Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.
Oncode Institute, Leiden, The Netherlands.
Sci Rep. 2021 Jan 14;11(1):1312. doi: 10.1038/s41598-020-79218-w.
Members of the solute carrier (SLC) transporter protein family are increasingly recognized as therapeutic drug targets. The majority of drug screening assays for SLCs are based on the uptake of radiolabeled or fluorescent substrates. Thus, these approaches often have limitations that compromise on throughput or the physiological environment of the SLC. In this study, we report a novel application of an impedance-based biosensor, xCELLigence, to investigate dopamine transporter (DAT) activity via substrate-induced activation of G protein-coupled receptors (GPCRs). The resulting assay, which is coined the 'transporter activity through receptor activation' (TRACT) assay, is based on the hypothesis that DAT-mediated removal of extracellular dopamine directly affects the ability of dopamine to activate cognate membrane-bound GPCRs. In two human cell lines with heterologous DAT expression, dopamine-induced GPCR signaling was attenuated. Pharmacological inhibition or the absence of DAT restored the apparent potency of dopamine for GPCR activation. The inhibitory potencies for DAT inhibitors GBR12909 (pIC = 6.2, 6.6) and cocaine (pIC = 6.3) were in line with values from reported orthogonal transport assays. Conclusively, this study demonstrates the novel use of label-free whole-cell biosensors to investigate DAT activity using GPCR activation as a readout. This holds promise for other SLCs that share their substrate with a GPCR.
溶质载体（SLC）转运蛋白家族的成员越来越被认为是治疗药物靶点。大多数 SLC 的药物筛选测定法基于放射性标记或荧光底物的摄取。因此，这些方法通常具有限制，这些限制会影响通量或 SLC 的生理环境。在这项研究中，我们报告了阻抗生物传感器 xCELLigence 的一种新应用，通过 G 蛋白偶联受体（GPCR）的底物诱导激活来研究多巴胺转运体（DAT）活性。由此产生的测定法被称为“通过受体激活的转运体活性（TRACT）测定法”，其基于 DAT 介导的细胞外多巴胺的去除直接影响多巴胺激活同源膜结合 GPCR 的能力的假设。在两种具有异源 DAT 表达的人细胞系中，多巴胺诱导的 GPCR 信号被减弱。DAT 的药理学抑制或不存在恢复了多巴胺对 GPCR 激活的明显效力。DAT 抑制剂 GBR12909（pIC = 6.2，6.6）和可卡因（pIC = 6.3）的抑制效力与报道的正交转运测定法中的值一致。总之，这项研究证明了使用 GPCR 激活作为读出信号，使用无标记全细胞生物传感器来研究 DAT 活性的新用途。这为其他与 GPCR 共享其底物的 SLC 提供了希望。
