Structural rearrangement of the intracellular domains during AMPA receptor activation

Abstract

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...

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

“…Desensitization after photoactivation of Y797AzF was strongly reduced. Comparing this to our recent FRET data suggesting the M4 (as assayed from the C-terminal movement) is likely to move during gating, desensitization and without pore opening (Zachariassen et al, 2016), a wider role for peripheral interactions to modulate AMPAR function appears likely. We did not detect crosslinking either within or between subunits for Y797AzF.…”

Cellular encoding of Cy dyes for single-molecule imaging

“…The emitted fluorescence was collected using a broad-band mirror and focused onto an iXon3 888 EM/CCD camera (Andor Inc., South Windsor, CT). The illuminated area on the coverslip was ~2500 µm (Zachariassen et al, 2016). A region of the oocyte was selected to focus the sample and then adjust the TIRF angle.…”

“…A common strategy to incorporate fluorophores into target proteins is the fusion to fluorescent proteins, such as GFP or one of its spectral variants (Miranda et al, 2013; Zachariassen et al, 2016). This approach has the built-in convenience of encoding but their positioning within the target protein can be limited by their large size, which may impact protein function or trafficking.…”

Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin

“…It is also possible that, upon agonist binding, stabilization of the active state by TARP may be achieved, not only through extracellular interactions but also through further altering interactions within the membrane domains and even the CT. Indeed, a recent study employing dynamic spectroscopy suggested that the CT and the intracellular linker between M1 and M2 move during AMPAR activation (Zachariassen et al, 2016).…”