Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT3 receptor responses in cell suspension based on aequorin bioluminescence caused by Ca2+ influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT3 receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT3A receptor or the human heteromeric 5-HT3A/B receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca2+-sensitive dyes. The potencies of two 5-HT3A receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT3A/B receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT3 receptors that is well suited for high-throughput screening.