Glutamate-receptor ion channels (iGluRs) participate in brain functions that range from fast synaptic transmission to activity-dependent changes that underlie certain forms of learning and memory. These receptors are also implicated in a variety of excitotoxic pathologies and neurodegenerative diseases. The AMPA subtype of iGluRs (AMPARs) gives rise to the fast component of excitatory postsynaptic currents (EPSCs) at virtually all brain synapses examined, but there are few direct measurements of the properties of individual channel molecules. In addition, there are no direct structural data on the determinants of protein-protein interactions that influence localization of the receptors at synapses. We propose a series of biophysical studies that will characterize the unitary properties of AMPARs by analyzing single-channel currents through native and recombinant channels. The studies of recombinant channels will be combined with crystallographic investigations of the ligand-binding domain of the GluR2 subunit (GluR2-S1S2). Other crystallographic studies will further define the molecular determinants of AMPAR interactions with important trafficking, scaffolding, and cytoskeletal proteins. The proposal has four main goals. 1.) In Aim 1 we will determine the kinetics of native AMPARs in one channel patches from cerebellar neurons in situ. The results will provide information crucial to evaluating the impact of receptor properties on synaptic transmission. 2.) Aim 2 will employ single-channel recording to elucidate the molecular mechanisms underlying the effect of mutations that reduce steady-state desensitization of AMPARs and determine to what extent the mutations also alter activation gating. 3.) In Aim 3, single-channel recording and x-ray crystallography will be used to understand how detailed interactions within the binding cleft influence the stability of binding cleft closure and in turn the affinity and efficacy of receptor agonists. 4.) The number and location of AMPARs at synapses are key determinants of the gain and fidelity of information transfer in the brain.
Aim 4 will use x-ray crystallography to determine the structural basis of important protein-protein interactions that regulate receptor trafficking.
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