A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry.
Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.
Scott
Clay W
CW
Lead Discovery Department, AstraZeneca Pharmaceuticals LP, Wilmington, DE 19810, USA. clay.scott@astrazeneca.com
Wilkins
Deidre E
DE
Trivedi
Shephali
S
Crankshaw
Denis J
DJ
D000873:Anthracenes / Q000494:pharmacology; D002219:Carbamates / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D051658:KCNQ2 Potassium Channel; D007700:Kinetics; D010655:Phenylenediamines / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011189:Potassium Chloride / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012413:Rubidium / Q000032:analysis / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D019789:Tetraethylammonium / Q000494:pharmacology; D014162:Transfection
C112297:10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone; D000873:Anthracenes; D002219:Carbamates; D007211:Indoles; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D010655:Phenylenediamines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011725:Pyridines; D000644:Quaternary Ammonium Compounds; C101866:ezogabine; D019789:Tetraethylammonium; D011189:Potassium Chloride; C022799:clofilium; C061017:linopirdine; D012413:Rubidium