TY - ABST

T1 - Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

AU - Zachariassen, Linda Grønborg

AU - Katchan, Mila

AU - Plested, Andrew

AU - Pickering, Darryl S

AU - Kristensen, Anders Skov

PY - 2014/2/16

Y1 - 2014/2/16

N2 - AMPA receptors (AMPARs) are glutamate-gated cation channels that mediatethe 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 mechanismunderlying 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 aconstruct (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 of0.11 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 FRETefficiency is dependent on functional state of GluA2-6Y-10C and hereby indi- cates that the intracellular CTD undergoes conformational changes during re-ceptor signaling.

AB - AMPA receptors (AMPARs) are glutamate-gated cation channels that mediatethe 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 mechanismunderlying 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 aconstruct (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 of0.11 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 FRETefficiency is dependent on functional state of GluA2-6Y-10C and hereby indi- cates that the intracellular CTD undergoes conformational changes during re-ceptor signaling.

M3 - Conference abstract for conference

ER -