Dynamics of the Cytoplasmic Region of an AMPA-Subtype Glutamate Receptor Revealed by State Dependent FRET

Abstract

AMPA receptors (AMPARs) are glutamate-gated ion channels, which mediate fast excitatory neurotransmission in the central nervous system. Extensive crystallographic studies of the extracellular domains and, more recently, crystal structures and single particle EM reconstructions of full-length receptors have set the framework for further investigations of receptor conformational dynamics, gating mechanism and regulation. However, intracellular regions are either truncated or not resolved in these structures. Further, the conformational transitions of the intracellular domains during receptor gating have not been investigated.In present study, we have explored single and double fusions of cyan and yellow variants of green fluorescent protein (CFP and YFP, respectively) at intracellular sites of AMPAR to enable measurement of conformational changes using Fluorescence Resonance Energy Transfer (FRET) in live cells. The fluorescent fusions retain wild-type receptor expression and kinetic properties. Fluorescence Lifetime Imaging (FLIM) showed ligand-dependent FRET efficiency. Conformational rearrangements accompanying receptor function were measured using a Patch Clamp Fluorometry (PCF) setup on live HEK 293 cells in real time. Our results suggest that FRET efficiency is dependent on the functional state of the receptor and allosteric modulation by Cyclothiazide, an AMPA receptor desensitisation blocker. Thus the intracellular sites undergo conformational rearrangements during receptor function.