Structure-function studies on the mechanism of AMPA receptor desensitization

描述（由申请人提供）：离子型谷氨酸受体包括一个大家族的配体门控离子通道，其负责哺乳动物中枢神经系统中的大多数兴奋性神经传递。AMPA受体亚家族介导毫秒级的快速兴奋性突触信号传导。在谷氨酸持续存在的情况下，AMPA受体迅速而深刻地脱敏，从而在某些突触处形成突触后反应。关于离子通道孔在脱敏期间如何关闭而谷氨酸仍然与受体结合的机制细节缺失，并且结论性答案需要关于全长受体的高分辨率结构信息， 脱敏状态。本申请概述了使用已证实的策略，以确定一个原子结构的全长AMPA受体的复杂性与高亲和力激动剂的X射线晶体学。该项目的初步结果包括AMPA受体与多种不同激动剂的复合物的成功结晶和来自几种晶体形式的衍射数据。这些晶体的衍射极限需要通过广泛优化结构和结晶条件来改善。将在电生理学实验中监测各种结构修饰对门控和脱敏的影响。结晶试验将不限于使用去污剂/蛋白质胶束的经典方法，还包括允许在富含脂质的环境中结晶的新技术，例如双胶束和无规立方相结晶。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。在脱敏状态的AMPA受体的新的X-射线结构模型的已知的封闭的，非脱敏状态的结构的比较将揭示潜在的受体脱敏推定的构象变化。这些预测的重排将通过功能实验进行测试，以确认从结构发现中得出的机理概念。该应用的预期见解不仅将提高我们在分子水平上对AMPA受体脱敏的理解，而且还为设计新型治疗剂铺平了道路，这些治疗剂可增强或减少脱敏，用于癫痫发作，神经和精神疾病的潜在治疗。

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.