Substrate specificity of transport mediated by the N411K PCFT mutant. Influx of tritiated PCFT substrates was assessed at a concentration far below the influx Kt for PCFT-WT, 0.5 µM (A) and at least an order of magnitude higher than the influx Kt for PCFT-WT, 50 µM (5 µM for pemetrexed) (B). Transport at the high concentration reflects the influx Vmax. Since a change in Vmax should result in a comparable proportional change in influx, irrespective of the substrate concentration, a discrepancy in the percentage change between the low and high substrate concentrations reflects an additional change in the influx Kt. The data shown are percentages of influx activity in relation to PCFT-WT ± SEM from 3 independent experiments. *Comparing the differences between the percentage reduction in transport of MTX (P < .01), pemetrexed (not significant), and folic acid (P <. 01) at the low vs high concentrations. **Comparing the difference between 5-formylTHF and 5-methylTHF influx at the high concentration (P < .05). ***Comparing influx of MTX (P < .005) and folic acid (P < .005) with transport of pemetrexed at the low substrate concentration.
