Structural elucidation of ionotropic glutamate receptor states with cryoelectron microscopy

 描述（由申请人提供）：本项目的目标是使用低温电子显微镜（cryo-EM）技术的新进展，以高分辨率阐明离子型谷氨酸受体（iGluR）离子通道的一系列功能状态的结构，并使用互补的功能和生化测定来验证我们的发现和假设，这些发现和假设由我们解决的cryo-EM模型驱动。iGluR负责大多数兴奋性神经传递，并且涉及广泛的病理学，从神经退行性疾病和精神障碍到癫痫和中风诱导的创伤。目前靶向指示这些不同疾病状态的iGluR功能状态范围的治疗剂的设计受到缺乏描述闭合通道状态之外的iGluR的信息的阻碍。这项研究将通过使用基于cryo-EM的方法直接解决对这些功能状态的新观点的需求，这将允许克服分析iGluR状态的晶体学障碍。使用最先进的冷冻-EM技术，将建立iGluR的高分辨率冷冻-EM。利用基于荧光的构建体筛选策略，将鉴定用于分离iGluR功能中的单独结构状态的新靶标。即，辅助蛋白如何与iGluR相互作用/调节iGluR和iGluR门控将被靶向。冷冻EM分析后，将使用先进的电生理技术和生化询问来探测数据。该项目中使用的方法将作为研究跨离子通道家族的功能状态的基础，并可能为靶向iGluR状态的新分子的基于结构的药物设计提供急需的模板。

 DESCRIPTION (provided by applicant): The goal of this project is to use new advances in cryo-electron microscopy (cryo-EM) technology to elucidate the structures of, at high-resolution, a range of functional states of ionotropic glutamate receptor (iGluR) ion channels, and to use complementary functional and biochemical assays to validate our findings and hypotheses driven by our solved cryo-EM models. iGluRs are responsible for the majority of excitatory neurotransmission, and are implicated in a wide range of pathologies, from neurodegenerative diseases and psychiatric disorders, to epilepsy and stroke-induced trauma. Current design of therapeutics targeting the range of iGluR functional states that dictate these different disease states is hindered by a lack of information describing iGluRs outside of a closed-channel state. This research will directly address the need for new views into these functional states by using cryo-EM based methods, which will allow crystallographic barriers in analyzing iGluR states to be overcome. Using state-of-the-art cryo-EM techniques, high-resolution cryo-EM of iGluRs will be established. With fluorescence-based construct screening strategies, new targets for isolating separate structural states in iGluR function will be identified. Namely, how auxiliary proteins interact with/regulate iGluRs and iGluR gating will be targeted. After cryo-EM analysis, data will be probed using advanced electrophysiological techniques and biochemical interrogation. The methods used in this project will serve as a foundation for investigating functional states across ion channel families, and may provide a much-needed template for structure-based drug design of new molecules targeting iGluR states.