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.

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3

“…These results suggest that this aspartate residue (Asp-473 in rOCT and Asp-478 in hOCT3) is an absolute requirement for OCT3-mediated transport and may participate in the stabilization of the positively charged cationic moiety of MPP ϩ in particular, and OCT substrates in general. It has been observed previously that, in OCT1 and OCT2, Asp-475 is critical for affinity and substrate selectivity (28,35,37). Additionally, the observed reduction of MPP ϩ uptake in the charge replacement mutation (Asp to Glu), in both OCT3 (Fig.…”

“…2), in support of the hypothesis that this TM11 Asp functions in all OCTs as the singular binding site for the cationic region of transported substrates (32). Although some models of substrate binding have been generated, in particular of spermidine binding to hOCT2 (37), questions regarding the exact role of this conserved residue in substrate recognition persist. In particular, the present study suggests that simply the presence of a negative charge in that position is not sufficient to accomplish substrate binding and translocation, because the substitution of Glu for Asp results in a complete loss of function as well (Fig.…”

“…5, A-G, and Fig. 6, B Each mutation in rat or human OCT3 represents one of many residues that purportedly contribute to determining substrate selectivity in OCTs (21,22,28,29,32,34,35,37). Therefore, it is unlikely that any one mutant can completely reproduce the blocker potency profile of OCT1, and we cannot discard the possibility that residues other than the ones investigated here play critical roles in shaping OCT3-substrate interactions.…”

“…Additionally, the observed reduction of MPP ϩ uptake in the charge replacement mutation (Asp to Glu), in both OCT3 (Fig. 2) as well as OCT1 and OCT2 (35,37), further implies that this aspartate residue may not only contribute to an electrostatic stabilization of positively charged substrates, but also that the steric and spatial orientation of this interaction is critical for the transport mechanism of both the rat and human OCT3.…”

“…A Conserved Aspartate in TM11 is Essential for OCT3 Transport Activity-OCT1 and OCT2 have been well characterized biochemically in numerous mutagenesis studies that have identified an aspartate residue (Asp-475 in rOCT1) in predicted transmembrane helix 11 (TM11) to be critically important for determining affinity and selectivity of substrates in OCT1 and OCT2 (28,35,37). The mechanism of substrate recognition and transport in OCT3, on the other hand, is less understood as the set of biochemical data collected in systems expressing OCT3 is, at present, extremely limited.…”