Stereoselective Transport of Amethopterin Enantiomers by the Proton-coupled Folate Transporter

Summary:Stereoselective transport of methotrexate (l-amethopterin, l-MTX) and its antipode (d-amethopterin, d-MTX) by the proton-coupled folate transporter (PCFT) was examined using PCFT-expressing HEK293 cells (PCFT-HEK293 cells). Uptake of both l-MTX and d-MTX was pH-dependent and decreased with an increase in the extracellular pH from 5.0 to 7.4. The initial uptake rate of l-MTX into PCFT-HEK293 cells followed Michaelis-Menten kinetics with a Km value of approximately 5.0 μM. Dixon plots revealed that l-MTX uptake was inhibited competitively by unlabeled l-MTX, d-MTX, and folic acid (FA), with Ki values of approximately 3.6, 180, and 2.1 μM, respectively. The initial uptake rate of d-MTX into PCFT-HEK293 cells also followed Michaelis-Menten kinetics with a Km value of 211 μM. The Vmax value of d-MTX was similar to that of l-MTX. The present study revealed that the transport of MTX enantiomers by PCFT is highly stereoselective with the uptake clearance of l-MTX being approximately 40-fold greater than that of d-MTX. It was also revealed that this high stereoselectivity results from the difference in Km values, and not Vmax values, between the enantiomers. The observed stereoselectivity was consistent with the differences in the intestinal absorption of MTX enantiomers in humans.