Abstract
The functional characteristics of human proton coupled folate transporter (hPCFT)/heme carrier protein (HCP) 1 were investigated. hPCFT/HCP1 expressed transiently in human embryonic kidney 293 cells mediated the transport of folate at an acidic extracellular pH of 5.5 in a manner independent of Na+ and insensitive to membrane potential, but its transport activity was absent at near-neutral pH. Folate transport mediated by hPCFT/hHCP1 at pH 5.5 was saturable with a Km of 1.67 μM and extensively inhibited by reduced folates, such as folinate, 5-methyltetrahydrofolate, and methotrexate (MTX). Sulfobro-mophthalein and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid were also found to be potent inhibitors of hPCFT/hHCP1, but hemin was found to exhibit only minimal inhibitory effect. When expressed stably as a protein fused with green fluorescent protein (GFP-hPCFT/HCP1) in MDCKII cells, GFP-hPCFT/HCP1 was mainly localized at the apical membrane, and the cellular accumulation of MTX was higher from the apical side than from the basal side. These functional features of hPCFT/HCP1 are consistent with those of the well characterized carrier-mediated folate transport system in the small intestine, suggesting that hPCFT/HCP1 is responsible for the intestinal absorption of folate and also MTX. We also found that sulfasalazine is a potent inhibitor of hPCFT/HCP1, which would interfere with the intestinal absorption of MTX when coadministered in therapy for rheumatoid arthritis as well as folate.
Footnotes
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This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and by a Grant-in-Aid for Research in Nagoya City University.
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Y.N. and K.I. contributed equally to this work.
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A portion of this work was previously presented: Nakai Y, Inoue K, Hatakeyama M, Hayashi Y, and Yuasa H (2006) Molecular identification and functional characterization of a pH-sensitive folate transporter in human intestine, in 21st Annual Meeting of the Japanese Society for the Study of Xenobiotics; 2006 Nov 29–Dec 1; Tokyo, Japan. p 277, Japanese Society for the Study of Xenobiotics, Tokyo, Japan.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.122606.
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ABBREVIATIONS: MTX, methotrexate; DMARD, disease-modifying antirheumatic drug; RFC, reduced folate carrier; PCFT, proton-coupled folate transporter; HCP, heme carrier protein; hPCFT, human proton-coupled folate transporter; NSAID, nonsteroidal anti-inflammatory drug; HEK, human embryonic kidney; PCR, polymerase chain reaction; GFP, green fluorescent protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; TPP, thiamine pyrophosphate; SSZ, sulfasalazine; mPCFT/HCP1, mouse ortholog of PCFT/HCP1; OAT, organic anion transporter.
- Received March 12, 2007.
- Accepted May 1, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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Functional Characterization of Human Proton-Coupled Folate Transporter/Heme Carrier Protein 1 Heterologously Expressed in Mammalian Cells as a Folate Transporter
