Elucidation of the cell-and organ-specific transport mechanisms of organic anion transporters

在小肠、肾脏和肝脏等上皮组织中，多种具有不同特征的有机阴离子转运体分布于刷状缘和基底膜，在药物的单向转运（吸收或分泌）中起重要作用。然而，有机阴离子转运蛋白的质膜靶向机制尚未完全清楚。本研究以有机阴离子转运蛋白OAT-K为模型转运蛋白，构建了在OAT-K N端融合增强型绿色荧光蛋白（EGFP）的表达载体。在MDCK细胞中稳定表达EGFP-OAT-K融合蛋白，并检测其生物合成和细胞表面靶向性。我们的数据表明，OAT-K是通过翻译后加工修饰的，其C-末端部分靶向顶膜。OAT-K的加工发生在内质网，其N-末端部分通过蛋白酶体途径降解。其他有机阴离子转运蛋白OAT 1和OAT 3在人肾脏中大量表达，因此我们通过启动子分析研究了其表达调控机制。结果表明，OAT 3基因启动子区存在cAMP反应元件。转录因子CREB-1和ATF-1通过cAMP反应元件调节组成型和诱导型表达，而OAT 1基因被证明受肝细胞核因子4α调节。我们还成功克隆了刷状缘膜上表达的H^+/有机阳离子反向转运蛋白MATE 1和MATE 2-K，并对其组织分布和功能特性进行了研究，为进一步比较刷状缘型和基底外侧型有机离子转运蛋白的膜靶向机制奠定了基础。

In the epithelial tissues such as small intestine, kidney and liver, various organic anion transporters with distinct characteristics were localized to the brush-border and basolateral membranes, and play an important role in determining the unidirectional transport (absorption or secretion) of drugs. However, the mechanism of plasma membrane targeting of organic anion transporters was not fully understood. In the present study, organic anion transporter OAT-K was selected as a model transporter, and we constructed the expression vector in which enhanced green fluorescent protein (EGFP) was fused to the N-terminus of OAT-K. EGFP-OAT-K fusion protein was stably expressed in MDCK cells and its biosynthesis and cell surface targeting were examined. Our data showed that OAT-K was modified by post-translational processing and its C-terminal portion was targeted to the apical membrane. It was suggested that processing of OAT-K occurred at the endoplasmic reticulum and that its N-terminal portion was degraded by proteasomal pathway.Other organic anion transporters, OAT1 and OAT3, are abundantly expressed in the human kidney, and therefore we examined the mechanisms concerning their regulation of expression by promoter analysis. It was demonstrated that a cAMP-response element exists in the promoter region of OAT3 gene. Transcription factors, CREB-1 and ATF-1, regulate the constitutive and inducible expression via a cAMP-response element, while OAT1 gene was shown to be regulated by hepatocyte nuclear factor-4α. We also succeeded in cloning of MATE1 and MATE2-K, the H^+/organic cation antiporters expressed in the brush-border membrane, and demonstrated their tissue distribution and functional characteristics.We will compare the mechanisms of membrane targeting between brush-border type and basolateral type of organic ion transporters in the future.