Abstract: AIM: To establish high-throughput potassium channel assay using FRET-based voltage sensor probes (VSPs).METHODS: RBL-2H3 Cells , which endogenously express high level Kir channels , were loaded with CC2-DMPE and DiSBAC2(3).The fluorescence response ratio of 460 and 570 nm before and after addition of high K+ was then detected.The response ratio can be used to evaluate membrane potential changes.RESULTS: The assay is robust as indicated by a Z' factor larger than0.6.The throughput is in the range of 10 000 data points per day , which is significantly higher than many other methods presently available for potassium channel.In 102 compounds screened with VSPs , 11 compounds were found to change the fluorescence ratio dosedependently.CONCLUSION: The assay allows semiautomated screening of compounds for potassium channel activity on 384-well plates and is sufficiently rapid for testing a large number of compounds.

Key words: fluorescence resonance energy transfer, voltage sensor probes, potassium channel blockers, high throughput screening