Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Inappropriate activation of these neurotransmitter receptors plays a role in the pathology of a number of neurodegenerative diseases, notably stroke and epilepsy. Analysis of the transmembrane topology has led to the realization that each subunit is made of several nearly independent modules. The module that binds glutamate, the ligand binding domain (LBD) can be produced in large quantities in bacteria as a soluble protein, which has allowed its structure to be determined. The LBDs of AMPA and kainate receptors bind agonists and antagonists with approximately the same affinity as the intact receptor and serve as an excellent system for studying the binding domain and its role in the function of the intact protein. Our previous work has focused on the structure and dynamics of the GluA2 LBD (AMPA receptor) and has defined the changes upon binding partial agonists, antagonists and allosteric regulators using NMR spectroscopy and crystallography, which in turn has improved our understanding of the structural basis of channel activation. Despite this progress, we know little about how AMPA and kainate receptors, despite their structural similarity, have such divergent kinetic properties. We have recently optimized the expression and purification of a kainate receptor (GluK2) LBD for study by NMR spectroscopy. Building upon this work and our previous results describing changes in hydrogen bonding and methyl group dynamics upon binding agonists, we will study mutations that alter the kinetic properties of GluA2 to resemble those of GluK2 and vice versa. This work will employ NMR spectroscopy, crystallography, isothermal titration calorimetry, whole cell recording, and thermal denaturation. This will allow us to determine how the binding domain controls kinetic properties and thus shapes synaptic currents for the different physiological properties of kainate and AMPA receptors. The second part of the project will investigate the mechanisms by which partial agonists activate glutamate receptors. Our previous work with AMPA receptors indicated that partial agonists may activate the channel by inducing transitions to a conformation of the LBD characterized by a full lobe closure, but with lower probability than full agonists. Kainate receptors have a wider range of partial agonists that may operate by different mechanisms. The characterization of partial agonism in kainate receptors, combined with high field NMR spectroscopy designed to study conformations of low probability, will allow us to dissect the mechanism of partial agonist binding and relate it to our previous results from single channel recording. These studies will use a range of biophysical techniques designed to understand the structural basis of the kinetic properties of AMPA and kainate receptors. This will provide essential information for development of drugs directed toward these essential neurotransmitter receptors.
Over-activity of AMPA receptors, which mediate the majority of excitatory synaptic transmission in the CNS, has been implicated in contributing to the pathological effects of stroke and epilepsy, and enhancement of the activity of AMPA receptors has been shown to be beneficial in increasing cognition. The goal of these studies is to understand the structure, function and dynamics of medically relevant AMPA (GluA2) and kainate (GluK2) receptors in terms of the structural correlates of the complex functional properties. These studies are important for expanding the targets on these receptors for drug design as well as understanding and modeling synaptic activity.
Showing the most recent 10 out of 18 publications
