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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Functional Characterization of Human Proton-Coupled Folate Transporter/Heme Carrier Protein 1 Heterologously Expressed in Mammalian Cells as a Folate Transporter

Yasuhiro Nakai, Katsuhisa Inoue, Naoki Abe, Mai Hatakeyama, Kin-ya Ohta, Masaki Otagiri, Yayoi Hayashi and Hiroaki Yuasa
Journal of Pharmacology and Experimental Therapeutics August 2007, 322 (2) 469-476; DOI: https://doi.org/10.1124/jpet.107.122606
Yasuhiro Nakai
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Katsuhisa Inoue
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Naoki Abe
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Mai Hatakeyama
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Kin-ya Ohta
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Masaki Otagiri
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Yayoi Hayashi
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Hiroaki Yuasa
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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

  • 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.

  • Y.N. and K.I. contributed equally to this work.

  • 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.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.107.122606.

  • 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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Journal of Pharmacology and Experimental Therapeutics: 381 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 381, Issue 2
1 May 2022
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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Functional Characterization of Human Proton-Coupled Folate Transporter/Heme Carrier Protein 1 Heterologously Expressed in Mammalian Cells as a Folate Transporter

Yasuhiro Nakai, Katsuhisa Inoue, Naoki Abe, Mai Hatakeyama, Kin-ya Ohta, Masaki Otagiri, Yayoi Hayashi and Hiroaki Yuasa
Journal of Pharmacology and Experimental Therapeutics August 1, 2007, 322 (2) 469-476; DOI: https://doi.org/10.1124/jpet.107.122606

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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Functional Characterization of Human Proton-Coupled Folate Transporter/Heme Carrier Protein 1 Heterologously Expressed in Mammalian Cells as a Folate Transporter

Yasuhiro Nakai, Katsuhisa Inoue, Naoki Abe, Mai Hatakeyama, Kin-ya Ohta, Masaki Otagiri, Yayoi Hayashi and Hiroaki Yuasa
Journal of Pharmacology and Experimental Therapeutics August 1, 2007, 322 (2) 469-476; DOI: https://doi.org/10.1124/jpet.107.122606
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