RT Journal Article SR Electronic T1 Functional and Structural Relevance of Conserved Positively Charged Lysine Residues in Organic Anion Transporting Polypeptide 1B3 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 400 OP 406 DO 10.1124/mol.111.071282 VO 80 IS 3 A1 Kathrin Mandery A1 Heinrich Sticht A1 Krystyna Bujok A1 Ingrid Schmidt A1 Christina Fahrmayr A1 Bettina Balk A1 Martin F. Fromm A1 Hartmut Glaeser YR 2011 UL http://molpharm.aspetjournals.org/content/80/3/400.abstract AB The human organic anion transporting polypeptide 1B3 (OATP1B3), located in the basolateral membrane of hepatocytes, mediates the uptake of endogenous substrates such as taurocholate and drugs from blood into hepatocytes. The transport activity of OATP1B3 is influenced by positively charged amino acids, which are facing the central pore. Molecular modeling was performed to select conserved positively charged amino acids, which may influence transport activity and anchoring of OATP1B3 in the plasma membrane. The modeling revealed that Lys361 faces the pore, and Lys399 is oriented to the plasma membrane. Therefore, the mutants L361>A, L361>R, L399>A, and L399>R were generated using site-directed mutagenesis to investigate the impact of the positive charges on transport activity and anchoring in the membrane. Transport kinetic analyses for the substrates sulfobromophthalein and taurocholate showed a loss of function for the L361>A mutant, whereas the transport activity was maintained by the L361>R mutant, indicating that the positive charge at position 361 is important for transport activity of OATP1B3. Comparative modeling with OATP1A2 and OATP2B1 revealed that the pore size around this lysine residue is larger in OATP1A2 and smaller in OATP2B1 compared with OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of L399>A and L399>R was decreased to 16 and 72% compared with wild-type OATP1B3 (p < 0.001), respectively, indicating that the positive charge of lysine at position 399 is necessary for an unimpaired cell surface expression. Furthermore, we provide a summary of amino acids, which influence the transport activity of OATP1B3.