Structural elucidation of ionotropic glutamate receptor states with cryoelectron microscopy

 描述(申请人提供)：该项目的目标是利用低温电子显微镜(Cryo-EM)技术的新进展，以高分辨率阐明一系列离子亲离子谷氨酸受体(IGluR)离子通道的结构，并使用互补的功能和生化分析来验证我们的发现和由我们解决的低温电子显微镜模型驱动的假设。IGluRs负责大多数兴奋性神经传递，并与从神经退行性疾病和精神障碍到癫痫和中风引起的创伤等广泛的病理过程有关。目前针对指示这些不同疾病状态的iGluR功能状态范围的治疗设计，由于缺乏描述封闭通道状态之外的iGluR的信息而受到阻碍。这项研究将通过使用基于冷冻-EM的方法直接解决对这些功能状态的新观点的需求，这将使分析iGluR状态的晶体障碍被克服。利用最先进的低温EM技术，将建立高分辨率的iGluRs低温EM。通过基于荧光的构建筛选策略，将识别分离iGluR功能中独立结构状态的新靶点。也就是说，辅助蛋白如何与iGluR和iGluR门控相互作用/调节将成为靶点。在冷冻-EM分析之后，将使用先进的电生理技术和生化询问来探测数据。本项目中使用的方法将作为研究离子通道家族功能状态的基础，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.