Role of the amino acid 45 residue in reduced folate carrier function and ion-dependent transport as characterized by site-directed mutagenesis

In previous reports, an E45K mutation in reduced folate carrier (RFC1) resu lted in marked substrate-specific changes in folate binding and the inducti on of an obligatory inorganic anion requirement for carrier function. In th is study, site-directed mutagenesis was employed to further characterize th e role of glutamate-45 in carrier function by replacement with glutamine, a rginine, aspartate, leucine, or tryptophan followed by tranfection of the m utated cDNAs into the MTX(r)A line, which lacks a functional endogenous car rier. Alterations in transport function with amino acid substitutions at th is residue were not charge related. Hence, E45Q, E45R, and E45K all 1) incr eased carrier affinity for 5-formyltetrahydrofolate similar to 4-fold, 2) i ncreased affinity for folic acid similar to 6- to 10-fold, 3) did not chang e affinity for 5- methyltetrahydrofolate, and 4) except for E45R decreased affinity for methotrexate (2- to 3-fold). In contrast, mutations E45D, E45L , and E45W generally reduced affinity for all these folates except for foli c acid. Finally, chloride-dependent influx was only noted in the E45R mutan t. These data further substantiate the important role that glutamate-45 pla ys in the selectivity of binding of folates to RFC1 and establish that it i s the addition of a positive charge at this site and not the loss of a nega tive charge that results in the induced anion dependence. These and other s tudies indicate that mutations in the first transmembrane domain can have a markedly selective impact on the affinity of RFC1 for folate compounds and in particularly a highly salutary effect on binding of the oxidized folate , folic acid.