pH dependence of methotrexate transport by the reduced folate carrier and the folate receptor in L1210 leukemia cells. Further evidence for a third route mediated at low pH.

F2-MTX'A is an L1210 leukemia cell line with a functional detect in the reduced folate carrier and high level expression of folate receptor beta. The pH dependence of methotrexate (MTX) influx by folate receptor beta in F2-MTX'A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by folate receptor beta had a pH optimum of 6.5, whereas influx mediated by the reduced folate carrier showed a pH optimum of 7.5. Increased folate receptor beta-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTX'A cells proceeded by another mechanism. This transport route became active at pH < 7.5, operated optimally at pH 6.0 to 6.5, and, unlike folate receptor beta-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a Ki of 5.3 microM and a Vmax of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a Ki of 148 microM, and had similar relative affinities for folic acid, leucovorin, and 5 methyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTX'A cells, optimal at low pH and distinct from the reduced folate carrier or the folate receptor.