Changes in FRET efficiency between fluorescent α7 subunits upon co-transfection with RIC-3. Pixel based FRET was used to monitor assembly between α7-Venus and α7-Cerulean nAChR subunits. RIC-3 concentration was varied as molar ratio to total α7 plasmid: 0:1 (negative control), 0.02:1, 0.1:1, 1:1, and 5:1. Confocal images of transfected HEK293T cells showing α7-Cerulean (A, D, G), α7-Venus (B, E, H) and FRET efficiency (C, F, I) expression. FRET efficiency was higher in cells expressing RIC-3 at a 1:1 ratio to α7 (F) than cells not expressing RIC-3 (C) or cells expressing RIC-3 at a 5:1 ratio to α7 (I). (J) Summary plot showing that FRET efficiencies increased significantly (p < 0.0001, Kruskal-Wallis rank sum test) with increasing concentrations of RIC-3 relative to α7 but went to baseline at 5:1 ratio. There was a significant increase of FRET efficiency at 0.1:1 RIC-3 to α7 (p = 0.0004, Wilcoxon rank sum test) as compared to no RIC-3 cells and at 1:1 RIC-3 relative to α7 (p < 0.0001, Wilcoxon rank sum test). Numbers inside the plot represent the number of cells analyzed. (K) Control experiments validating the FRET measurements. α4YFP β2CFP shows significantly greater FRET efficiency than α4CFP β2YFP. This is expected because the transfection ratio favours an (α4)3(β2)2 stoichiometry  and the combination with the more acceptors (YFP) theoretically would have greater FRET efficiency. GYFP GCFP (GluClβ-YFP GluClα-CFP) are heteromeric glutamate-gated chloride channels found in invertebrates and show high FRET. Although they are members of the cys-loop family of receptors, they are not expected to assemble with any of the nicotinic receptors. Our negative control experiment, α4YFP GCFP (GluClα-CFP) shows very little FRET. A positive control experiment with RIC-3 and α7V α7C (α7-Venus α7-Cerulean) showing significant levels of FRET. Numbers inside the bars represent the number of cells analyzed.
Dau et al. BMC Neuroscience 2013 14:47 doi:10.1186/1471-2202-14-47