Received for publication March 13, 2007.
Revised April 18, 2007.
Accepted for publication May 1, 2007.

Functional characterization of human PCFT/HCP1 heterologously expressed in mammalian cells as a folate transporter

Abstract

The functional characteristics of human proton coupled folate transporter/heme carrier protein 1 (hPCFT/HCP1) were investigated. hPCFT/HCP1 expressed transiently in HEK293 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, as well as by MTX. sulfobromophthalein 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 additionally 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.

Key words: HCP1, PCFT, folate, methotrexate, sulfasalazine, transporter

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