Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor live cell imaging reagent / dye LiveReceptor AMPAR | Funakoshi

Specific labeling regent for synaptic AMPA type glutamate receptor
Live Receptor AMPAR

LiveReceptor AMPAR is the reagent which can specifically label AMPA-type glutamate receptors (AMPARs) with fluorescein dye on living neurons.
This reagent is based on a unique chemical reaction and can accomplish labeling of AMPARs keeping its ligand-binding pockets and receptor functions.
LiveReceptor AMPAR is a powerful tool to investigate physiological functions of AMPAR by monitoring receptor trafficking of endogenous AMPARs on live cells. No complicated protocol and genetic manipulation are required.

Generally, fluorescein direct conjugated ligand has been used for labeling receptors, however, such ligands easiliy detach from ligand binding pocket, and then signal will be faded out. (See below image)

Our "LiveReceptor" has a linker of acyl imidazole between fluorescein and ligand. While ligand is binding to receptor, nucleophillic amino acids attacks acyl imidazole and changes its chemical structure. By this method, receptors are directly labeled and keep fluorescence even after the ligand is detached from binding pocket.

This product is commercialized by the research result of Professor Hamachi, at Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University.

* PFQX : 6-pyrrolyl-7-trifluoromethyl-quinoxaline-2,3-dione. This is affinity ligand for AMPAR.

Fig.1 Live cell imaging of labelled endogenous
AMPARs in cultured neurons Cultured hippocampal neurons were treated with 1 μM of LiveReceptor AMPAR (in left) or Fluorescein-conjugated ligand as negative control (in right) for 1 hour at 17C and washed out three times with the basal medium. Dendritic spin-like punctual structures were observed on live cells by specifically LiveReceptor. (scal bars, 10 μm)

Fig.2 Live cell imaging of labelled endogenous AMPARs in cultured slice tissue
Acutely prepared hippocampal slices were treated with 1 μM of LiveReceptor AMPAR for 1 hour at 17C in the absence (in left) or presence (in right) of 10 μM NBQX, a potent inhibitor of AMPAR, and washed out three times with the basal medium. Dendritic spin-like punctual structures were observed on live cells and the signal was clearly disappeared by NBQX. (scal bars, 5 μm)

Fig.3 Staining of AMPARs in deep tissue in cultured slice tissue
Cultured slice tissue of mouse was stained by LiveReceptor AMPAR and fixed. After fixation, stained by anti-GluA2/3 antibody. x-y dimension shows a good concordance between LiveReceptor AMPAR and anti-GluA2/3 antibody.
However, x-z dimension shows that anti-GluA2/3 antibody stained the surface of slice tissue only. LiveReceptor AMPAR penetrates into deep tissue and can label them. (Black arrowhead : Top of sliced tissue / White arrowhead : Bottom of sliced tissue)

Please down load the movie from here.