(A) Contour plots (top) and histograms (bottom) of FRET distributions obtained with WT bMRP1 in the following conditions (from left to right): apo, + LTC4 (10 µM), + ATP (5 mM), + ATP/LTC4 (5 mM/10 µM), + Vi/ATP/LTC4 (1 mM/5 mM/10 µM). Shown in the right column are data for the E1454Q mutant in the presence of ATP/LTC4 (5 mM/10 µM). The time-dependent changes in the contour plots were due to fluorophore photobleaching, which depopulated FRET-active molecules over time. Time points after photobleaching were excluded from subsequent analysis. The histograms represent the cumulative FRET distributions over the entire 1.25 s time window. Overlaid on the histograms are fitted distributions by the five-state model with mean FRET values of 0.92 (magenta, OF), 0.80 (yellow, IF1), 0.63 (green, IF2), 0.42 (blue, IF3), and 0.23 (orange, IF4). n denotes the number of molecules analyzed. (B) Relative occupancy of the IF1 state in the presence of increasing concentrations of LTC4. Data are fitted to a dose-response function with the Hill equation, yielding an EC50 of 0.32 ± 0.17 µM. (C) Relative occupancy of the OF state in the presence of increasing concentrations of ATP. Data are fitted to a dose-response function with the Hill equation, yielding an EC50 of 0.05 ± 0.02 mM. Data are represented as mean ± SEM.