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Measuring Serotonin Binding to Its Receptors In Vitro via Charge Transfer to ANAP
Olivia G Brado1, Aspen T Hawkins1, Adam D Hill1
1Department of Chemistry, Trinity College, Hartford, CT 06106, USA.
International Journal of Molecular Sciences
|November 27, 2025
Summary
We developed a new fluorescence method to measure serotonin (5-HT) binding to its receptors. This technique uses a special amino acid (ANAP) to track 5-HT interactions in real-time for better understanding of neurotransmitter signaling.
Area of Science:
- Biochemistry
- Neuroscience
- Molecular Biology
Background:
- Serotonin (5-HT) is a crucial neurotransmitter regulating numerous physiological processes.
- Understanding 5-HT receptor interactions is vital for neuroscience and drug development.
- Existing methods for measuring 5-HT binding have limitations in real-time cellular analysis.
Purpose of the Study:
- To develop and validate a novel in vitro fluorescence-based assay for quantifying 5-HT binding.
- To investigate the biophysical mechanism of 5-HT binding to its receptors using this new method.
- To demonstrate the utility of this assay for studying 5-HT receptor function in a cellular context.
Main Methods:
- Incorporation of a fluorescent non-canonical amino acid, ANAP, into the ligand binding site of 5-HT receptors.
- Utilizing time-resolved photoluminescence and transient absorption spectroscopy to analyze ANAP fluorescence quenching.
- Expressing ANAP-tagged 5-HT3A receptors in HEK293T cells and assessing functionality via whole-cell patch clamp.
- Employing density functional theory (DFT) calculations to elucidate the quenching mechanism.
Main Results:
- ANAP fluorescence was quenched by 5-HT and dopamine, indicating ligand interaction.
- 5-HT-induced ANAP quenching occurs via a nanosecond-timescale charge-transfer process.
- ANAP-tagged 5-HT3A receptors were functional and showed concentration-dependent fluorescence quenching upon 5-HT addition.
- The method successfully measured 5-HT binding to receptors in intact cells and isolated membranes.
Conclusions:
- A novel, robust fluorescence-based method for measuring 5-HT binding has been established.
- This technique provides real-time insights into 5-HT-receptor interactions at the molecular level.
- The assay is broadly applicable for studying various 5-HT binding proteins, including receptors and transporters.
