Figure 5

FRET efficiency measurements show that RIC-3 interacts with α7 and β2 nicotinic subunits. (A-I) Pixel based FRET was used to monitor assembly between CFP-RIC-3 and a fluorescently tagged nicotinic receptor subunit (either α7-Venus or β2YFP). (J) Summary data showing significantly higher levels of FRET efficiency between CFP-RIC-3 and α7-Venus at 1:1 ratio as compared to 5:1 ratio (p = 0.007, Welch two sample t-test). There was significantly greater FRET efficiency between CFP-RIC-3 and β2YFP at 0.2:1 (p = 0.003, Wilcoxon signed rank test post-hoc analysis) and 0.5:1 (p < 0.0001, Wilcoxon signed rank test post-hoc analysis) ratios as compared to 5:1 ratio. Also there was significantly greater FRET efficiency between CFP-RIC-3 and β2YFP at 0.2:1 (p = 0.009, Wilcoxon signed rank test post-hoc analysis) and 0.5:1 (p = 0.002, Wilcoxon signed rank test post-hoc analysis) ratios as compared to 1:1 ratio. No significant (NS) FRET efficiency could be detected between CFP-RIC-3 and α4YFP (p = 0.55, Kruskal-Wallis rank sum test) even though there was a similar trend as β2YFP. Therefore, equimolar RIC-3 interacts with α7 while RIC-3 optimally interacts with β2 at a 0.5:1 molar ratio.