5-HT_FAsTR: a Versatile, Label-free, High-throughput, Fluorescence-based Microplate Assay to Quantify Serotonin Transport and Release

Overview

Journal Sci Rep

Specialty Science

Date 2024 Mar 20

PMID 38504103

Authors

Lina Bukowski

Markus Emanuel Strom

Jens Lindengren Andersen

Jannick Bang Maesen

Lin Tian

Steffen Sinning

Affiliations

Soon will be listed here.

Abstract

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.

References

Amphoux A, Vialou V, Drescher E, Bruss M, Mannoury la Cour C, Rochat C . Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain. Neuropharmacology. 2006; 50(8):941-52. DOI: 10.1016/j.neuropharm.2006.01.005. View

Klein Herenbrink C, Fullerton Stoier J, Reith W, Dagra A, Gregorek M, Cola R . Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors. Commun Biol. 2022; 5(1):578. PMC: 9187629. DOI: 10.1038/s42003-022-03488-5. View

Bi X, Beck C, Gong Y . Genetically Encoded Fluorescent Indicators for Imaging Brain Chemistry. Biosensors (Basel). 2021; 11(4). PMC: 8069469. DOI: 10.3390/bios11040116. View

Henry L, Field J, Adkins E, Parnas M, Vaughan R, Zou M . Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants. J Biol Chem. 2005; 281(4):2012-23. DOI: 10.1074/jbc.M505055200. View

Schwartz J, Blakely R, DeFelice L . Binding and transport in norepinephrine transporters. Real-time, spatially resolved analysis in single cells using a fluorescent substrate. J Biol Chem. 2002; 278(11):9768-77. DOI: 10.1074/jbc.M209824200. View

Hilber B, Scholze P, Dorostkar M, Sandtner W, Holy M, Boehm S . Serotonin-transporter mediated efflux: a pharmacological analysis of amphetamines and non-amphetamines. Neuropharmacology. 2005; 49(6):811-9. DOI: 10.1016/j.neuropharm.2005.08.008. View

Baumann M, Ayestas Jr M, Partilla J, Sink J, Shulgin A, Daley P . The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue. Neuropsychopharmacology. 2011; 37(5):1192-203. PMC: 3306880. DOI: 10.1038/npp.2011.304. View

Cheng Y, Prusoff W . Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973; 22(23):3099-108. DOI: 10.1016/0006-2952(73)90196-2. View

Kristensen A, Andersen J, Jorgensen T, Sorensen L, Eriksen J, Loland C . SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev. 2011; 63(3):585-640. DOI: 10.1124/pr.108.000869. View

10.

Schneider C, Rasband W, Eliceiri K . NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7):671-5. PMC: 5554542. DOI: 10.1038/nmeth.2089. View

11.

Pereira D, Schmitz Y, Meszaros J, Merchant P, Hu G, Li S . Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum. Nat Neurosci. 2016; 19(4):578-86. PMC: 4853199. DOI: 10.1038/nn.4252. View

12.

Wall S, Gu H, Rudnick G . Biogenic amine flux mediated by cloned transporters stably expressed in cultured cell lines: amphetamine specificity for inhibition and efflux. Mol Pharmacol. 1995; 47(3):544-50. View

13.

Rannversson H, Wilson P, Kristensen K, Sinning S, Kristensen A, Stromgaard K . Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation. J Biol Chem. 2015; 290(23):14582-94. PMC: 4505525. DOI: 10.1074/jbc.M114.629071. View

14.

Mayer F, Luf A, Nagy C, Holy M, Schmid R, Freissmuth M . Application of a Combined Approach to Identify New Psychoactive Street Drugs and Decipher Their Mechanisms at Monoamine Transporters. Curr Top Behav Neurosci. 2016; 32:333-350. DOI: 10.1007/7854_2016_63. View

15.

Marvin J, Borghuis B, Tian L, Cichon J, Harnett M, Akerboom J . An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods. 2013; 10(2):162-70. PMC: 4469972. DOI: 10.1038/nmeth.2333. View

16.

Koepsell H . Organic Cation Transporters in Health and Disease. Pharmacol Rev. 2019; 72(1):253-319. DOI: 10.1124/pr.118.015578. View

17.

Davis B, Nagarajan A, Forrest L, Singh S . Mechanism of Paroxetine (Paxil) Inhibition of the Serotonin Transporter. Sci Rep. 2016; 6:23789. PMC: 4817154. DOI: 10.1038/srep23789. View

18.

Jaronczyk M, Walory J . Novel Molecular Targets of Antidepressants. Molecules. 2022; 27(2). PMC: 8778443. DOI: 10.3390/molecules27020533. View

19.

Sabatini B, Tian L . Imaging Neurotransmitter and Neuromodulator Dynamics In Vivo with Genetically Encoded Indicators. Neuron. 2020; 108(1):17-32. DOI: 10.1016/j.neuron.2020.09.036. View

20.

Fowler A, Seifert N, Acker V, Woehrle T, Kilpert C, de Saizieu A . A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro. J Biomol Screen. 2006; 11(8):1027-34. DOI: 10.1177/1087057106294698. View