Structural rearrangement of the intracellular domains during AMPA receptor activation
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Structural rearrangement of the intracellular domains during AMPA receptor activation. / Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang; Pickering, Darryl S; Plested, Andrew; Kristensen, Anders Skov.
In: Proceedings of the National Academy of Sciences USA (PNAS), Vol. 113, No. 27, 2016, p. E3950-E3959.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Structural rearrangement of the intracellular domains during AMPA receptor activation
AU - Zachariassen, Linda Grønborg
AU - Katchan, Ljudmila
AU - Jensen, Anna Guldvang
AU - Pickering, Darryl S
AU - Plested, Andrew
AU - Kristensen, Anders Skov
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
U2 - 10.1073/pnas.1601747113
DO - 10.1073/pnas.1601747113
M3 - Journal article
C2 - 27313205
VL - 113
SP - E3950-E3959
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 27
ER -
ID: 163106851