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Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Publication date 2015
Author Jack Thomas, Simonin Jonathan, Ruepp Marc-David, Thompson Andrew J., Gertsch Jürg, Lochner Martin (corresponding author),
Project Synthetic Neurochemistry - Introduction of Biophysical Tools into Ion Channels Using Chemical Approaches
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Original article (peer-reviewed)

Journal Neuropharmacology
Volume (Issue) 90
Page(s) 63 - 73
Title of proceedings Neuropharmacology
DOI 10.1016/j.neuropharm.2014.11.007

Abstract

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP;1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron,tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with cocrystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo life imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology including FP and ligand competition to life imaging of 5-HT3 expressing tissues.
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