Abstract
Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are responsible for the hepatic uptake of organic anions. They share similar sequences and structures with 12 putative transmembrane domains (TMs). Their substrate specificities are very broad and overlap each other, whereas each transporter specifically recognizes certain substrates. Because the homology of the amino acid sequence in the latter part of OATP1B1 and OATP1B3 is relatively low, to determine which TMs in the latter part of OATP1B1 are important for its substrate recognition, we constructed several cell lines expressing chimeric transporters in which some TMs of OATP1B1 were substituted with those of OATP1B3, and we investigated the transport kinetics of estrone-3-sulfate (E-sul; a substrate preferentially accepted by OATP1B1) and estradiol-17β-d-glucuronide (EG; a substrate accepted by both transporters). As the number of substituted TMs at the N terminus with those of OATP1B3 increased, the Km value of E-sul greatly increased and its uptake clearance decreased. The substitution of TM7 or TM9 of OATP1B1 with that of OATP1B3 (named 1B1-TM7 or 1B1-TM9) did not change the transport kinetics of EG, whereas the Km value of E-sul in 1B1-TM9 increased 7.4-fold. Conversely, the substitution of TM8 resulted in an 18-fold increase in the Km value of E-sul and abolished the transporter-mediated uptake of EG. These results suggest that TM8 in OATP1B1 is critical for the substrate recognition of both E-sul and EG and that TM9 is important for the recognition of E-sul, whereas it is interchangeable with that of OATP1B3 for EG transport.
Footnotes
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This study was supported by a grant-in-aid for scientific research (A) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [Grant KAKENHI: 20249008].
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doi:10.1124/jpet.108.148411.
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ABBREVIATIONS: OATP, organic anion transporting polypeptide; TM, transmembrane domain; EG, estradiol-17β-d-glucuronide; E-sul, estrone-3-sulfate; SN-38, 7-ethyl-10-hydroxycamptothecin; CCK-8, cholecystokinin octapeptide; HEK, human embryonic kidney; PBS, phosphate-buffered saline; TTBS, Tris-buffered saline containing 0.05% Tween 20.
- Received November 7, 2008.
- Accepted February 23, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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