Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are “gold standard” because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the “mix & measure” assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca⁺² measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compard the IC₅₀ values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca⁺² mobilization assays can be an alternate to radioligand binding assays.