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.T he α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (iGluRs) reside in postsynaptic membranes in the central nervous system (1). Like the other major iGluR subtypes, the N-methyl-Daspartate receptors (NMDARs) and kainate receptors, AMPARs are ligand-gated cation channels formed by hetero-or homotetrameric assembly of the GluA1 to GluA4 subunits into a membranespanning receptor with an integral ion channel. The energy from binding of the excitatory neurotransmitter glutamate at extracellular sites triggers AMPARs to passage through functional states that can include ion channel activation, deactivation, and desensitization. The timing and interplay between the necessarily distinct conformations of these states determine both basal transmission and short-term plasticity of the postsynapse (2-4).Structures of AMPARs captured in various functional states using X-ray crystallography and cryoelectron microscopy have recently become available (5-9). Overall, the structures confirm three distinct structural layers (Fig. 1). The amino-terminal domain (ATD) and the ligand-binding domain (LBD), both distal to the membrane, and the transmembrane ion channel are each formed from four copies of the respective domain from each subunit. However, a fourth region of the receptor extends from the intracellular side of the membrane. This part of the receptor includes intracellular loops between the transmembrane helices and the variable C-terminal domain (CTD), and remains entirely unresolved in the presently available AMPAR structures.The GluA2 structures and previous results from biochemical, biophysical, and crystallographic studies of isolated subunits and fu...

Polyamine toxins from spiders and wasps are potent open-channel blockers of ionotropic glutamate (iGlu) receptors. It is well-established that secondary amino groups in the polyamine moiety of these toxins are key to both selectivity and potency at iGlu receptors, still some native spider polyamine toxins comprise both N-methyl and N-hydroxy functionalities. Here, we investigate the effect of both N-methylation and N-hydroxylation of spider polyamine toxins by the synthesis and biological evaluation of the naturally occurring N-methylated argiopinines and pseudoargiopinines I and II, N-hydroxylated Agel-489 and Agel-505, as well as N-methylated analogues of the NMDA and AMPA iGlu receptor subtype selective antagonists ArgTX-93 and ArgTX-48. Efficient synthetic strategies for the synthesis of target compounds were developed, and evaluation of biological activity at AMPA and NMDA receptors identified highly potent and in some cases very selective ligands.