中文摘要：

目的研究人肝脏特异的有机阴离子转运蛋白基因OATP-C分子膜外区保守的49位阳离子赖氨酸,对其有机阴离子底物摄取能力的影响。方法从人肝脏mRNA中克隆OATP-C野生型基因全长cDNA序列,通过定点突变将高度保守的第49位赖氨酸残基突变为中性氨基酸-苏氨酸,构建其C端的GFP融合蛋白的真核表达载体,转染HEK293细胞,观察突变体的细胞膜定位能力及底物摄取功能的变化。结果OATP-C野生型及突变体基因均能定位表达于HEK293细胞质膜;[^3H]硫酸盐雌酮底物摄取实验显示,突变体5min底物摄取量较野生型下降74.18%,浓度依赖的[^3H]硫酸盐雌酮底物摄取动力学分析表明,突变体Km增高和vmax降低。结论OATP-C蛋白膜外区第49位赖氨酸是其关键的功能氨基酸,是其重要的功能结构单位。

英文摘要：

Objective To investigate the effect of the mutant of organic anion transporting polypeptide-C （OATP-C） positive ion lysine-49 localized on the polypeptide extracellular loop in impairing uptaking ability of its substrate. Methods Full length cDNA of the wild type OATP-C was cloned from human liver mRNA by RTPCR. Then, the highly conserved lysine-49 residues were mutated to threonine by site-directed mutagenesis technique to construct the eukaryotic expression vectors of C terminal GFP fusion protein. Targeting ability and its uptaking substrate capability of cell plasma membrane were observed by transfecting those constructs into HEK293 cells. Results Both wild-type OATP-C protein and mutant were highly expressed on HEK293 cell plasma membrane. [ 3H ] estrone sulfate substrate uptaking technique showed that the amount of substrate uptaken by mutant at 5 min had decreased by 74.18% , compared with the wild type OATP-C protein. [^3H] estrone sulfate substrate uptaking dynamics demonstrated that Km value increased but V max decreased in the mutant. Conclusion The conserved positive ion lysine-49 on extracellular loop of OATP-C may be a determinant amino acid in uptaking organic anion substrates.