The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical antifolates such as methotrexate (Mtx) and pemetrexed into mammalian cells. RFC is characterized by 12 stretches of mostly hydrophobic, alpha helix-promoting amino acids, internally oriented N- and C-termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. In order to understand the molecular mechanism of folate and anti-folate transport, it is essential to identify key amino acids or domains involved in substrate binding and membrane translocation. By co-expression and N-hydroxysuccinimide Mtx radioaffinity labeling of hRFC TMD1-6 and TMD7-12 half molecules, a critical substrate binding domain was previously localized to within TMDs 7-12 (J. Biol. Chem. 279: 46755, 2004). Localization of this binding domain was further refined to TMDs 9-12 by enzymic digestion with endoproteinase GluC and LysC. A transport competent cysteine (Cys)-less hRFC was used as a template to prepare 42 consecutive single Cys-substituted mutants from Leu379 to Asp420, including all of TMD11 and the highly conserved connecting loop domain between TMDs 10 and 11. The Cys mutant hRFC constructs were transfected into RFC null Chinese-hamster ovary (CHO) cells. All 42 single Cys-substituted hRFC constructs were expressed in CHO cells and mutant hRFC proteins were detected on Western blots. With the exception of Phe400Cys and Gly401Cys hRFCs, all Cys mutants showed high level transport activity when assayed with radioactive Mtx. Treatment with the small, water soluble thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulfonate (MTSES), had no effect on the hRFC activity for Cys-less hRFC or the majority of the Cys-substituted mutants. However, MTSES significantly inhibited (>40%) Mtx uptake by the Thr408Cys and Thr412Cys hRFC mutants. Loss of transport activity by Thr408Cys and Thr412Cys hRFCs was largely abolished in the presence of excess leucovorin [(6R,S)-5-formyltetrahydrofolate], another hRFC transport substrate. Our results strongly suggest that Thr408 and Thr412, localized in TMD11, are aqueous accessible and that TMD11 is likely a structural and/or functional component of the putative hRFC transmembrane channel for anionic folate and antifolate substrates.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]