title: Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 Is Critical for the Function of Organic Anion Transporting Polypeptide 2B1.
abstract: Organic anion transporting polypeptide 2B1 (OATP2B1), which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrates, most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, such as maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates. There are totally six tryptophan residues in OATP2B1. However, little is known about their role in the function and expression of OATP2B1. Our results show that, while W272, W276, and W277 located at the border of extracellular loop 3 and transmembrane domain 6 exhibit a moderate effect on the surface expression of OATP2B1, W611 located at the middle of transmembrane domain 11 plays a critical role in the function of OATP2B1. The tryptophan-to-alanine mutation of W611 changes the kinetic characteristics of OATP2B1-mediated estrone-3-sulfate (E3S) transport radically, from a monophasic saturation curve (with Kmand Vmaxvalues being of 7.1 ± 1.1 μM and 182 ± 7 pmol/normalized mg/min, respectively) to a linear curve. Replacing alanine with a phenylalanine will rescue most of OATP2B1's function, suggesting that the aromatic side chain of residue 611 is very important. However, hydrogen-bond forming and positively charged groups at this position are not favorable. The important role of W611 is not substrate-dependent. Molecular modeling indicates that the side chain of W611 faces toward the substrate translocation pathway and might interact with substrates directly. Taken together, our findings reveal that W611 is critical for the function of OATP2B1.
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Jialin
authors: J
authors: Bian
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Meng
authors: M
authors: Jin
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Mei
authors: M
authors: Yue
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Meiyu
authors: M
authors: Wang
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Hongjian
authors: H
authors: Zhang
authors: Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University , Suzhou 215123, China.
authors: Chunshan
authors: C
authors: Gui
mesh_terms: D019921:Biotinylation; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D027361:Organic Anion Transporters / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D053719:Tandem Mass Spectrometry; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D027361:Organic Anion Transporters; C450859:SLCO2B1 protein, human; D014364:Tryptophan
keywords: OATP2B1; molecular modeling; transmembrane domain; transporter; tryptophan residue