Using fluorescence to measure real-time binding of serotonin to its receptors.

Abstract

Neurotransmitter binding assays that can be performed on or near physiological time scales would greatly enhance our understanding of nervous signaling. We set out to create one such assay to measure the binding of serotonin (5-hydroxytryptamine, 5-HT) to intact, fluorescently-tagged receptors. Tryptophan quenches the small organic fluorophores Prodan and bimane via photo-induced electron transfer. As serotonin shares a common indole structure with tryptophan, we reasoned that it may also quench the Prodan-based fluorescent amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP). In solution, serotonin quenches ANAP in the mM range. We used amber stop-codon suppression to express ionotropic 5-HT3A serotonin receptors tagged with ANAP in the serotonin binding site in HEK293T cells. To ensure that our protein constructs were not prematurely truncated at the amber stop codon, we included a downstream mOrange protein tag in the cytoplasmic loop between the M3 and M4 transmembrane helices. We used a microscope-coupled spectrograph to acquire fluorescence emission spectra from ANAP-tagged channels and found that the addition of 5-HT reduced ANAP fluorescence in a dose-dependent fashion at concentrations in the low µM range. Based on our findings, we believe this strategy offers great promise for the detection of 5-HT binding to functional receptors in intact cells. Such measurements can be paired with voltage clamp to study the binding-activity relationship of 5-HT3A and may also offer insights into the function of metabotropic serotonin receptors.