The widely used iGluSnFR consists of a circularly permutedenhanced green fluorescent protein (cpEGFP) fused to a glutamate binding protein (GluBP) from a bacterium.[3] When GluBP binds a glutamate molecule, it changes its shape, pulling the EGFP barrel together, increasing fluorescence. A specific peptide segment (PDGFR) is included to bring the sensor to the outside of the cell membrane.[4] In the more recent version by Aggarwal et al. (2022),[1] researchers introduced iGluSnFR to two additional anchoring domains, a glycosylphostidylinositol (GPI) anchor, and a modified form of the cytosolic -cterminal domain of Stargazin with a PDZ ligand.
History
The first genetically encoded fluorescent glutamate sensors (FLIPE, GluSnFR and SuperGluSnFR) were constructed by attaching cyan-fluorescent protein (CFP) and yellow-fluorescent protein (YFP) to a bacterial glutamate binding protein (GluBP).[5][6]Glutamate binding changed the distance between CFP and YFP, changing the efficiency of energy transfer (FRET) between the two fluorophores.[7][8] A breakthrough in visualizing glutamate release was achieved with iGluSnFR, a single-fluorophore glutamate sensor based on EGFP producing a ~5‑fold increase in fluorescence.[3] To measure synaptic transmission at high frequencies, novel iGluSnFR variants with accelerated kinetics have recently been developed.[9][10]
