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Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

Departments of Medicine and Molecular Pharmacology and Cancer Research Center, Albert Einstein College of Medicine, Bronx, New York

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; K_t, 45 nmol/L) than methotrexate (MTX; K_t, 1 µmol/L) with comparable V_max values. The K_i values for folic acid, ZD9331, and ZD1694 were 400–600 nmol/L, and the K_i values for PT523, PT632, and trimetrexate were >50 µmol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx K_t was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 µmol/L), but V_max was decreased 4.5-fold. There was no difference in the K_t for PMX (1.2 µmol/L) or the K_i for folic acid (130 µmol/L) or PT523 ( 0.2 µmol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl^– (HepG2-B > R5), Na⁺ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

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