Förster Resonance Energy Transfer (FRET) Analysis of the C-Terminal Domain of Fp-Tagged Homomeric and Heteromeric AMPARs Upon Phosphorylation

Abstract

AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are tetrameric assemblies of GluA1 to GluA4 subunits. Recently Cryo-EM and X-ray crystal structures of homomeric and heteromeric AMPARs has been provided; showing a highly modular architecture with an extracellular domain containing four glutamate binding sites coupled to a transmembrane domain containing the central ion channel. AMPARs also contain an intracellular domain (ICD) formed of C-terminals from each subunit. The ICD is important for interactions with intracellular proteins and contains multiple regulatory phosphorylation sites, but its structural and mechanistic role is poorly understood as the ICD is not resolved in present structures. We have used a Förster Resonance Energy Transfer (FRET) approach to study intramolecular distances and dynamics of the ICD in homomeric and heteromeric AMPARs by insertion of fluorescent proteins (FPs) at various intracellular positions in the GluA1 and GluA2 subunits. Using fluorescence life-time imaging (FLIM) to determine FRET efficiencies between FPs or a membrane dye, we determine relative distances within the ICD and to the membrane. Furthermore, we investigate the effect of phosphorylation mimicking mutations in the GluA1 subunit on FRET and identify mutation-induced changes that indicate phosphorylation state of the ICD can change its structure. Our preliminary data provides new insight into the structural architecture and potential role of the ICD.