Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier

The reduced folate carrier (RFC1) plays a major role in the delivery of fol ates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane do mains. This article explores the role of these residues in transport functi on by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines t ransfected with R131L, R155L, or R366L all lacked activity, despite high le vels of protein expression in the plasma membrane, suggesting the crucial r ole of these amino acid residues in RFC1 function. In several mutant carrie rs, R26L, R42L, and K332L, there was little or no change in the influx K-t value for MTX or influx K-i value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was mos t prominent for K404L, which had 11- and 4-fold increases in influx K-i for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. Th e marked influx stimulation observed with wild-type carrier when extracellu lar chloride was decreased was significantly diminished when influx was med iated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a majo r site of inhibition by chloride in the wild-type carrier. These studies in dicate the important role that some positively charged residues within tran smembrane domains of RFC1 play in RFC1 function.