title: Functional characterization of human proton-coupled folate transporter/heme carrier protein 1 heterologously expressed in mammalian cells as a folate transporter.
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 K(m) of 1.67 microM 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.
authors.affiliation: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nakai
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Inoue
authors.forename: Naoki
authors.initials: N
authors.lastname: Abe
authors.forename: Mai
authors.initials: M
authors.lastname: Hatakeyama
authors.forename: Kin-ya
authors.initials: KY
authors.lastname: Ohta
authors.forename: Masaki
authors.initials: M
authors.lastname: Otagiri
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yuasa
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D018501:Antirheumatic Agents / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004008:Diclofenac / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008727:Methotrexate / Q000378:metabolism*; D018613:Microscopy, Confocal; D009550:Nigericin / Q000494:pharmacology; D058979:Proton-Coupled Folate Transporter; D012460:Sulfasalazine / Q000494:pharmacology; D014162:Transfection
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D018501:Antirheumatic Agents; D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D004008:Diclofenac; D049452:Green Fluorescent Proteins; D012460:Sulfasalazine; D005492:Folic Acid; D009550:Nigericin; D007213:Indomethacin; D008727:Methotrexate