Establishment Of An Early Screening Technique For ?-aminobutyric Acid Receptors Based On Fluorescence In Central Nervous System

Objective:Although ion channels are attractive targets for the discovery of new drugs,the systematic screening of ion channel target drugs is still challenging due to the lack of effective and low-cost detection methods for high-throughput or medium-throughput screening.In the early screening of new drug discovery,the combination of fluorescence imaging microplate reader and voltage-sensitive dye may be a promising tool for screening multi-target ion channel blockers.In this study,we optimized and verified the effect of using voltage-sensitive dyes to detect compounds on the stable expression of GABAA receptor cells of different subtypes.Our results indicate that this voltage-sensitive dye-based detection can provide a sensitive and quantitative method to study the effect of functional drugs on GABAA receptor subtypes,thereby achieving medium-throughput,low cost,and multiple targets early screening.Methods:In this study,HEK-293 cell lines stably expressing GABAA receptors of different subtypes were established,including ?1?2?2,?2?3?2,?3?3?3,?4?3?2,?5?3?2,?6?2?2,and then PCR and manual patch clamp were used to confirm the functional expression of the target genes.HEK-293 cell lines expressing GABAA receptors were cultured in 96-well plates for 24 hours,then the medium was removed,and voltage-sensitive dyes and test compounds were added to the cells,and the plates were transferred to a microplate reader for fluorescence signal detection.Results:First,we calculated the EC50 value of y-aminobutyric acid and the IC50 value of Bicuculline for a variety of GABAA receptor subtypes,generated corresponding dose response curves,and tested the effects of a series of known compounds.As a strong indicator of the reliability of the experimental method,the calculation of the Z' value shows that the measurement method developed in this study has good reproducibility.In addition,the results of the test compounds are also consistent with previous reports in the literature.Conclusion:Through the above research,we proved that the method is simple,fast and reliable,which provides a new technical method for multi-target screening of GABAA receptors in the early stage of drug discovery.