Abstract
Calcium-activated chloride channels (CaCCs) are essential for epithelial secretion, neuronal transmission, and smooth muscle function. Among the Anoctamin family, Anoctamin 1 (ANO1) and Anoctamin 2 (ANO2) are classical CaCCs proteins. ANO1 has been identified as a potential therapeutic target due to its involvement in diseases such as cancer and cystic fibrosis. However, current high-throughput screening (HTS) systems face limitations in achieving subtype-specific detection and optimizing screening strategies. Stable HTS cell models expressing ANO1 or ANO2 were constructed via lentiviral transduction in Fischer mouse thyroid (FRT) cells. The models were validated using flow cytometry, Reverse Transcription Polymerase Chain Reaction(RT-PCR), and YFP-H148Q/I152L-based iodide fluorescence quenching assays. Patch-clamp electrophysiology was employed to characterize ANO1 and ANO2 current properties. Although these electrophysiological features have been previously reported, their application in HTS workflows had not been systematically evaluated. ANO1 displayed notable current rundown under sustained stimulation with high Ca(2+) or agonist concentrations, whereas ANO2 maintained stable currents under identical conditions. Based on these findings, an optimized screening strategy was developed, incorporating agonist concentration gradients and the timing of inhibitor application. This approach improved the specificity and reliability of modulator detection. A robust and functionally validated cell-based HTS platform for CaCCs modulator discovery was established. By integrating the electrophysiological characteristics of ANO1 into the screening design, the optimized strategy enhances the accuracy of identifying selective ANO1 modulators. This work provides a methodological basis for future mechanism-driven screening of CaCCs-targeted compounds.