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

ArticleinJournal of Pharmacology and Experimental Therapeutics 322(2):469-76 · September 2007with4 Reads
DOI: 10.1124/jpet.107.122606 · Source: PubMed
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.
    • "For proton co-transporters, like those expressed in small intestinal epithelium, optimal transporting activity is usually observed at acidic pH condition which is close to physiological conditions in the intestine. The proton-coupled folate transporter (PCFT), encoded by SLC46A1, incorporates folate by coupling a proton co-transport down the proton gradient at pH 5.5, however, this transport activity is absent at neutral pH (Nakai et al., 2007). Peptide transporters 1 and 2 (PEPT1 and PEPT2, encoded by SLC15A1 and SLC15A2) are similar examples, which mediate absorption small peptides and peptide-like drugs from the intestine and kidney respectively with symport of a proton (Liu et al., 1995; Liang et al., 1995). "
    [Show abstract] [Hide abstract] ABSTRACT: Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles.
    Article · Mar 2015
    • "The initial set of experiments was conducted using the Caco-2 cell as an intestinal epithelial cell model, in which PCFT has been suggested to be responsible for folate uptake. Caco-2 cells were preincubated with myricetin (100 mM) for 1 h and, then, the uptake of folate was evaluated in its absence in the medium under an acidic condition of pH 5.5, where the H þ -dependent folate transporter can operate efficiently [12,13]. As shown inFig. "
    [Show abstract] [Hide abstract] ABSTRACT: Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled folate transporter (PCFT), the uptake of folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 μM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of folate uptake, which was attributable to a reduction in the maximum transport rate (Vmax). Although the affinity of the transporter for folate was increased at the same time as indicated by a reduction in the Michaelis constant (Km), the change in Km was overwhelmed in extent by that in Vmax. Consistent with the finding, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in Vmax and Km by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of folate and also antifolate drugs. Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
    Article · Feb 2014
    • "The transport properties of PCFT have been characterized in transfected cell lines and in oocytes microinjected with PCFT cRNAs (complementary RNAs) (Qiu et al., 2006; Deng et al., 2009). In human embryonic kidney 293 cells, transport activity was maximal at pH 4.5 (Nakai et al., 2007), although it was appreciable up to pH 6.5 (). With further increased pH, there is a dramatic loss of transport activity such that, above pH 7, transport is very low. "
    [Show abstract] [Hide abstract] ABSTRACT: This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (SLC19A1) and the proton-coupled folate transporter (PCFT) (SLC46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of heath disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer such as methotrexate and pralatrexate are transported by RFC and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties, and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases.
    Full-text · Article · Jan 2014
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