Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

Linda Grønborg Zachariassen, Mila Katchan, Andrew Plested, Darryl S Pickering, Anders Skov Kristensen

Research output: Contribution to conferenceConference abstract for conferenceResearchpeer-review

Abstract

AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate
the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are formed
by homo- or heterotetramers of GluA1 to GluA4 sub- units. A recent X-ray crystal structure of a
full-length homomeric GluA2 AM- PAR has allowed unique insight into AMPAR molecular structure and
provides an improved framework for beginning to understand the structural mechanism
underlying receptor function, regulation and pharmacological modulation.
In the present study, we have explored dual insertion of cyan and yellow variants (CFP and YFP,
respectively) of green fluorescent protein at various positions in the GluA2 AMPAR subunit to
enable measurements of intra- receptor conformational changes using Fo¨ rster Resonance Energy
Transfer (FRET) in live cells. We identify dual CFP/YFP-tagged GluA2 subunit con- structs that
retain function and display intrareceptor FRET. This includes a
construct (GluA2-6Y-10C) containing YFP in the intracellular loop between the M1 and M2
membrane-embedded segments and CFP inserted in the C-ter- minal domain (CTD). GluA2-6Y-10C displays
FRET with an efficiency of
0.11 while retaining wild-type receptor expression and kinetic properties. We have used
GluA2-6Y-10C to study conformational changes in homomeric GluA2 receptors during receptor
activation. Our results show that the FRET
efficiency is dependent on functional state of GluA2-6Y-10C and hereby indi- cates that the
intracellular CTD undergoes conformational changes during re-
ceptor signaling.
Original languageEnglish
Publication date16 Feb 2014
Number of pages1
Publication statusPublished - 16 Feb 2014

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