%0 Journal Article %A Kathrin Mandery %A Heinrich Sticht %A Krystyna Bujok %A Ingrid Schmidt %A Christina Fahrmayr %A Bettina Balk %A Martin F Fromm %A Hartmut Glaeser %T Functional and structural relevance of conserved positively charged lysine residues in organic anion transporting polypeptide 1B3 %D 2011 %R 10.1124/mol.111.071282 %J Molecular Pharmacology %P mol.111.071282 %X 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 Lys361>Ala, Lys361>Arg, Lys399>Ala and Lys399>Arg were generated using site-directed mutagenesis in order 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 Lys361>Ala mutant, whereas the transport activity was maintained by the Lys361>Arg 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 to OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of Lys399>Ala and Lys399>Arg was decreased to 16% and 72% compared to 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. %U https://molpharm.aspetjournals.org/content/molpharm/early/2011/06/03/mol.111.071282.full.pdf