TABLE 2

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⁺
Transporter	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⁺.