Abstract：
Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogues used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of co-expressed ENT1. We created a cell model where ENT1 was removed from HEK293 cells using CRISPR/cas9 (ENT1KO cells); this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by qPCR. Wildtype HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 {plus minus} 0.06 and 1.50 {plus minus} 0.22 pmol/µl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 µM), while hypoxanthine was the only nucleobase with a sub-millimolar affinity (320 µM). A range of nucleoside/nucleobase analogues were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 µM for ticagrelor to 2,300 µM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. Significance Statement We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.
摘要：
平衡核苷转运蛋白（ENT）介导临床使用的内源性核苷和核苷类似物的跨膜通量。主要亚型，ENT1已被很好地表征。然而,另一个子类型，ENT2由于其相对低的表达和共表达的ENT1的混杂影响而在其天然环境中被较少地表征。我们创建了一个细胞模型，其中使用CRISPR/cas9（ENT1KO细胞）从HEK293细胞中去除ENT1；该细胞系具有ENT2作为唯一的功能性嘌呤转运蛋白。通过测量[3H]2-氯腺苷摄取来评估转运蛋白功能。ENT1蛋白基于[3H]硝基苄基硫代肌苷的结合进行定量，免疫印迹法检测ENT1/ENT2蛋白。通过qPCR检查参与嘌呤代谢的相关转运蛋白和酶的表达变化。野生型HEK293细胞和ENT1KO细胞具有相似的SLC29A2/ENT2转录物/蛋白表达和ENT2介导的[3H]2-氯腺苷转运活性(Vmax值为1.02{正负}0.06和1.50{正负}0.22pmol/µl/s,分别)。在测试的内源性核苷/核碱基中，腺苷对ENT2具有最高的亲和力（Ki）（2.6µM），而次黄嘌呤是唯一具有亚毫摩尔亲和力（320µM）的核碱基。在此模型中，还测试了一系列核苷/核碱基类似物对ENT2的亲和力，亲和力（Ki）范围从8.6µM的替格瑞洛到2,300µM的6-巯基嘌呤。我们的数据表明，从这些细胞中去除内源性ENT1不会改变ENT2的表达或功能。该细胞系应被证明可用于分析通过ENT2起作用的新型药物并研究ENT2调节。重要性陈述我们已经创建了一种细胞系,由此可以在没有ENT1的混杂影响的情况下详细研究内源性ENT2。ENT1的缺失对ENT2的表达和功能没有影响。这种新型细胞系将为研究药物与ENT2的相互作用以及ENT2表达和功能的细胞调节提供理想的模型。
