定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）来实现机械理解异聚 GlyR 与 gephryin 结合这将建立在过去同聚体的冷冻电镜研究的基础上。该提案的具体目标是：首先，表征亚基特异性结构。已知异源 GlyR 可以调节亚基组成、离子传导和细胞内调节。二、研究神经递质结合口袋与通道之间的构象耦合第三，探索在跨膜域内结合的配体的变构效应。申请人在表达和纯化异质GlyR方面取得了重大进展。异聚 GlyR 并进行初步冷冻电镜研究，得到 2.25-2.5 Å 冷冻电镜在完成该项目时，申请人将接受膜蛋白方面的培训。生物化学、冷冻电镜、电生理学和 EPR 该项目将在 Sudha 进行。查克拉帕尼位于凯斯西储大学的实验室最近取得了重大进展。对同聚 GlyR 和其他配体门控离子的冷冻电镜研究对该领域做出了贡献可用资源包括配备 Titan Krios 显微镜和实验室的冷冻电镜设施。实验室的几位成员在冷冻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。