Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3
Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3
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DOI：
10.1111/j.1476-5381.2009.00568.x
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发表时间：
2010-02-01
影响因子：
7.3
通讯作者：
Koenig, J.
中科院分区：
文献类型：
--
作者：
Glaeser, H.;Mandery, K.;Koenig, J.
背景与目的：有机阴离子转运多肽1B 3（OATP 1B 3）（SLCO 1B 3）介导内源性底物（如雌酮-3-硫酸酯）和药物（如普伐他汀）从血液进入肝细胞。基于结构的OATP 1B 3建模表明，具有正静电势的孔有助于转运机制。因此，我们研究了OATP 1B 3介导的摄取磺溴酞（BSP）和普伐他汀的保守的带正电荷的氨基酸的作用。实验方法：OATP 1B 3中的残基Lys 28，Lys 41和Arg 580被丙氨酸，精氨酸，谷氨酰胺，甘氨酸或赖氨酸取代。利用免疫荧光、免疫印迹分析和细胞摄取测定，研究了这些突变体对蛋白表达和转运活性的影响。关键结果：免疫荧光显示，所有突变体均定位于质膜，Arg 580> Ala和Arg 580> Lys突变体部分滞留于细胞内。Lys 41> Ala、Lys 41> Gln、Lys 41> Gly、Arg 580> Gly和Arg 580> Lys显示BSP和普伐他汀的转运显著降低。BSP转运的动力学分析显示，对于Lys 41> Ala（野生型：190 +/- 8，Lys 41> Ala：16 +/- 4 pmol（mg蛋白）-1 min-1，P < 0.001），归一化为细胞表面蛋白表达的V-max显著降低，而Lys 41> Arg和Arg 580> Lys（103 +/- 8和123 +/- 14 pmol（mg蛋白）-1 min-1，P > 0.05）的V-max没有显著变化。这表明，在位置41和580的正电荷是重要的BSP的运输活动。结构建模表明，Lys 41的带正电荷的侧链在孔内是柔性的。Arg 580的定位是由相邻的Glu 74和Asn 77残基决定的，动力学分析证实了Glu 74> Ala.Conclusions和影响：我们证明了保守的带正电荷的氨基酸Lys 41和Arg 580是OATP 1B 3转运活性的关键。
Background and purpose: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.Experimental approach: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.Key results: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.Conclusions and implications:We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.