Structure, function and dynamics of a glutamate receptor

描述（由申请人提供）：嗜凉性谷氨酸受体控制多种正常的神经元过程，包括学习和记忆。此外，这些重要的神经递质受体的激活与许多神经退行性疾病有关，特别是中风和癫痫。因此，针对谷氨酸受体的药物将具有相当大的治疗价值。对跨膜拓扑结构的分析表明，每个亚基都是由一系列模块组成的，结合谷氨酸的模块可以在细菌中作为可溶性蛋白（S1S2结构域）产生。S1S2结构域结合激动剂和拮抗剂的亲和力与完整受体大致相同，是研究结合结构域的一个很好的系统。E. Gouaux和合作者已经解决了GluR2亚基的S1S2结构域的晶体结构。我们使用核磁共振光谱来表征GluR2 S1S2结构域的动态性质。不幸的是，由于其快速动力学和低通道电导，GluR2受体的功能难以研究。本提案的目的是将结构和动力学研究扩展到谷氨酸受体亚基（GluRS），该亚基形成更适合详细动力学测量的通道。核磁共振和荧光研究将表征一系列完整和部分激动剂存在下的结构和动力学。从晶体结构和同源性模型可以看出，S1S2结构域是一种配体结合后关闭的双分子体结构。我们的假设是通道电导是由单个亚基的叶瓣闭合调节的，并且大范围的构象动力学或片内动力学都有助于电导水平的变化。开放通道爆发中的打开和关闭与S1S2结构域的内部动力学在相同的时间尺度上。单通道电导和速率常数将被确定在同一系列激动剂中的同源GluRS受体通道，以确定激动剂和受体构象之间的原子水平相互作用的明显差异是否会不同地影响通道动力学。将内部动力学与通道的单通道特性进行比较，可以确定这些动力学是否在离子通道功能的控制中起作用。最后一个目标将包括核磁共振和突变S1S2结构域的单通道记录，以确定叶间界面在激动剂亲和力和功效控制中的作用。虽然同质GLURS受体通道可能局限于少数神经元，但GLUR2/GLUR3异质受体通道可能代表了新皮层中大量的突触后AMPA受体。该结果将揭示谷氨酸受体重要亚基的结合位点，并为进一步的药物开发提供重要信息。

DESCRIPTION (provided by applicant): lonotropic glutamate receptors control a variety of normal neuronal processes including learning and memory. In addition, activation of these important neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. Thus, drugs targeted toward glutamate receptors would be of considerable therapeutic value. Analysis of the transmembrane topology led to the realization that each subunit is made of a series of modules, and the module that binds glutamate can be produced in bacteria as a soluble protein (S1S2 domain). The S1S2 domain binds agonists and antagonists with approximately the same affinity as the intact receptor and serves as an excellent system for studying the binding domain. The crystal structure of the S1S2 domain of the GluR2 subunit has been solved by E. Gouaux and collaborators. We have used NMR spectroscopy to characterize the dynamic properties of the GluR2 S1S2 domain. Unfortunately, the function of the GluR2 receptor is difficult to study due to its rapid kinetics and low channel conductance. The purpose of this proposal is to extend the structural and dynamic studies to a glutamate receptor subunit (GluRS) that forms channels more suitable for detailed kinetic measurements. NMR and fluorescence studies will characterize the structure and dynamics in the presence of a series of full and partial agonists. As shown by crystal structures and homology models, the S1S2 domain is a bilobe structure that closes upon ligand binding. Our hypothesis is that the channel conductance is modulated by lobe closure in individual subunits and that either large-scale conformational dynamics or intralobe dynamics contribute to changes in conductance levels. Openings and closings within an open channel burst are on the same timescale as the internal dynamics of the S1S2 domain. Single channel conductances and rate constants will be determined for homomeric GluRS receptor-channels in the same series of agonists to determine if clear differences in atomic level interactions between agonist and receptor conformation differentially affect channel kinetics. Comparison of the internal dynamics with the single channel properties of the channels should allow us to determine if these dynamics play a role in the control of ion channel function. The last aim will include NMR and single channel recordings from mutated S1S2 domains to determine the role of the interlobe interface in the control of agonist affinity and efficacy. Although homomeric GLURS receptor-channels are likely to be confined to a small number of neurons, GLUR2/GLUR3 heteromeric receptor-channels may represent a significant number of postsynaptic AMPA receptors in the neocortex. The results will shed light on the binding site of an important glutamate receptor subunit and provide essential information for further drug development.