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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function.

In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in MTXrA-E45K cells was characterized by a 12- and 3.6-fold decrease in influx Vmax and Kt respectively, relative to L1210 cells. The folic acid influx Ki in L1210 cells was more than 50-fold greater than that of MTX based upon inhibition of 5-CHO-THF influx. In comparison, the mutated carrier had comparable affinities for folic acid and MTX in MTXrA-E45K cells due to a 7-fold decrease in the folic acid influx Ki and 7-fold increase in the MTX influx Ki. Transport via native RFC1 is inhibited by a variety of anions in L1210 cells associated with an increase in influx Kt. However, influx of 5-CHO-THF in MTXrA-E45K cells in a HEPES buffer (9 mM chloride) was decreased by 70% due to a 3-fold fall in the Vmax. In the complete absence of chloride (K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10% that in HBS buffer. 5-CHO-THF influx was restored by addition of chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP which were all inhibitory over a broad range of concentrations. The data suggest that substitution of a positive for a negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when cells are placed under selective pressure with antifolates that utilize RFC1 as the major route of entry into mammalian cells.[1]


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