The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABA_A receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [³H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [³H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (K_m and V_max values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na⁺-dependent [³H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [³H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies.

中文翻译：

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[ ³ H] ATPCA作为研究甜菜碱/ GABA转运蛋白1和肌酸转运蛋白的功能作用的底物的药理学表征

甜菜碱/γ-氨基丁酸（GABA）转运蛋白1（BGT1）是参与终止GABA能神经传递的四个GABA转运蛋白（GAT）之一。尽管提示可能与癫痫发作的治疗有关，但是BGT1在大脑中的确切功能重要性仍然难以捉摸。这部分是由于缺乏可用于探测其功能的有效和选择性的药理学工具化合物。我们先前曾报道过鉴定2-氨基-1,4,5,6-四氢嘧啶-5-羧酸（ATPCA），它是BGT1相对于GAT1 / GAT3的选择性底物，也是GABA _A受体的激动剂。为了提供对BGT1的新功能见解，我们在此介绍the化类似物的合成和药理学表征，[ ³H] ATPCA。在细胞系中表达的重组转运蛋白上使用传统的吸收检测方法，[ ³ H] ATPCA在四个GAT中显示出对BGT1的显着选择性（K _m和V _max值分别为21μM和3.6 nmol ATPCA /（min×mg蛋白） ），但也发现它是肌酸转运蛋白（CreaT）的底物。在小鼠皮质细胞培养物中的实验中，我们在神经元中观察到了Na ⁺依赖性的[ ³ H] ATPCA摄取，但在星形胶质细胞中却没有。GABA，ATPCA和非竞争性BGT1选择性抑制剂可抑制神经元摄取，表明神经元中的功能性BGT1。总之，我们报告[ ³H] ATPCA作为BGT1和CreaT的新型放射性底物。放射性配体的双重活性使其最适合用于重组研究。