Abstract

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (∼2.5 μM), only [³H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 μM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx K_m was ∼300 μM; the raltitrexed influx K_i was ∼70 μM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.