NFRET measurements of the effects of nicotine and β2 M3–M4 loop mutations on α4β2 nAChR stoichiometry. (A) Representative confocal images of Neuro-2a cells expressing a TG/TGN marker (GalT-eCFP) and α4-eGFP plus α4-mcherry plus β2wt nAChRs. FP tags (FP = any FP) were used to obtain the trans-Golgi ROI and to separately measure pixel-based NFRET values for either the whole cell section or for the TG/TGN. Bars, 5 µm. (B) Frequency plots of NFRET values (x axis) versus number of pixels (y axis) for α4-eGFP plus α4-mcherry coexpressed with β2wt/β2-L349M/β2-365AAQA368 subunits. Pixel-based NFRET values were separately obtained in the presence or absence of 0.1 µM nicotine (nic) as indicated. Column labels show the ROI for NFRET measurements (whole cell section or TG/TGN), and row labels show the type of β2 subunit (β2wt/β2-L349M/β2-365AAQA368) coexpressed with α4-eGFP plus α4-mcherry. Frequency histograms (black) were fitted to two Gaussian components (green) with low (α4)2(β2)3 and high (α4)3(β2)2 mean NFRET values. Overall fits (red) are shown for each graph; data are quantified in Table I.
