Molecular Determinants of Substrate Selectivity in OCT3 (SLC22A3)

Abstract

Organic cation transporters (OCTs) are polyspecific, facilitative transporters with major roles in metabolite and drug clearance. OCT1 and OCT2 are expressed in hepatocytes and proximal tubular cells, whereas OCT3 is in astrocytes. OCT1 and OCT2 have been well-studied, but the pharmacology and structure-function relationships of OCT3 remain obscure. Previously, we screened polyamine compounds for transport and competition against model substrate methyl-4-phenylpyridinium (MPP+) in OCT-expressing Xenopus oocytes, and established that polyamines are transported substrates for OCTs, and that hydrophobicity is a principal requirement for recognition of polycationic substrates and inhibitors. OCT3 exhibited significantly higher requirements for hydrophobicity than other isoforms. Here, we investigated the implications of key residues in shaping the distinct selectivity profile of rat and human OCT3. Homology modeling identified a putative hydrophobic cleft in OCTs capable of accommodating a wide variety of structures and that all isoforms share a conserved Asp within this pocket (D478). Replacement of this residue with Arg, Asn or even Glu drastically reduced MPP+ uptake, suggesting that this Asp residue is obligatory for OCT3-mediated transport, and may help stabilize positively charged substrates. Several key residues within the OCT1 and OCT2 binding pocket are not conserved in OCT3, and these differences might account for more stringent requirements in OCT3. Mutations at these sites in OCT3 to the corresponding residue in OCT1 and OCT2 (L166F, F450L and E451Q) intrinsically decreased MPP+ uptake rate. Potent OCT1 and OCT2 blockers tetraethylammonium (TEA) and 1,10-diaminodecane (DA10) interact weakly with wild-type OCT3. There was no enhancement of TEA or DA10 block in mutant OCT3; rather, MPP+ uptake rate increased in presence of these compounds. These results suggest that these residues may help stabilize substrates within the transporter and imply a more complex interaction between substrates than simple competition for a single site.