title: Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function.
abstract: Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln(420). A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser(135) was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC.
authors.affiliation: Department of Medicine, Albert Einstein College of Medicine, and the Albert Einstein Cancer Research Center, Bronx, New York 10461, USA.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Wang
authors.forename: Arun
authors.initials: A
authors.lastname: Rajgopal
authors.forename: I David
authors.initials: ID
authors.lastname: Goldman
authors.forename: Rongbao
authors.initials: R
authors.lastname: Zhao
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018388:Codon, Terminator / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D058978:Reduced Folate Carrier Protein; D014162:Transfection
chemical_list: D018388:Codon, Terminator; D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D005492:Folic Acid; D008727:Methotrexate