Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells

This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-β (F2-MTX^rA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-β and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 μM MTX in F2-MTX^rA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-β exhibited an influx K_t one-third, and an influx V_max one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTX^rA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTX^rA cells. The inhibitory effects of these anions on transport in F2-MTX^rA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTX^rA cells. Substitution of Cl^- with isethionate or NO₃^- partially restored influx in the latter cells, whereas SO₄^2- was inhibitory. Anions enhanced MTX binding to folate receptor-β with isethionate > SO₄^2- > Cl^-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTX^rA cells, respectively. The data indicate that folate receptor-β-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.