Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

Abstract

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.

Stereoselective Recognition of Amethopterin Enantiomers by the Rat Proton-Coupled Folate Transporter

“…The K m value (2.14 µM) determined for L-MTX in the current study was found to be similar to those reported previously in a range of different systems, including 0.6-2.6 µM in HepG2 cells transfected with rPCFT cDNA, 21) 5.7 µM in Xenopus oocytes injected with rPCFT cRNA, 22) and 1.0-4.6 µM in HepG2 cells transfected with mouse PCFT cDNA. 21) The Dixon plot showed that the uptake of L-MTX was inhibited in a competitive manner by unlabeled L-MTX, which gave a K i value of 1.25 µM.…”

“…21) The Dixon plot showed that the uptake of L-MTX was inhibited in a competitive manner by unlabeled L-MTX, which gave a K i value of 1.25 µM. It is noteworthy that this K i value is almost equal to the K m value (2.14 µM) of L-MTX.…”

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

“…For >60 y, folate analogs that inhibit intracellular folate-using enzymes, termed antifolates, have been developed to treat a variety of cancer types and inflammatory diseases (8)(9)(10)(11)(12)(13)(14). More recently, the emphasis in folatemediated drug therapy has shifted to obtain a better understanding of transport mechanisms of antifolates because dose-limiting toxicities arise from their transport via RFC, and possibly PCFT, into normal cells (6,11,(15)(16)(17). To date, antifolates approved for clinical use are transported primarily via RFC, although one of the most recently developed antifolates, pemetrexed (PMX), may also transit through PCFT (6,11,(18)(19)(20)(21).…”

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

“…There are two N-glycosylation consensus sites (Asn-58 and Asn-68) in the hPCFT loop domain between transmembrane domains (TMDs) 1 and 2. Based on results with substituted cysteine-scanning mutagenesis of a Cys-less hPCFT and accessibilities to thiol-reactive agents (27) and on studies of hemagglutinin (HA) epitope accessibilities to epitope-specific antibody (28,29), hPCFT has 12 TMDs with N and C termini directed to the cytosol. Structurally and/or functionally important residues have been identified in hPCFT and include (with possible roles) Glu-185 (which is required for proton coupling) (30), His-247 and Ser-172 (which modulate folate and proton access to a high affinity binding site) (31), His-281 (which is important for substrate binding) (31), Arg-376 (which impacts proton and substrate binding) (12), and Asp-156 (which contributes to PCFT protein stability) (13) (Fig.…”