Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier

A phenylalanine substitution for serine in the reduced folate carrier at re sidue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cel l line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folat e source. The transport characteristics of the mutated carrier were studied by transfection into the MTX(r)A line, which lacks endogenous RFC1 functio n. The level of expression of carrier in the cell lines studied was determi ned by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF). In flux of 5-CH3-THF and 5-CHO-THF mediated by RFC1-S309F was 20- and 7-fold g reater than that of MTX, respectively. Consistent with the influx differenc e between 5-CHO-THF and MTX, the growth requirement (EC50) for 5-CHO-THF in MTX(r)A-S309F cells was decreased by a factor of 9, while the MTX IC50 was reduced by a factor of only similar to 2 as compared with the recipient MT X(r)A cells. The decrease in 5-CH3-THF influx mediated by the mutated carri er was attributed to a decrease in the mobility of the 5-CH3-THF-carrier co mplex, since the influx K-t was essentially unchanged. However, the reducti on in 5-CHO-THF and MTX influx was attributed to decreases in both carrier affinity and V-max, although the decline in the MTX influx V-max appeared t o be much greater than for 5-CHO-THF. The inhibitory effect of chloride on S-CHO-THF influx observed for L1210 cells was eliminated in the MTX(r)A-S30 9F line. This study represents another example of a single mutation in RFC1 that markedly impairs MTX influx but partially preserves transport of redu ced folates when cells are selected with 5-CHO-THF as the available folate substrate. The data indicate that residues in the predicted eighth transmem brane domain of RFC1 can play an important role in the selectivity of folat e binding and the mobility of the carrier-substrate complex. (C) 1999 Elsev ier Science Inc.