Modulation of AMPA receptor desensitization by nootropic drugs and endogenous proteins
Abstract
AMPA receptors play a central role in basal excitatory synaptic transmission as well as synaptic maturation and plasticity. An important characteristic of AMPA receptors (AMPARs) is the rapid and profound desensitization that occurs within milliseconds of glutamate binding; however, the mechanisms controlling desensitization of native AMPARs are incompletely understood. We have applied patch-clamp recording techniques in cultured neurons and heterologous cells to (1) test a model of AMPAR desensitization based on the crystal structure of the extracellular domains and (2) determine whether proteins associated with synaptic AMPARs affect desensitization and/or other channel properties. A key finding from crystallization of the extracellular domains of the receptor is that the benzothiadiazine positive modulator cyclothiazide, which blocks AMPAR desensitization, binds at and stabilizes the dimer interface formed by adjacent subunits. This and other data suggested a model in which desensitization involves a conformational change disrupting the dimer interface. Our data from intact hippocampal AMPAR indicate the benzothiadiazines prevent the conformational change to the desensitized state, and, conversely, cannot bind desensitized AMPARs. The transmembrane AMPAR regulatory protein (TARP) stargazin (γ2) serves multiple roles in trafficking and stabilizing synaptic AMPARs and may be incorporated as an auxiliary subunit. Transfection of hippocampal neurons with stargazin produced two distinct effects on AMPAR functional properties without affecting surface receptor density: reduced glutamate-evoked desensitization and increased efficacy of the partial agonist kainate. Kinetic and dose-response analyses suggest that stargazin affects glutamate desensitization through an allosteric interaction that destabilizes the desensitized state of the receptor, and that potentiation of kainate responses reflects increased efficacy rather than altered affinity. Stargazin's effects were also observed in HEK 293 cells transfected with various heteromeric and homomeric AMPARs, with subunit-dependent effects on glutamate desensitization, kainate efficacy, and trafficking. We show that two regions of stargazin mediate its functional effects: the C-terminal intracellular domain appears more important for effects on glutamate-evoked desensitization and receptor trafficking, while the first extracellular domain makes a larger contribution to altered kainate efficacy. These data indicate that TARPs directly modulate channel function and, as auxiliary subunits of AMPARs, contribute to functional heterogeneity of the desensitization properties of neuronal AMPARs.
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- OSU Dissertations [11222]