Structure, function and dynamics of a glutamate receptor

描述(申请人提供)：嗜离子谷氨酸受体控制各种正常的神经元过程，包括学习和记忆。此外，这些重要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。对跨膜拓扑的分析导致了这样的认识，即每个亚基都由一系列模块组成，而与谷氨酸结合的模块可以在细菌中以可溶性蛋白质的形式产生(S1S2结构域)。S1S2结构域以与完整受体大致相同的亲和力结合激动剂和拮抗剂，是研究结合结构域的良好系统。E.Gouaux和他的合作者已经解决了GluR2亚单位S1S2结构域的晶体结构。我们用核磁共振波谱表征了GluR2 S1S2结构域的动态性质。不幸的是，由于GluR2受体的快速动力学和低通道电导，其功能很难研究。这项建议的目的是将结构和动力学研究扩展到谷氨酸受体亚单位(GluRS)，它形成的通道更适合于详细的动力学测量。核磁共振和荧光研究将在一系列完全和部分激动剂存在的情况下表征结构和动力学。晶体结构和同源模型表明，S1S2结构域是一种双体结构，在配体结合时关闭。我们的假设是，通道电导受到单个亚基中叶关闭的调制，大尺度构象动力学或大叶内动力学有助于电导水平的变化。开放通道猝发中的开启和关闭与S1S2域的内部动力学在相同的时间尺度上。将测定同一系列激动剂中同构体GluRS受体通道的单通道电导和速率常数，以确定激动剂和受体构象之间原子水平相互作用的明显差异是否会对通道动力学产生不同影响。通过将内部动力学与通道的单通道特性进行比较，我们可以确定这些动力学是否在离子通道功能的控制中发挥作用。最后一个目标将包括从突变的S1S2结构域进行核磁共振和单通道记录，以确定叶间界面在控制激动剂亲和力和有效性中的作用。虽然同构体GLURS受体通道可能仅限于少数神经元，但GLUR2/GLUR3异构体受体通道可能代表新皮质中大量突触后AMPA受体。这一结果将揭示一个重要的谷氨酸受体亚单位的结合部位，并为进一步的药物开发提供必要的信息。

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.