Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1).
The hepatic uptake transporters OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) mediate the uptake of endogenous metabolites and drugs from blood into hepatocytes. Alterations of transport function are accompanied with variations in drug plasma concentrations and the risk of adverse drug effects. Thus, knowledge on amino acids determining substrate recognition or transport kinetics are important to predict alterations in transport kinetics. Therefore, we analyzed the charged amino acids His54 and Tyr169, both located at the extracellular entry of the predicted transmembrane pore of OATP1B1. Based on a computational analysis we established HEK293 cell lines overexpressing the mutant OATP1B1 proteins HEK-OATP1B1p.H54Q, -p.H54A, -p.Y169H and -p.Y169A and analyzed protein expression, localization and transport kinetics of the four OATP1B1 substrates bromosulfophthalein, estradiaol-17β-glucuronide, taurocholate and pravastatin. Consequences on transport were detected for all mutants and these were different for each amino acid exchange and for each substrate tested. For example, the exchange H54Q resulted in reduced transport for BSP (78% of wildtype OATP1B1 transport at 0.05μM, P<0.01) with reduced affinity to this substrate (Kmvalue increases from 0.76μM to 8.04μM) but in stimulated E217βG transport (138% compared to wildtype transport at 10μM, P<0.001). Investigating amino acid exchanges located at the extracellular entry of the transport pore of the OATP1B1 protein we demonstrated that these residues are involved in modulating transport kinetics and this participation strongly depends on the substrate and not on the physicochemical character of the investigated amino acid.
Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
Moritz
M
Gruetz
Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
Heinrich
H
Sticht
Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
Hartmut
H
Glaeser
Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
Martin F
MF
Fromm
Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany. Electronic address: joerg.koenig@fau.de.
Jörg
J
König
D000595:Amino Acid Sequence; D001692:Biological Transport; D004958:Estradiol / Q000031:analogs & derivatives* / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017035:Pravastatin / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013448:Sulfobromophthalein / Q000737:chemistry*; D013656:Taurocholic Acid / Q000737:chemistry*; D019076:Transgenes
D027381:Liver-Specific Organic Anion Transporter 1; D011994:Recombinant Proteins; C503999:SLCO1B1 protein, human; D013448:Sulfobromophthalein; C025483:estradiol-17 beta-glucuronide; D004958:Estradiol; D013656:Taurocholic Acid; D017035:Pravastatin
Drug transport; Hepatocyte; Membrane transport; Site-directed mutagenesis; Transport kinetics; Transporter