Abstract by Parisa Razaee

AMPA receptors (AMPARs), represent a subgroup of ionotropic glutamate receptors (iGluRs) that play a crucial role in facilitating the predominant excitatory neurotransmission within the mammalian central nervous system (CNS). These receptors operate as homo- and heterotetramers, composed of the subunits GluA1-4. The number and specificity of postsynaptic AMPAR subtypes during synaptic activity impact the formation and consolidation of memory in the brain. However, the poor ability to dissect the AMPAR subtypes in synapses is an obstacle. 

In the present study, the biophysical technique of Förster Resonance Energy Transfer (FRET) is utilized to differentiate AMPAR subtypes. FRET involves a non-radiative transfer of energy from a donor fluorophore to an acceptor fluorophore. FRET efficiency is highly sensitive to the distance between the donor and acceptor, decreasing steeply as the distance increases, with an inverse relationship to the sixth power of their separation, making it a valuable tool for measuring intramolecular distances within macromolecules like AMPARs, provided that appropriate donor and acceptor fluorophores are incorporated into the receptor subunits. 

In the current investigation, the genetic encoding of cyan fluorescence protein (CFP) and HALO domain was examined as a technique for incorporating FRET donors and acceptors into the AMPAR subunits GluA1-3. A systematic screening was conducted to identify permissive region within the receptor subunits suitable for the insertion of CFP and HALO domain. An insertion site was identified between the receptor SP and the N-terminal domain. Utilizing this site, several FRET-enabled constructs of the AMPAR were developed. A distinct strategy was employed based on Fluorescence-lifetime imaging microscopy (FLIM) to achieve intrareceptor FRET in these constructs. A subtype-dependent alteration in FRET was observed upon introducing different subunit combinations of AMPAR to the HEK293T cells. 

In the second part of this study, the genetic encoding of AMPARs with self-labeling protein (SLP) such as SNAP and HALO domains was examined. In this strategy, by using cell impermeable labels for SLP domains we were able to selectively focus on expressed AMPARs in the extracellular region of the cell membrane, while by using CFP all expressed AMPARs send fluorescence signals. We observed di-heterotetrameric subtypes of GluA1/2 and GluA2/3 on the membrane, whereas GluA1 and GluA3 did not form a di-heterotetramer subtype. We examined the expression of DNA constructs encoding AMPAR subunits with SNAP and HALO domains in hippocampal and cortical neurons. The primary neurons expressed exogenously introduced plasmid DNAs encoding dual AMPAR constructs. These methodologies provide the potential to create FRET constructs by incorporating fluorophores into the area of the AMPARs, allowing differentiation between subtypes. They also offer a means for future studies to distinguish these subtypes before and during long-term potentiation (LTP), enabling investigation into their involvement in memory formation.