Structure, function and dynamics of a glutamate receptor

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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 GLURSreceptor-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.