title: Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2.
abstract: Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Kyohei
authors.initials: K
authors.lastname: Higashi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Imamura
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fudo
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uemura
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; Amine Pharma Research Institute, Innovation Plaza at Chiba University, Chiba, Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Saiki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Tyuji
authors.initials: T
authors.lastname: Hoshino
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Toida
authors.affiliation: Faculty of Pharmacy, Chiba Institute of Science, Chiba, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kashiwagi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; Amine Pharma Research Institute, Innovation Plaza at Chiba University, Chiba, Japan.
authors.forename: Kazuei
authors.initials: K
authors.lastname: Igarashi
mesh_terms: D000376:Agmatine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D011073:Polyamines / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011700:Putrescine / Q000378:metabolism
chemical_list: D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D011073:Polyamines; C491013:SLC22A2 protein, human; D000376:Agmatine; D011700:Putrescine