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°C in a monoexponential manner with t1/2=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 t1/2 at 50mM potassium was 5.1min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC50=0.5μM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC50s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC50s=2.3 and 7.1μM, respectively) and XE991 (IC50s=0.3 and 0.9μM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC50 = 27μM). 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.