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GASTROINTESTINAL, 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

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan (Y.N., K.I., N.A., M.H., K.O., H.Y.); Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (M.O.); and Department of Biopharmaceutics, College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan (Y.H.)

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 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.

Address correspondence to: Dr. Hiroaki Yuasa, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. E-mail: yuasa{at}phar.nagoya-cu.ac.jp

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