title: Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1.
abstract: The liver-specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone-3-sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site-directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1-mediated estrone-3-sulfate transport. Comparison of the kinetics of estrone-3-sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [D-penicillamine(2,5)]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Chunshan
authors.initials: C
authors.lastname: Gui
authors.forename: Bruno
authors.initials: B
authors.lastname: Hagenbuch
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D002460:Cell Line; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D027361:Organic Anion Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013379:Substrate Specificity / Q000235:genetics*
chemical_list: D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; D029362:Organic Anion Transporters, Sodium-Independent; D011993:Recombinant Fusion Proteins; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D004970:Estrone; C017296:estrone sulfate