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-HT(3)) 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-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/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 Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/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-HT(3) receptors that is well suited for high-throughput screening.
Walstab
Jutta
J
Institute of Pharmacology and Toxicology, University of Bonn, 53113 Bonn, Germany.
Combrink
Sandra
S
Brüss
Michael
M
Göthert
Manfred
M
Niesler
Beate
B
Bönisch
Heinz
H
D000331:Aequorin / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands; D049408:Luminescent Agents / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D011719:Pyrazines / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection
D007093:Imidazoles; D008024:Ligands; D049408:Luminescent Agents; D011719:Pyrazines; D044406:Receptors, Serotonin, 5-HT3; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium