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.
Highlights
Polyamines are present at millimolar concentrations in both prokaryotic and eukaryotic cells and play important roles in cell growth and differentiation [1,2]
We studied the properties of putrescine, agmatine and spermidine uptake by human organic cation transporter 2 (hOCT2) in detail, and found that the active center of putrescine, agmatine and spermidine uptake are located on a-helices 9 to 12
The level of hOCT2 mutants on plasma membrane was nearly equal, confirming that these amino acid residues are involved in the recognition of substrates. These results indicate that recognition sites of putrescine, agmatine and spermidine on hOCT2 strongly overlap, concordant with the finding that the three amines are transported with similar affinity and velocity
Summary
Polyamines (putrescine, spermidine and spermine) are present at millimolar concentrations in both prokaryotic and eukaryotic cells and play important roles in cell growth and differentiation [1,2]. In Escherichia coli, we far reported the properties of several polyamine transport systems [3,4,5,6] Those are spermidine-preferential and putrescine specific uptake systems as well as PotE (involved in the excretion of putrescine by a putrescine-ornithine antiporter activity) and CadB (involved in the excretion of cadaverine by a cadaverine-lysine antiporter activity). It has been reported that PuuP functions as a putrescine transporter when putrescine is used as an energy source under glucose starvation [10,11] In this case, it was necessary to accumulate high concentrations of putrescine in cells [11]. Since the polyamine metabolizing enzyme spermidine/spermine N1-acetyltransferase is not present in yeast, this likely explains why five kinds of excretion proteins are present
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