Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark.
Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, 95616-8635, USA.
Sci Rep. 2024 Mar 19;14(1):6541. doi: 10.1038/s41598-024-56712-z.
The neurotransmitter serotonin plays a pivotal role in mood and depression. It also acts as a vasoconstrictor within blood vessels and is the main neurotransmitter in the gastrointestinal system. In neurotransmission, released serotonin is taken up by serotonin transporters, which are principal targets of antidepressants and the psychostimulant, ecstasy. The investigation of serotonin transporters have relied almost exclusively on the use of radiolabeled serotonin in heterogenous end-point assays. Here we adapt the genetically encoded fluorescent biosensor, iSeroSnFR, to establish and validate the Serotonin (5-HT) Fluorescence Assay for Transport and Release (5-HT_FAsTR) for functional and pharmacological studies of serotonin transport and release. We demonstrate the applicability of the method for the study of a neuronal, high-affinity, low-capacity serotonin transporter (SERT) as well as an extraneuronal low-affinity, high-capacity organic cation transporter and mutants thereof. 5HT_FAsTR offers an accessible, versatile and reliable semi-homogenous assay format that only relies on a fluorescence plate reader for repeated, real-time measurements of serotonin influx and efflux. 5HT_FAsTR accelerates and democratizes functional characterization and pharmacological studies of serotonin transporters and genetic variants thereof in disease states such as depression, anxiety and ADHD.
神经递质 5-羟色胺在情绪和抑郁中起着关键作用。它在血管中也充当血管收缩剂，并且是胃肠道系统中的主要神经递质。在神经传递中，释放的 5-羟色胺被 5-羟色胺转运蛋白摄取，而 5-羟色胺转运蛋白是抗抑郁药和致幻剂摇头丸的主要靶点。对 5-羟色胺转运蛋白的研究几乎完全依赖于使用放射性标记的 5-羟色胺进行异质终点测定。在这里，我们改编了基因编码的荧光生物传感器 iSeroSnFR，以建立和验证 5-羟色胺（5-HT）荧光转运和释放测定（5-HT_FAsTR），用于 5-羟色胺转运和释放的功能和药理学研究。我们证明了该方法适用于研究神经元高亲和力、低容量 5-羟色胺转运蛋白（SERT）以及非神经元低亲和力、高容量有机阳离子转运蛋白及其突变体。5HT_FAsTR 提供了一种易于使用、多功能且可靠的半同质测定格式，仅依赖荧光平板读数器即可进行重复的实时测量 5-羟色胺内流和外流。5HT_FAsTR 加速了功能表征和药理学研究，并使 5-羟色胺转运体及其在疾病状态（如抑郁症、焦虑症和 ADHD）中的遗传变异民主化。
