Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.
Br J Pharmacol. 2010 Feb 1;159(3):698-708. doi: 10.1111/j.1476-5381.2009.00568.x. Epub 2010 Jan 22.
Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin.
Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated.
Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V(max) normalized to cell surface protein expression for Lys41>Ala (wild type: 190 +/- 8, Lys41>Ala:16 +/- 4 pmol (mg protein)(-1) min(-1), P < 0.001), whereas V(max) of Lys41>Arg and Arg580>Lys (103 +/- 8 and 123 +/- 14 pmol (mg protein)(-1) min(-1), P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala.
We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.
有机阴离子转运多肽 1B3（OATP1B3）（SLCO1B3）介导内源性底物（如雌酮-3-硫酸盐）和药物（如普伐他汀）从血液摄取到肝细胞内。基于结构的 OATP1B3 模型表明，带正电荷的孔有助于转运机制。因此，我们研究了 OATP1B3 介导的磺溴酞（BSP）和普伐他汀摄取过程中保守的带正电荷氨基酸的作用。
用丙氨酸、精氨酸、谷氨酰胺、甘氨酸或赖氨酸替换 OATP1B3 中的赖氨酸 28、赖氨酸 41 和精氨酸 580。利用免疫荧光、免疫印迹分析和细胞摄取实验，研究这些突变对蛋白表达和转运活性的影响。
免疫荧光显示所有突变体均定位于质膜，Arg580>Ala 和 Arg580>Lys 突变体部分保留在细胞内。Lys41>Ala、Lys41>Gln、Lys41>Gly、Arg580>Gly 和 Arg580>Lys 对 BSP 和普伐他汀的转运明显减少。BSP 转运的动力学分析显示，Lys41>Ala 的 Vmax 与细胞表面蛋白表达归一化值显著降低（野生型：190±8，Lys41>Ala：16±4 pmol/（mg 蛋白）-1 min-1，P<0.001），而 Lys41>Arg 和 Arg580>Lys 的 Vmax 没有显著变化（103±8 和 123±14 pmol/（mg 蛋白）-1 min-1，P>0.05）。这表明 41 位和 580 位的正电荷对 BSP 的转运活性很重要。结构模型表明，Lys41 的正电荷侧链在孔内具有柔性。Arg580 的取向由相邻的残基 Glu74 和 Asn77 确定，这一点通过 Glu74>Ala 的动力学分析得到了证实。
我们证明了保守的带正电荷氨基酸 Lys41 和 Arg580 对 OATP1B3 的转运活性至关重要。
