RESUMO
This study revealed the importance of serine 318 (S318) residue for proton-coupled folate transporter (PCFT, SLC46A1) functioning. Substitution of S318 with arginine or lysine impaired transport of methotrexate (MTX), but substitution with alanine (has a simple side chain structure), or cysteine (structurally similar to serine), had no significant effect on MTX transport. The initial uptake rate of MTX by S318A and S318C mutant at pH 5.0, followed by Michaelis-Menten kinetics with a Km value of approximately 2.3 µM (for S318A) and 2.9 µM (for S318C), was similar to that of the wild-type. The normalized Vmax value of the S318A mutant, calculated by dividing the Vmax value by the Western blot protein band's relative intensity, was approximately 2-fold greater than that of the wild-type. The normalized Vmax value of the S318C mutant was approximately 0.8-fold smaller than the wild-type. Results obtained showed that the substitution of S318 with basic amino acid residues results in the loss of transport activity, even though PCFT mutants are expressed at the cell membrane. Alternatively, the substitution of S318 with neutral amino acids did not significantly affect the transport function of PCFT.