Figure 4. Carrier-protein dependent transport of R123 through biological membranes.

(A–D) HEK-293 MSRII cells were singly transduced with BacMam viruses carrying human CDS' for OATP1A2, OATP1B3, OATP2B1 and OATP1B1 and incubated for 48 hours to allow protein expression. Cells were exposed to 0–2 µM R123, in the presence (open circles) or absence (closed circles) of 10 nM specific inhibitor for 120, 300, 420 and 600 seconds at 37°C, and intracellular fluorescence determined: Inhibitors were ketoconazole (OATP1A2), rifamycin (OATP1B1/3) and montelukast (OATP2B1). Data points correspond to the rate of uptake obtained from linear regressions over three time points with duplicate values at each concentration. (E) Naive MDCKII cells (open circles), or MDCKII cells with the CDS for human ABCB1 stably integrated (closed circles) were loaded with varying concentrations of R123 and efflux measured over 7.5 minutes. For all panels, inset images represent Western blot analysis for naïve and transfected cells (top row), plus actin loading control (bottom row), demonstrating high-level expression of the requisite human transporter in each case.