FIGURE 6.

Rate of efflux of rhodamine 123 or NBD-cyclosporine A by D164C/D805C mutant P-gp was restored when grown in the presence of CsA. P-gp virus-transduced HeLa cells grown in the presence or absence of 6.25 μm CsA were studied for rate of Rh123 or NBD-CsA efflux. 300,000 cells were de-energized before loading with Rh123 or NBD-CsA as described under “Experimental Procedures”. The efflux of the substrates was initiated by resuspending cells in PBS with 50 mm glucose at 37 °C and was monitored over time by flow cytometry. The accumulation of Rh123 at 0 min is taken as 100%, and the values are calculated as the percentage of remaining fluorescence inside the cells. These values represent the average from two independent experiments. The t½ of Rh123 efflux for cysless-WT is 1.8 min versus 15 min for D164C/D805C (absence of CsA), which is restored to levels similar to cysless-WT when the cells were grown in the presence of CsA. The right panel shows efflux of NBD-CsA; t½ of NBD-CsA efflux for cysless-WT is 4.2 min, whereas the rate cannot be determined for D164C/D805C as the total efflux was <50% (in the absence of CsA). The t½ was restored to levels similar to cysless-WT when the cells were grown in the presence of CsA.