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 ϩ 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 M and extensively inhibited by reduced folates, such as folinate, 5-methyltetrahydrofolate, and methotrexate (MTX). Sulfobromophthalein and 4,4Ј-diisothiocyanostilbene-2,2Ј-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.

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…Immunoreactive proteins were detected on an Odyssey infrared imager. The figure shows putative tetrameric ((hPCFT) 4 ), trimeric ((hPCFT) 3 ), and dimeric ((hPCFT) 2 ) species, as described under "Results. "…”

“…Because mammalian cells cannot synthesize folates de novo, membrane transport of extracellular folates is essential. Three primary transport routes are involved in folate internalization, including the reduced folate carrier (RFC), 4 the proton-coupled folate transporter (PCFT), and the folate receptors. These transporters differ in terms of mechanism, and each system typically plays a unique role in mediating folate transport across epithelia and into systemic tissues (2).…”

“…The human PCFT (hPCFT; SLC46A1) is a proton-folate symporter that functions optimally at acidic pH by coupling the downhill flow of protons to the uphill transport of folates (3)(4)(5). The role of hPCFT in intestinal folate absorption was established by demonstrating loss-of-function mutations in hPCFT in patients with the rare autosomal inherited disorder, hereditary folate malabsorption (HFM) (5).…”

A mouse model of hereditary folate malabsorption: deletion of the PCFT gene leads to systemic folate deficiency

“…2 Three mammalian folate transporter systems have been described to date in a variety of tissues: (1) the bidirectional reduced folate carrier 1 (RFC1), also known as SLC19A1, 3,4 (2) the glycosyl-phosphatidylinositol-anchored folate receptors (FOLR1, FOLR2, and FOLR4) and one secreted receptor in humans without a mouse homolog (FOLR3), 5 and (3) the human proton coupled folate transporter (PCFT). [6][7][8] The RFC1 transporter is expressed ubiquitously, including the brush-border membrane of epithelial cells in the small intestine. 9 Although RFC1 is necessary for folate transport in erythroid cells, its involvement in intestinal folate uptake has not been confirmed.…”

Folic acid uptake by the human syncytiotrophoblast is affected by gestational diabetes, hyperleptinemia, and TNF-α

“…The concentrations of folate analogs of SITS and of TPP used in the characterization of FA uptake were chosen based on previous work from our group (30,32,43) and others (7,35,44).…”