Conformational Changes In Glutamate Receptor

说明(申请人提供)：嗜冷谷氨酸受体是哺乳动物中枢神经系统中的主要兴奋性神经递质受体，与体内许多广泛的影响有关。谷氨酸与胞外结构域的结合启动了一系列构象变化，最终形成阳离子选择性跨膜通道，然后由于受体的脱敏而关闭。分离的AMPA亚型(LBD)配体结合域的晶体结构使人们能够了解LBD的结构变化；然而，这些结构缺乏跨膜片段，而跨膜片段是蛋白质的主要功能部分。为了更全面地了解激动剂结合是如何耦合到通道激活和脱敏的，有必要研究跨膜片段存在时LBD的变化。这可以通过使用荧光共振能量转移(FRET)来实现。为了测量构象变化，可以在特定的位置标记修饰的AMPA受体。利用这种修饰的受体，我们建议通过建立标记有供体和受体荧光团的蛋白质上特定位点之间的距离变化来研究构象变化。第一个特定的目的是确定配体结合区的裂隙闭合是否是通过各种配体和L650和Y450位点的突变来控制受体激活的主要控制因素，L650和Y450位点诱导了广泛的激活。第二个具体目的是通过检验脱敏的二聚体界面假说来确定与脱敏相关的构象变化，该假说被认为是由于谷氨酸受体二聚体中两个亚基之间的界面分离而导致的，以缓解裂隙闭合引起的跨膜片段上的压力。通过使用这些技术、配体和突变体，将在完整的受体中研究与通道激活和脱敏耦合的激动剂结合。在健康问题上，谷氨酸受体是通过离子受体进行快速兴奋性突触传递的主要媒介，也参与更复杂的神经元过程，如学习和记忆。谷氨酸介导的兴奋性毒性与各种神经损伤和疾病有关，包括缺血、缺氧、中风、低血糖、癫痫、亨廷顿病、肌萎缩侧索硬化症、甲状腺机能亢进和阿尔茨海默病。更好地了解结构-功能相关性并确定受体功能的分子基础是设计和开发下一代药物的关键一步，这些药物可以改变这一重要受体的功能，从而有助于上述临床疾病的治疗。

DESCRIPTION (provided by applicant): lonotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system and are implicated in a number of wide-ranging effects in the body. Glutamate binding to an extracellular domain initiates a series of conformation changes that ends in the formation of a cation selective transmembrane channel, which then closes due to desensitization of the receptor. The crystal structures of the isolated ligand binding domain of the AMPA subtype (LBD) gives knowledge into the structural changes in the LBD; however, these structures lack transmembrane segments, the primary functional part of the protein. To gain a more complete understanding of how agonist binding is coupled to channel activation and desensitization, it is necessary to study changes in the LBD in the presence of the transmembrane segments. This can be achieved by using fluorescence resonance energy transfer (FRET). To measure conformational changes modified AMPA receptor can be tagged at specific sites. Using this modified receptor, we propose to study the conformational changes by establishing distance changes between specific sites on the protein tagged with donor and acceptor fluorophores. The first specific aim is to establish whether cleft closure in the ligand binding domain is the primary control of receptor activation using various ligands and mutations at the L650 and Y450 sites, which induces a wide range of activations. The second specific aim is to determine the conformational changes associated with desensitization by testing the dimer interface hypothesis for desensitization, which is thought to be caused by the separation of the interface between two subunits in the glutamate receptor dimer to relieve stress on the transmembrane segments induced by cleft closure. Through the use of these techniques, ligands, and mutants, agonist binding coupled to channel activation and desensitization will be investigated in the full receptor. Regarding health issues, glutamate receptors are the primary mediators of fast excitatory synaptic transmission via ionotropic receptors and are also involved in more intricate neuronal processes, such as learning and memory. Glutamate-mediated excitotoxicity implicated in neuronal damage associated with various neurological insults and diseases, including ischemia, anoxia, stroke, hypoglycemia, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, lathyrisms, and Alzheimer's disease. Gaining a better understanding of structure-function correlations and determining the molecular basis of the receptor function is a key step towards designing and developing the next generation of drugs that can alter the function of this important receptor and hence aid in the treatment of the clinical conditions listed above.