Cloning/characterization of the canine organic anion transporting polypeptide 1b4 (Oatp1b4) and classification of the canine OATP/SLCO members

Abstract

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076 bp, encoding a 692-amino acid protein with a molecular mass of ∼ 85 kDa. The Oatp1b4 gene is approximately 61 kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine^2,5]enkephalin (DPDPE), estradiol-17β-glucuronide (E17βG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17βG and estrone-3-sulfate transports were monophasic with K_m values of 5 ± 1 µM and 33 ± 4 µM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

Introduction

Organic anion transporting polypeptides (humans: OATP, other species: Oatp) are members of a large superfamily of mainly polyspecific uptake transporters (Hagenbuch and Meier, 2003) that are expressed in various organs. In humans there are 11 OATPs that have been classified into 6 different families (Hagenbuch and Meier, 2004). Family 1B contains the two liver-specific OATP1B1 (Abe et al., 1999, Hsiang et al., 1999, König et al., 2000b) and OATP1B3 (Abe et al., 2001, König et al., 2000a) that are involved in the elimination of a broad range of xenobiotics including numerous drugs (Hagenbuch and Gui, 2008, König et al., 2006). Mice and rats have only a single transporter in the OATP1B family that is the ortholog of human OATP1B1 and OATP1B3 which arose through gene duplication after divergence of the rodents (Hagenbuch and Meier, 2004). Similarly, comparison of the predicted Oatps in the genomes of the dog, the cow and the horse suggest that there is only a single Oatp in family 1B while in rhesus monkeys, a species much closer related to humans, both Oatp1b1 and Oatp1b3 orthologs have been found. Besides the transporters in mice and rats, Oatps have been cloned from cow (Geyer et al., 2004), horse (Brown et al., 2007) and quail (Nakao et al., 2006). In the dog, a species that is frequently used during preclinical safety and pharmacokinetic studies (Abbott et al., 2004, Hartman et al., 1996, Shen et al., 2006), the OATP1B1/OATP1B3 ortholog has only been predicted based on the DNA sequence that became available through the dog genome project (Lindblad-Toh et al., 2005). However, in order to compare results obtained in dogs to other species, in particular to humans, it is important to isolate and characterize the dog liver OATP1B family member.

Here we report the isolation and characterization of a canine organic anion transporting polypeptide that based on the phylogenetic relationship to other OATPs/Oatps is called Oatp1b4. After its isolation from a dog liver cDNA library, dog Oatp1b4 was functionally characterized using transient expression in human embryonic kidney (HEK293) cells.