Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.
Mol Pharmacol. 2011 Sep;80(3):400-6. doi: 10.1124/mol.111.071282. Epub 2011 Jun 3.
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.
人有机阴离子转运多肽 1B3（OATP1B3）位于肝细胞的基底外侧膜，介导内源性底物如牛磺胆酸盐和药物从血液进入肝细胞。OATP1B3 的转运活性受面向中央孔的正电荷氨基酸的影响。进行了分子建模以选择保守的正电荷氨基酸，这些氨基酸可能影响 OATP1B3 在质膜中的转运活性和锚定。建模表明 Lys361 面向孔，Lys399 朝向质膜。因此，使用定点突变生成了 L361>A、L361>R、L399>A 和 L399>R 突变体，以研究正电荷对转运活性和膜锚定的影响。对底物磺溴酞和牛磺胆酸盐的转运动力学分析表明，L361>A 突变体失去功能，而 L361>R 突变体保持转运活性，表明位置 361 的正电荷对于 OATP1B3 的转运活性很重要。与 OATP1A2 和 OATP2B1 的比较建模表明，与 OATP1B3 相比，该赖氨酸残基周围的孔大小在 OATP1A2 中较大，在 OATP2B1 中较小，这可能与各自的底物谱有关。与野生型 OATP1B3 相比，L399>A 和 L399>R 的细胞表面表达分别降低至 16%和 72%（p<0.001），表明位置 399 的赖氨酸的正电荷对于未受损的细胞表面表达是必需的。此外，我们提供了影响 OATP1B3 转运活性的氨基酸的总结。
