Influence on the substrate concentration used for uptake measurement on affinity of TBuA+ for inhibition of MPP+ uptake mediated by rOCT1 wild-type and two rOCT1 mutants
Initial uptake of different concentrations of MPP+ into HEK293 cells stably transfected with rOCT1, rOCT1(Y222F), or rOCT1(D475E) was measured in the presence of different concentrations of TBuA+, and the IC50 values for TBuA+ inhibition were determined by fitting the Hill equation to data of individual experiments. Mean ± S.D. values of three or four experiments are shown. The compiled uptake measurements and the calculated Hill coefficients are shown in Fig. 2.
Transporter | IC50 Values for Inhibition of MPP+ Uptake by TBuA+ Using Different Concentrations of MPP+ | ||
---|---|---|---|
100 nM MPP+ | 12.5 nM MPP+ | 0.25 nM MPP+ | |
rOCT1 | 1.14 ± 0.04 µM | 0.26 ± 0.01 µM##,■■ | 0.57 ± 0.07 µM# |
rOCT1(Y222F) | 0.30 ± 0.03 µM*** | 0.27 ± 0.02 µM | 0.36 ± 0.14 µM |
rOCT1(D475E) | 0.72 ± 0.13 µM** | 0.17 ± 0.04 µM**,∆∆,••• | 6.80 ± 0.68 µM***,### |
WT, wild-type.
ANOVA with post hoc Tukey test:
↵** P < 0.01;
↵*** P < 0.001 for difference to rOCT1 measured with the same concentration of MPP+;
↵# P < 0.05;
↵### P < 0.001 for difference to the IC50 value obtained with 100 nM MPP+;
↵••• P < 0.001, for difference to the IC50 value obtained with 0.25 nM MPP+. Student’s t test:
↵∆∆ P < 0.01 for difference to the IC50 value obtained with 100 nM MPP+;
↵■■ P < 0.01 for difference to the IC50 value obtained with 0.25 nM MPP+.