Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Enhancers Using IonWorks HT

Abstract

Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput electrophysiological assay by using the IonWorks platform. Screening of 20 CHO-K1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at —10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at —80 mV and depolarize to —10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC50s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation ( R2 > 0.9) between the 2 assays, thus demonstrating the highly predictive nature of the IonWorks assay. ( Journal of Biomolecular Screening 2007;1059-1067)