Structural rearrangement of the intracellular domains during AMPA receptor activation

Linda Grønborg Zachariassen; Ljudmila Katchan; Anna Guldvang Jensen; Darryl S Pickering; Andrew Plested; Anders Skov Kristensen

doi:10.1073/pnas.1601747113

Structural rearrangement of the intracellular domains during AMPA receptor activation

Authors:

Zachariassen, Linda Grønborg ;
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Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
Katchan, Ljudmila ;
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Leibniz Institute for Molecular Pharmacology
Jensen, Anna Guldvang ;
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IF-Administration, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet
Pickering, Darryl S ;
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0000-0001-8687-6094
Molecular and Cellular Pharmacology, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
Plested, Andrew ;
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Leibniz Institute for Molecular Pharmacology
Kristensen, Anders Skov
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Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet

DOI:

10.1073/pnas.1601747113

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.

Type:

Journal article

Language:

English

Published in:

Proceedings of the National Academy of Sciences Usa (pnas), 2016, Vol 113, Issue 27

Keywords:

Journal Article

Main Research Area:

Medical science

Publication Status:

Published

Review type:

Peer Review

Submission year:

2016

Scientific Level:

Scientific

ID:

2305892375

Structural rearrangement of the intracellular domains during AMPA receptor activation

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