The relationship between transporter function and NFRET. (A) Scatter plot of the ratio and mean (peripheral NFRET)/(perinuclear NFRET) for all pixels in the ROIs versus the percent function of each construct as compared with nonfluorescent wild-type mGAT1 in [³H]GABA uptake assays. Data points for constructs are individually labeled and omit the mGAT1 prefix, which is common to all. Constructs with ≥75% wild-type function are represented by solid symbols. Constructs that showed ≤65% wild-type function but good whole cell NFRET are represented by half-filled symbols. mGAT1XFP45, which demonstrated poor function and poor whole cell NFRET, is represented by an open symbol. (B) The proportions of each Gaussian component of the total peripheral ROI NFRET signal for each construct are plotted in stacked columns. Each column represents a single construct design and appears on the x axis at the percentage of wild-type [³H]GABA uptake it exhibits in uptake assays.