RT Journal Article SR Electronic T1 Role of the Amino Acid 45 Residue in Reduced Folate Carrier Function and Ion-Dependent Transport as Characterized by Site-Directed Mutagenesis JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 317 OP 323 VO 57 IS 2 A1 Rongbao Zhao A1 Feng Gao A1 Pi Jun Wang A1 I. David Goldman YR 2000 UL http://molpharm.aspetjournals.org/content/57/2/317.abstract AB In previous reports, an E45K mutation in reduced folate carrier (RFC1) resulted in marked substrate-specific changes in folate binding and the induction of an obligatory inorganic anion requirement for carrier function. In this study, site-directed mutagenesis was employed to further characterize the role of glutamate-45 in carrier function by replacement with glutamine, arginine, aspartate, leucine, or tryptophan followed by tranfection of the mutated cDNAs into the MTXrA line, which lacks a functional endogenous carrier. Alterations in transport function with amino acid substitutions at this residue were not charge related. Hence, E45Q, E45R, and E45K all 1) increased carrier affinity for 5-formyltetrahydrofolate ∼4-fold, 2) increased affinity for folic acid ∼6- to 10-fold, 3) did not change 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 folic acid. Finally, chloride-dependent influx was only noted in the E45R mutant. These data further substantiate the important role that glutamate-45 plays in the selectivity of binding of folates to RFC1 and establish that it is the addition of a positive charge at this site and not the loss of a negative charge that results in the induced anion dependence. These and other studies 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. The American Society for Pharmacology and Experimental Therapeutics