Mutational Analysis of the Functional Role of Conserved Arginine and Lysine Residues in Transmembrane Domains of the Murine Reduced Folate Carrier

Abstract

The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, 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 most 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. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated 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 major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.

Resistance to antifolates

“…Ser309Phe (eighth TMD) and Glu404Leu (11th TMD) mutations also produced changes in anion sensitivity. Influx was independent of chloride over a broad range of chloride concentrations with a simulatory effect below 50 mm (Zhao et al, 1999a;Sharina et al, 2001). …”

Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates

“…The TMD2 exofacial loop boundary region has been slightly modified from a previous report (30) based on biotin maleimide and stibenedisulfonate maleimide labeling results for the hamster (37) and human (data not shown) transporters as described in the text. Structurally or functionally important amino acids, as determined from published mutagenesis studies (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), are shown as red circles. Deletion of residues 204 -217 was previously found (14) to abolish transport by hRFC.…”

“…Replacement of Arg-133 in hRFC (with Glu, His, or Leu) or Arg-373 in hamster RFC (with His, Gln, Asn, or Ala) or substitution of leucine at the homologous positions in murine RFC (Arg-131 and Arg-373, respectively) significantly inhibited Mtx transport activity (20,23,26). However, replacement of arginine at these positions with a positively charged lysine was reasonably well tolerated (20,26).…”

“…However, replacement of the deleted segments with nonhomologous 73-or 84-amino acid segments of the structurally analogous thiamine transporter, SLC19A2, restored transport, although maximal activity had an absolute requirement for the conserved 204 -214 peptide. From mutant studies, highly conserved amino acids in or flanking the TMDs including Gly-44, Glu-45, Ser-46, Ile-48, Asp-88, Val-106, Trp-107, Ser-127, Ala-132, Arg-133, Ser-313, Arg-373, and Lys-411 were implicated as functionally or structurally important (the numbers refer to the positions in the hRFC molecule) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Most recently, by co-expression and N-hydroxysuccinimide Mtx radioaffinity labeling of hRFC TMD1-6 and TMD7-12 half-molecules combined with 2-nitro-5-thiocyanatobenzoic acid cleavage adjacent to cysteine residues, a substrate binding region was localized to between amino acids 394 and 457, corresponding to TMDs 11 and 12 (30).…”

The H+‐dependent reduced folate carrier 1 of humans and the sodium‐dependent methotrexate carrier‐1 of the rat are orthologs

“…However, of the 33 amino acids that have been identified as important for Mtx transport by RFC1 [14][15][16][17][18][19] only Ser 317 was exchanged for an alanine in rat MTX-1 (Fig. 1).…”