Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23.
Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23.
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DOI：
10.1007/s11010-011-0748-y
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发表时间：
2011-06
影响因子：
4.3
通讯作者：
Stoll, James
中科院分区：
文献类型：
--
作者：
Bennett, Katie M.;Liu, Jun;Hoelting, Courtney;Stoll, James
有机阳离子转运蛋白（OCT、SLC22）家族是多特异性跨膜蛋白家族，负责多种组织中许多阳离子药物、毒素和内源代谢物的摄取或排泄。许多 OCT 先前已被表征，但有许多孤儿基因的功能仍然未知。在本研究中，克隆并表征了两种新型大鼠 SLC22 基因：SLC22A17 (BOCT1) 和 SLC22A23 (BOCT2)。 Northern blot分析显示BOCT1和BOCT2 mRNA在多种组织中表达。 BOCT1在脑、初级神经元和脑内皮细胞中强烈表达，其中在脉络丛中表达最高。 BOCT2 在大脑和肝脏中也大量表达。为了表征这些基因的产物，使用 PCR 技术从大鼠血脑屏障 cDNA 文库中分离出 BOCT1 cDNA，并从大鼠脑毛细血管和培养的神经元中分离出 BOCT2 cDNA。将表达 BOCT1 和 BOCT2 的质粒以及 OCT1 和 OCTN2 的对照 cDNA 转染至 HEK-293 细胞中。通过蛋白质印迹和荧光显微镜验证重组细胞表面蛋白。使用放射性同位素摄取测定评估 BOCT1 和 BOCT2 的转运活性。表达 OCT1 和 OCTN2 的细胞分别转运经典底物 1-甲基-4-苯基吡啶鎓 (MPP+) 和肉毒碱。然而，表达 BOCT1 和 BOCT2 的细胞并未表现出对这些底物或许多其他 SLC22 底物的转运活性。相对于其他 OCT，这些新的家族成员具有非保守的氨基末端，这可能会妨碍典型的 SLC22 转运功能。
The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood–brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenylpyridinium (MPP+) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.
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