Figure 6

Effect of acute nicotine treatment on FRET efficiency and protein expression of α4CFP and β2YFP subunits in the presence or absence of RIC-3. Equimolar amounts of α4CFP and β2YFP cDNA were transfected either with (1:1 ratio of RIC-3 to nAChR subunit) or without RIC-3. Cells were incubated in various nicotine concentrations (0, 0.1, 1 and 10 μM) at 37°C 30 min before imaging. (A) In the absence of RIC-3 there was a significant progressive increase in FRET efficiency between α4CFP and β2YFP subunits, signifying receptor assembly, at all concentrations of nicotine (p = 0.002, Kruskal-Wallis rank sum test). With RIC-3 there was no change in FRET efficiency between α4CFP and β2YFP subunits with increasing concentrations of nicotine. Significant difference levels comparing groups of nicotine concentrations relative to no nicotine control: *, p = 0.03, **, p = 0.002 (Wilcoxon rank sum tests). Quantification of mean α4CFP (B) and β2YFP (C) fluorescence intensities per cell showed no change with increasing nicotine concentrations whether RIC-3 (at 1:1 ratio) was present or absent. However, the mean α4CFP and β2YFP fluorescence intensities with RIC-3 was significantly greater than without RIC-3 at all nicotine concentrations (p < 0.0001, RIC-3 factor, two-way ANOVA; for both α4CFP and β2YFP subunits). For pairwise comparison of α4CFP and β2YFP fluorescence between no RIC-3 and RIC-3 coexpression at each nicotine concentration significance levels are reported as p values (post-hoc pairwise Tukey’s HSD tests).