Amino Acid Residues in Transmembrane Domain 10 of Organic Anion Transporting Polypeptide 1B3 Are Critical for Cholecystokinin Octapeptide Transport

Abstract

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multi-specific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain twelve transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide . To determine which structural domains/residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells and determined uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in dramatically reduced CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using sitedirected mutagenesis, we identified three key residues within TM10, namely Y537, S545 and T550. When we replaced these residues by the corresponding amino acid residues found in OATP1B1, CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showedthat the K m values for CCK-8 transport in the TM10-replacement and triple mutant were only 1.3 and 1.1 μM, respectively as compared to 16.3 μM for wild-type OATP1B3. Similarly, the V max values dropped from 495.5 pmol/normalized mg/min for wild-type OATP1B3 to 13.3 and 19.0 for the TM10-replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545 and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.The mammalian liver is the major detoxification organ of the body and the hepatic sinusoidal membrane is equipped with transporters that mediate influx and efflux of endogenous and xenobiotic compounds. Uptake transporters belong to the superfamily of solute carriers (SLC) (1) which mediate the first step of the hepatic elimination by facilitating hepatic uptake of those compounds from the portal vein. In humans, organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate the sodium-independent transport of a wide range of † This work was supported by National Institute of Health grants RR021940, and GM077336. amphipathic organic compounds including numerous drugs and other xenobiotics (2). There are 11 human OATPs (3,4) with the polyspecific OATP1B3 and 1B1 being predominantly expressed at the basolateral membrane of hepatocytes (5-9), indicating their significant roles in the hepatic clearance of amphipathic organic compounds. OATP1B3 shares 80% amino acid sequence identity with OATP1B1. Recent findings for rat Oatp1a1 suggest that all OATPs comprise twelve putative transmembrane domains (TMs) (10). As expected from their high sequence similarity, OATP1B3 and 1B1 exhibit overlapping transport activities for an array of substances including bile salts, unconjugated and conjugated bilirubin, bromosulfophthalein (...

Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

“…TM10 has before been suggested to be important for OATP-mediated transport. Besides our results with OATP1B3, 15 cysteine-scanning mutagenesis experiments of rat Oatp2b1 indicated that TM10 is part of the substrate binding site. 19 Thus, TM10 seems to be important not only in the OATP1 subfamily but also in the OATP2 subfamily.…”

“…This is different from our findings with OATP1B3 where chimera 1B3_TM10 only impaired transport of CCK-8, whereas uptake of DPDPE and taurocholate was normal or even increased. 15 Helical wheel analysis of TM10 revealed that residue L545 is located on one side of TM10, whereas residues F546, L550, and S554 are located on the other side of the helix (Fig. 8).…”

“…8). According to our model for OATP1B3, 15 residue L545 faces the substrate translocation pathway, whereas residues F546, L550, and S554 face inside the helical bundle. Thus, only L545 of the quadruple mutant might interact with the substrate directly.…”

“…15. Plasmids containing the cDNAs of OATP1B1, OATP1B3, and chimeras or mutants were transiently transfected into HEK293 cells using Lipofectamine 2000 following the manufacturer's instruction.…”

“…11,13,14 In a previous study, we showed that the three amino acid residues Y537, S545, and T550 in TM10 of OATP1B3 are critical for CCK-8 transport. 15 A recently published study demonstrated that TM8 and TM9 are critical for substrate recognition for OATP1B1. 16 However, in this study, we extend the constructs to include TM10 and report our results that demonstrate the importance of TM10 for the function of OATP1B1.…”