Validation of a medium-throughput electrophysiological assay for KCNQ2/3 channel enhancers using IonWorks HT.
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 degrees 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. EC(50)s 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 (R(2) > 0.9) between the 2 assays, thus demonstrating the highly predictive nature of the IonWorks assay.
Jow
Flora
F
Discovery Neuroscience, Wyeth Research, Princeton, New Jersey 08543-8000, USA.
Shen
Ru
R
Chanda
Pranab
P
Tseng
Eugene
E
Zhang
Howard
H
Kennedy
Jeffrey
J
Dunlop
John
J
Bowlby
Mark R
MR
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods*; D051658:KCNQ2 Potassium Channel / Q000378:metabolism*; D051659:KCNQ3 Potassium Channel / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D012413:Rubidium / Q000378:metabolism; D014162:Transfection
D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D026902:Potassium Channel Blockers; D012413:Rubidium