FLIM-FRET Imaging of AMPA Receptors: New Principle for Subtype-Specific Elucidation
Rezaee, P.; Pedersen, M. G.; Zachariassen, L. G.; Staudt, M.; Herth, M. M.; Kristensen, A. S.
Show abstract
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory neurotransmission, and their subunit composition (GluA1-4) critically shapes synaptic strength and plasticity. Discriminating AMPAR subtypes at the molecular level remains challenging with conventional techniques. Here, we applied Forster resonance energy transfer (FRET) combined with fluorescence-lifetime imaging microscopy (FLIM) to resolve subtype-specific AMPAR assemblies. Cyan fluorescent protein (CFP) and HALO domain were genetically introduced into GluA1-3 subunits to make intrareceptor FRET pairs. Self-labeling protein (SLP) domains such as SNAP and HALO were used with cell-impermeable substrates to selectively label surface-expressed receptors. This approach identified di-heterotetrameric GluA1/2 and GluA2/3 assemblies at the HEK293T cell membrane, whereas GluA1/3 failed to form di-heteromers. These findings demonstrate the FLIM-FRET method as a tool for differentiating AMPAR subtypes in living cells, providing a foundation for studying subtype-specific receptor organization in excitatory synapses.
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