Structural rearrangement of the intracellular domains during AMPA receptor activation

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.