Development Of A High-Throughput Screening Model And Optimization Of Screening Strategy For Calcium-Activated Chloride Channel Modulators

Objective: Calcium activated chloride channel(CaCCs)are a class of ion channels involved in a variety of physiological effects,including the regulation of the control of and smoothing muscle contraction of epithelium secretion,sensory transformation,and neuronal excitement.Anoctamins1(Ano1)and Anoctamins2(Ano2),among the Anoctamins family of membrane proteins,are classical calcium-activated chloride channels,and targeted modulators of Ano1 are potential drug candidates for cancer,cystic fibrosis,hypertension,diarrhea,and asthma.At present,the screening of CaCCs regulators is still in its infancy,and the discovery of high specific regulators is mainly limited by the lack of screening strategies.Therefore,in this study,by constructing a CACCS regulator high-throughput cell screening model,studying and comparing the electrophysiological properties of Ano1 and Ano2.To optimize the screening strategy of CaCCs regulators according to the electrophysiological properties of Ano1,in order to achieve precise hits on Ano1-specific regulators and lay the foundation for the subsequent discovery of novel and efficient CaCCs regulators.Methods: The lentiviral expression vectors PLVX-Blasticidin-Ano1-EGFP,PLVX-Blasticidin-Ano2-EGFP and PLVX-Puro-YFP-H148Q/I152 L were constructed,lentivirally packaged and infiltrated Fischer rat thyroid follicular epithelial(FRT)cell line,Ano1-YFP-H148Q/I152 L stably transfected FRT cell line(Ano1 regulator screening model)and Ano2 stably transfected FRT cell line were constructed.Fluorescence inverted microscopy was used to observe the expression location of target genes in cells,flow cytometry was applied to detect the purity of stably transfected cells,and Western blot technique and RT-PCR were used to verify the expression of Ano1,Ano2 and YFP-H148Q/I152 L from protein level and gene level.Fluorescence quenching kinetics assay was performed to verify the functionality of the high-throughput cell screening model for CaCCs constructed in this experiment and to assess the Z’ factor.The electrophysiological properties of Ano1 and Ano2 were studied and compared using whole-cell and inside-out membrane clamp techniques,and the constructed high-throughput cell screening model for CaCCs was further applied to validate the electrophysiological properties of Ano1 and optimize the screening strategy of Ano1 modulators according to the electrophysiological properties of Ano1.Results: Ano1-YFP-H148Q/I152 L stably transfected FRT cell line(Ano1regulator screening model)and Ano2 stably transfected FRT cell line were established by applying lentiviral infection method,respectively.Cell fluorescence images showed that Ano1 and Ano2 fused EGFP were expressed on the cell membrane and YFP-H148Q/I152 L was stably expressed in the cytoplasm.The purity of each stably transfected cell line was >90% by flow cytometry,and the results of Western blot and RT-PCR experiments showed that Ano1,Ano2 and YFP-H148Q/I152 L were overexpressed in the corresponding FRT cells.The whole-cell and inside-out experiments showed that both Ano1 and Ano2 exhibited the classical current characteristics of CaCCs,and the calcium sensitivity of Ano1 was significantly higher than that of Ano2;the results of both whole-cell and inside-out experiments showed that Ano1 differed from Ano2 in that it showed the electrophysiological characteristics of rundown of the activated current under the condition of continuous activation of high concentration of calcium ions,i.e.,the channel opening was reduced or even closed.The screening strategy of Ano1 modulator was optimized according to the electrophysiological properties of Ano1 rundown,and the hitting efficiency of Ano1 agonist was improved by setting the concentration gradient of the small molecule to be screened through multiplicative dilution;the screening strategy for Ano1 inhibitor was optimized in the dosing sequence,and the appropriate concentration of Ano1 agonist was added in advance to make Ano1 open continuously during the whole screening process.The specificity of the screening of Ano1 inhibitors was improved by adding the inhibitor to be tested again.Conclusion: This experiment successfully constructed the cell model of CaCCs modulator high-throughput screening,which has good stability and sensitivity of high-throughput screening.This experiment clarified the electrophysiological properties of Ano1,and successfully found that the electrophysiological phenomenon of rundown occurs when a high concentration of agonist continuously activates Ano1.The regulator screening strategy was optimized based on this electrophysiological phenomenon,which solved the previous problems in screening Ano1 regulators by applying the yellow fluorescent protein(YFP)iodine ion fluorescence quenching method.