Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, no M3 receptor drugs with high speci city, high activity, and few side effects have been developed, and there is a lack of methods suitable for highthroughput screening of drugs with GPCRs. In this study, we established an e cient and sensitive drug cell screening model and method for targeting M3 receptors based on calcium-activated chloride channels (CaCCs). This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We veri ed that the model can sensitively detect changes in intracellular Ca 2+ concentration using uorescence quenching kinetics experiments, con rmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.

Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, no M3 receptor drugs with high specificity, high activity, and few side effects have been developed, and there is a lack of methods suitable for high-throughput screening of drugs with GPCRs. In this study, we established an efficient and sensitive drug cell screening model and method for targeting M3 receptors based on calcium-activated chloride channels (CaCCs). This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We verified that the model can sensitively detect changes in intracellular Ca2+ concentration using fluorescence quenching kinetics experiments, confirmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.