Functional role of serine 318 of the proton-coupled folate transporter in methotrexate transport |
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| Institution: | 1. Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, 980-8578, Japan;2. Division of Risk Assessment, National Institute of Health Sciences, Tonomachi 3-25-26, Kawasaki-ku, Kanagawa, 210-9501, Japan;3. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan;4. Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan;1. Central Institute for Experimental Animals, Kawasaki, Japan;2. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Japan;1. Department of Pharmacy, Fukuoka City Hospital, Fukuoka City Hospital Organization, Local Incorporated Administrative Agency, Fukuoka, Japan;2. Department of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan;3. Department of Pharmacy, Kyushu University Hospital, Fukuoka, Japan;1. Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo, 105–8512, Japan;2. School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba, 274–8555, Japan;1. Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo, 060-0812, Japan;2. Department of Pharmacy, Hokkaido University Hospital, Kita-14-jo, Nishi-5-chome, Kita-ku, Sapporo, 060-8648, Japan |
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| Abstract: | 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. |
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| Keywords: | Proton-coupled folate transporter Methotrexate Hereditary folate malabsorption HFM"} {"#name":"keyword" "$":{"id":"kwrd0030"} "$$":[{"#name":"text" "_":"Hereditary folate malabsorption PCFT"} {"#name":"keyword" "$":{"id":"kwrd0040"} "$$":[{"#name":"text" "_":"Proton-coupled folate transporter |
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