Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis

Abstract

Listen

Translate article

Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R–, K411E–, and K411L–hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22–36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H 4PteGlu) uptake levels for both the K411E– and K411L–hRFCs were also impaired (∼33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R–hRFC. While loss of MTX transport activity for the K411R–hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R–hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H 4PteGlu compared to K43-6 cells. The 5-CHO-H 4PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R–hRFC, however, this response was decreased for the K411E– and K411L–hRFCs. The preservation of low levels of transport for the K411E– and K411L–hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H 4PteGlu over MTX for K411R–hRFC.