Stereoselective Transport of Human His27- and Arg27-Reduced Folate Carrier

Abstract

1-4)The cDNA of the RFC has been cloned in mice, hamsters, and humans, and its transport characteristics have been revealed.5-11) RFC is expressed in the plasma membrane of a variety of different cells and transports reduced folates, with the maximal level of transport being observed at neutral pH. 2,4,12) In contrast, PCFT is expressed in the epithelial cells of a range of different tissues, including the liver, kidney, and placenta. Among these tissues, the expression of PCFT appears to be at its greatest in the small intestine, 3,13) where it is expressed in the apical membrane of epithelial cells and is involved in the absorption of reduced and oxidized folates using a H + gradient as its driving force. 12)Methotrexate (L-amethopterin, L-MTX) is used clinically as an antineoplastic and antirheumatic drug. Orally administered L-MTX is actively absorbed by PCFT in the small intestine, with the process being driven by a H + gradient, because its molecular structure is similar to that of folic acid (FA). D-Amethopterin (D-MTX) is the optical isomer of L-MTX, and the absorption of D-MTX from the human small intestine is significantly lower than that of L-MTX. It has been reported that oral absorption of L-MTX is approximately 40-fold greater than that of D-MTX. 14) In our previous studies using hPCFTexpressing HEK293 cells, the transport of the MTX enantiomers by PCFT was revealed to be highly stereoselective with the uptake clearance of L-MTX being approximately 40-fold greater than that of D-MTX. 15)RFC is expressed in the body to a much greater extent than PCFT, and is responsible for the uptake of reduced folates in a number of different cell types.16) It is well known that there are differences in the transport characteristics of PCFT and RFC. For example, PCFT-mediated transport occurs much more readily at acidic pH, whereas RFC-mediated transport is optimal at neutral pH. PCFT transports both oxidized and reduced folates, whereas RFC only transports reduced folates. As mentioned above, the transport of the different enantiomers of amethopterin by PCFT is highly stereoselective. Interestingly, however, the stereoselectivity of RFC-mediated transport has not yet been examined. Although the homology of the amino acid sequences between RFC and PCFT is only about 10%, both of these transporters have common substrates (i.e., reduced folates). With this in mind, we examined the stereoselective transport of RFC using the enantiomers of amethopterin, which possess a similar structure to that of the reduced folates.A single nucleotide polymorphism (SNP) of hRFC has been reported, involving the substitution of a guanine with an adenine (G80A), with the change resulting in the replacement of a histidine residue (H27) with an arginine (R27). 17)