Trapping the activation mechanism of AMPA-type glutamate receptors

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Throughout the brain, the fast excitatory synaptic transmission that underpins thought and deed is handled by the AMPA-subtype of ionotropic glutamate receptors (iGluRs). AMPA-receptors have rapid kinetics, activating and deactivating within hundreds of microseconds. Glutamate binding to each of the four subunits drives the opening of an integral transmembrane ion channel. To map conformational changes during activation in full-length AMPA receptors, we will use a recently-developed technique: chemical modification coupled to rapid solution exchange electrophysiology. This method allows us to activate receptors on the same timescale as those at native synapses, tracking their activity whilst chemically trapping them in conformations relevant to their signaling role in the brain. We will begin by examining the motions within individual subunits. Subsequently, we will extend our studies to newly-discovered intersubunit contacts between the ligand binding domains. Trapping ‘bridges’ formed by metal ions bound at introduced sites, or by disulfide bonds, will be employed in combination with a battery of pharmacological tools. We will thus directly elucidate the dynamics of the neurotransmitter-binding site in the context of the full-length receptor. To complement these functional studies, we will perform structural and biochemical studies on selected trapped mutants to investigate their conformations and multimeric arrangement. Finally, we will use the trapping bridges we develop as tools to define the positions occupied by different subunits in mixed complexes. These studies are expected to provide novel insights into the gating of AMPA channels during normal brain activity and in disease, and elucidate new mechanisms for the modulation of these channels by drugs.