Validation of a medium-throughput electrophysiological assay for KCNQ2/3 channel enhancers using IonWorks HT.
J Biomol Screen
; 12(8): 1059-67, 2007 Dec.
Article
em En
| MEDLINE
| ID: mdl-18087070
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 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Eletrofisiologia
/
Canal de Potássio KCNQ2
/
Canal de Potássio KCNQ3
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Biomol Screen
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Estados Unidos