Structure-function studies on the mechanism of AMPA receptor desensitization

说明(申请人提供)：离子型谷氨酸受体包括一大类配体门控离子通道，负责哺乳动物中枢神经系统的大部分兴奋性神经传递。AMPA受体亚家族在毫秒的时间尺度上介导快速兴奋性突触信号。在谷氨酸持续存在的情况下，AMPA受体迅速而深刻地脱敏，从而形成某些突触的突触后反应。在谷氨酸与受体结合的情况下，离子通道孔在脱敏过程中如何关闭的机制细节尚不清楚，而确凿的答案需要关于全长受体的高分辨率结构信息 不敏感的状态。本申请概述了通过X射线结晶学确定全长AMPA受体与高亲和力激动剂复合体中全长AMPA受体原子结构的成熟策略的使用。该项目的初步结果包括AMPA受体在各种不同激动剂的络合物中的成功结晶和几种晶型的衍射数据。这些晶体的衍射极限需要通过结构和结晶条件的广泛优化来提高。在电生理实验中，将监测各种结构修改对门控和脱敏的影响。结晶试验将不仅限于使用洗涤剂/Protei胶束的传统方法，还包括允许在富含脂质的环境中结晶的新技术，如双胶束和脂类立方相结晶。此外，将测试与来自构象特异性抗体的Fab片段的共结晶，以减少晶体中的构象异质性。将新的AMPA受体脱敏状态的X射线结构模型与已知的闭合非脱敏状态结构进行比较，将揭示受体脱敏潜在的构象变化。这些预测的重排将通过功能实验进行测试，以确认从结构发现得出的机械概念。这一应用不仅将在分子水平上加深我们对AMPA受体脱敏的理解，还将为设计新的治疗药物铺平道路，这些药物可以增强或减少脱敏作用，用于癫痫、神经和精神疾病的潜在治疗。 与公共健康相关：AMPA型谷氨酸受体是配体门控离子通道，在大脑中介导快速兴奋性突触后电流。增强AMPA受体脱敏的药物，在谷氨酸持续存在时导致通道关闭的一个鲜为人知的过程，有助于有效治疗癫痫发作、癫痫和兴奋性毒性相关疾病，如缺血性中风。这一应用的结构洞察将为以AMPA受体脱敏为靶点的治疗化合物的新设计提供分子蓝图。

DESCRIPTION (provided by applicant): Ionotropic glutamate receptors comprise a large family of ligand-gated ion channels which are responsible for the majority of excitatory neurotransmission in the mammalian central nervous system. The subfamily of AMPA receptors mediates fast excitatory synaptic signaling on a millisecond timescale. In the continuous presence of glutamate, AMPA receptors desensitize rapidly and profoundly, thereby shaping the postsynaptic response at certain synapses. Mechanistic details on how the ion channel pore closes during desensitization with glutamate remaining bound to the receptor are missing and conclusive answers require high resolution structural information about the full-length receptor in the desensitized state. This application outlines the use of proven strategies to determine an atomic structure of a full-length AMPA receptor in complex with a high affinity agonist by X-ray crystallography. Preliminary results from this project include the successful crystallization of AMPA receptors in complex with a variety of different agonists and diffraction data from several crystal forms. The diffraction limit of these crystals needs to be improved by extensive optimization of the construct and crystallization conditions. The effects of various construct modifications on gating and desensitization will be monitored in electrophysiological experiments. Crystallization trials will not be limited to classical methods using detergent/protei micelles but also include new techniques which allow crystallization in a lipid-rich environment, such as bicelle and lipidic cubic phase crystallization. Furthermore, co-crystallization with FAB fragments from conformation-specific antibodies will be tested to reduce conformational heterogeneity in the crystal. Comparison of the new X-ray structural model of the AMPA receptor in the desensitized state to the known closed, non-desensitized state structure will reveal putative conformational changes underlying receptor desensitization. These predicted rearrangements will be tested by functional experiments to confirm the mechanistic concepts concluded from the structural findings. The expected insights from this application will not only improve our understanding of AMPA receptor desensitization on a molecular level, but also pave the way for designing novel therapeutic agents which enhance or reduce desensitization for the potential treatment of seizures, neurological and psychiatric disorders. PUBLIC HEALTH RELEVANCE: AMPA-type glutamate receptors are ligand-gated ion channels mediating fast excitatory postsynaptic currents in the brain. Drugs enhancing AMPA receptor desensitization, a poorly understood process resulting in channel closure in sustained presence of glutamate, are useful for the effective treatment of seizures, epilepsy and excitotoxicity-related diseases, like ischemic stroke. The structural insights from this applicatio will provide a molecular blueprint for novel designs of therapeutic compounds targeting AMPA receptor desensitization.