Human intestinal folate transport: Cloning, expression, and distribution of complementary RNA

Background & Aims: Despite intensive investigations very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. Methods: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. Results: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pi = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 mu mol/L; maximum velocity = 128 +/- 3 fmol . h(-1). oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (K-i = 0.84 mu mol/L, 0.71 mu mol/L, and 10 mu mol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (K-i = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. Conclusions: These results represent the first molecular characterization of a human small intestinal folate carrier.