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bioRxiv · 10.1101/2025.10.01.679840

Measuring serotonin binding to its receptors via charge transfer to ANAP

Brado, O. G.·Hawkins, A. T.·Hill, A. D.·Puljung, M. C.
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Abstract

Serotonin (5-HT) is a vital intercellular messenger with diverse signaling functions throughout the human body. We have characterized and implemented a novel, in vitro fluorescence-based method of measuring 5-HT binding to gain a fuller understanding of the interactions between 5- HT and its receptors. This method involves expression of 5-HT receptor proteins in cultured cells with the fluorescent, non-canonical amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) incorporated into the ligand binding site. ANAP fluorescence was quenched in solution by both 5-HT and dopamine. Time-resolved photoluminescence and transient absorption spectroscopy confirmed that ANAP quenching by 5-HT occurs via a charge- transfer process that recovers through back-electron transfer on the nanosecond timescale. Supported by density functional theory calculations, this process likely involved a ANAP reduction by 5-HT. To test this method on intact receptors in a cellular context, we expressed 5- HT3A receptors (5-HT-gated ion channels) in HEK293T cells with ANAP inserted co- translationally into the transmitter binding site. Fluorescently labeled 5-HT3A receptors were functional and activated by 5-HT, as assessed by whole-cell patch clamp. Addition of 5-HT caused a concentration-dependent quenching of fluorescence from ANAP-tagged channels in intact cells and unroofed plasma membranes, demonstrating the utility of this method for measuring 5-HT binding to its receptors. Collectively, these results delineate a technique for measuring transmitter binding that can be widely adopted to explore 5-HT binding not only to 5- HT3 receptors, but to any 5-HT receptor, transporter, or binding protein in heterologous expression systems.

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Brado, O. G., Hawkins, A. T., Hill, A. D., Puljung, M. C.. 2025-10-03. Measuring serotonin binding to its receptors via charge transfer to ANAP. https://doi.org/10.1101/2025.10.01.679840

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