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        Characterization and Validation of Fluorescent Receptor Ligands: A Case Study of the Ionotropic Serotonin Receptor

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        The application of fluorescent receptor ligands has become widespread, incited by two important reasons. “Seeing is believing”—it is possible to visualize in real time in live cells ligand–receptor interactions, and to locate the receptors with subcellular precision allowing one to follow, e.g., internalization of the ligand–receptor complex. The high sensitivity of photon detection permits observation of on the one hand receptor–ligand interactions on cells with low, native receptor abundance, and on the other of individual fluorophores unveiling the stochastic properties of single ligand–receptor complexes.
        The major bottlenecks that impede extensive use of fluorescent ligands are due to possible dramatic changes of the pharmacological properties of a ligand upon chemical modification and fluorophore conjugation, aggravated by the observation that different fluorophores can provoke very dissimilar effects. This makes it virtually impossible to predict beforehand which labelling strategy to use to produce a fluorescent ligand with the desired qualities.
        Here, we focus on the design, synthesis, and evaluation of a high-affinity fluorescent antagonist for the ionotropic serotonin type-3 receptor.
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