TY - JOUR T1 - Concentrative transport of antifolates mediated by the proton-coupled folate transporter (PCFT, SLC46A1); augmentation by a HEPES buffer JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.117.110445 SP - mol.117.110445 AU - Rongbao Zhao AU - Mitra Najmi AU - Srinivas Aluri AU - David C Spray AU - I. David Goldman Y1 - 2018/01/01 UR - http://molpharm.aspetjournals.org/content/early/2018/01/11/mol.117.110445.abstract N2 - The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors where it delivers antifolates, particularly pemetrexed, into cancer cells. Studies on PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This paper addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. A HeLa-derived cell line was employed in which folate-specific transport was mediated exclusively by PCFT. There was a substantial chemical gradient for methotrexate at pH 7.0 which decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antifolate gradient correlated with an alkaline intracellular pH under the former (pH-7.85), but not the latter (pH-7.39), conditions and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was due to the activity of Na+H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger(s) or Na+/K+ ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 vs 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families. ER -