Figure 1.
Polyamine, agmatine, TEA and carnitine uptake activities by various organic cation transporters expressed in HEK293 cells.
(A) Polyamine and agmatine transport activities of HEK293 cells transfected with pCMV-XL4, pcDNA3.1(+) or pcDNA3 including a gene for hOCT or hMATE were measured as described in Materials and Methods. Activity of HEK293 cells transfected with the vector only was subtracted from the activity of HEK293 cells transfected with hOCT1, hOCT2, hOCT2-A, hOCT3, hOCTN1, hOCTN2, hMATE1 or hMATE2-K cDNA. (B) Time course of polyamine and agmatine uptake by hOCT2 or hOCT3. ○, pCMV6-XL4 plasmid containing hOCT2 or hOCT3 cDNA; •, pCMV6-XL4 vector. Data shown are the mean ± S.E. of triplicate determinations. (C) TEA and carnitine uptake activities by various organic cation transporters were measured as described in Materials and Methods. (D) Presence of hOCTs and hMATEs in kidney [35] and their polyamine and agmatine uptake activities are summarized.
Figure 2.
Determination of the Km and Vmax values of polyamine and agmatine uptake by hOCT2 and hOCT3.
(A and B) Polyamine and agmatine uptake activities were measured as described in the legend of Fig. 1 using various concentrations of substrates. The Km and Vmax values for putrescine, agmatine and spermidine by hOCT2 and hOCT3 determined by Lineweaver-Burk plot are also shown. (C) Spermidine uptake by hOCT2 was measured at various pHs, and the Km and Vmax values were calculated at pH 8.6.
Figure 3.
Effect of non-labeled polyamines, agmatine, γ-aminobutyric acid (GABA) and several amino acids on polyamine and agmatine uptake by hOCT2 and hOCT3.
Polyamine and agmatine transport activities were measured in the presence of a 20-fold concentration (10 mM) of non-labeled polyamines, agmatine, GABA and several amino acids, and are shown as percent activity of control (None). Putrescine, agmatine and spermidine uptake activity by HEK293 cells transfected with pCMV6-XL4/hOCT2 was 258, 257, 128 pmol/min/mg protein, respectively in the presence of 0.5 mM [14C]putrescine, [14C]agmatine or [14C]spermidne as the substrate. Agmatine uptake by hOCT3 was 223 pmol/min/mg protein. Data shown are the mean ± S.E. of triplicate determinations. *p<0.05; **p<0.01.
Figure 4.
Identification of amino acid residues in hOCT2 involved in putrescine, agmatine and spermidine uptake by site-directed mutagenesis.
(A) Putrescine, agmatine and spermidine uptake activities of hOCT2-myc-His mutants. Asp and Glu in hOCT2 were mutated to Asn and Gln, respectively. Putrescine, agmatine and spermidine uptake activities of the mutants are shown as percent activity of control (WT). Putrescine, agmatine and spermidine uptake by HEK293 cells transfected with pcDNA3.1/hOCT2-myc-His were 202, 205 and 120 pmol/min/mg protein, respectively. Values are means ± S.E. of triplicate determinations. *p<0.05; **p<0.01. (B) Expression level of hOCT2-myc-His mutant proteins in HEK293 cells. Western blot analysis was performed as described in the Materials and Methods. Relative amount of the expression level of hOCT2-myc-His mutant proteins on plasma membrane is shown as mean of triplicate determinations.
Figure 5.
Functional acidic amino acid residues in hOCT2.
Putative secondary structure models of hOCT2 and hOCT2-A are shown. Functional acidic amino acid residues in hOCT2 are shown in red, blue, green and violet. Asp427, Glu448, Glu456, Asp475 and Glu516 were involved in putrescine, agmatine and spermidine uptake activity (red), and Glu524 and Glu530 in putrescine and spermidine uptake (blue). Glu528 and Glu540 were involved in putrescine uptake activity (green), and Asp551 in spermidine uptake activity (violet).
Figure 6.
Modeling of spermidine binding to hOCT2.
(A) Amino acid residues involved in transport activity of the three amines (red) and of putrescine and spermidine (blue) are shown on α-helices 1 to 12, whose relative positions were constructed based on the structure model of hOCT2 [31]. (B) View of the model from the cytoplasmic aspect of hOCT2. The 12 α-helices (blue) of hOCT2, and spermidine (white, proton; orange, carbon; blue, nitrogen) bound to hOCT2 are shown. (C and D) Side view of the model, with the cytoplasmic aspect of the protein directed toward the bottom. Spermidine binding site on hOCT2 is shown in more detail together with the functional amino acids in D. The distance (Å) between the amino acid residues involved in the interaction with spermidine and the primary amino- or secondary amine-group of spermidine is shown.