Abstract
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 (pIC50 = 6.2, 6.6) and cocaine (pIC50 = 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.
Highlights
Members of the solute carrier (SLC) transporter protein family are increasingly recognized as therapeutic drug targets
Dopamine was less potent on U2OS-dopamine transporter (DAT) cells (pEC50 = 4.0 ± 0.2) compared to U2OS-mock cells (pEC50 = 5.3 ± 0.2) (Table 1), showing that the presence of DAT reduces the apparent potency of dopamine
A recently described high-throughput screening (HTS)-compatible label-free cell-based cytotoxicity assay for monocarboxylate transporter 1 (SLC16A1) showed promise for inhibitor screening studies[36], this method demands that a selective cytotoxic substrate is available for the SLC
Summary
Members of the solute carrier (SLC) transporter protein family are increasingly recognized as therapeutic drug targets. 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). 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. Cell-based assays using fluorescent membrane potential[8], pH-sensitive or calcium-sensing dyes, can achieve impressive throughput using platforms such as fluorescent imaging plate readers (FLIPR)[9], but may result in non-specific signals and require thorough signal validation Another approach based on electrophysiological measurements (SURFE2R) can attain increased screening capacity[10], these assays require electrogenicity of the SLC, valid for a minority only, and often use liposome or membrane preparations. In this work mammalian bone osteosarcoma (U2OS) cells with endogenous expression of both ENT1 and adenosine receptors were assessed using the xCELLigence technology
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