Substrate- and pH-Specific Antifolate Transport Mediated by Organic Anion-Transporting Polypeptide 2B1 (OATP2B1-SLCO2B1)
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy (M.V.); Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester (M.-H.C., M.F.R.); and Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York (R.Z., I.D.G.)
- Address correspondence to:
I. David Goldman, Albert Einstein Cancer Center, Chanin 2, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: i.david.goldman{at}einstein.yu.edu
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 Km was ∼300 μM; the raltitrexed influx Ki 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.
Footnotes
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This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA82621] (to I.D.G.); the National Institutes of Health National Eye Institute [Grant EY017732] (to M.F.R.); the National Institutes of Health National Institute of Diabetes and Kidney and Digestive Diseases [Grants DK083007, DK090728] (to M.F.R.); and the American Heart Association [Scientist Development Grant] (to M.H.C.).
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Data in this study contributed to the following dissertation: Visentin M (2011) Organic anion transporting polypeptide 2B1 (OATP2B1) as a new pH-dependent low-affinity folate transporter, Ph.D. thesis, University of Udine Graduation School of Medical Sciences, Udine, Italy.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- MTX
- methotrexate (4-amino-10-methyl-pteroylglutamic acid)
- BSP
- sulfobromophthalein
- RFC
- reduced folate carrier
- FCCP
- (4-(trifluoromethoxy)phenylhydrazone)
- OATP
- organic anion-transporting polypeptide
- PCR
- polymerase chain reaction
- PCFT
- proton-coupled folate transporter
- SLC
- solute carrier family
- (6S)5-formylTHF
- (6S)5-formyltetrahydrofolate
- (6S)5-methylTHF
- (6S)5-methyltetrahydrofolate
- MES
- 2-(N-morpholino)ethanesulfonic acid
- ORF
- open-reading frame
- IPMX
- pemetrexed current
- E3S
- estrone-3-sulfate
- I-V
- current-voltage.
- Received July 16, 2011.
- Accepted October 21, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
