Development and Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Openers Using QPatch HT

Abstract

The KCNQ2/3 channel has emerged as a drug target for a number of neurological disorders including pain and epilepsy. Known KCNQ2/3 openers have effects on two distinct biophysical properties of the channel: (1) a hyperpolarizing shift in the voltage dependence of channel activation (V(1/2)), and (2) an increase in channel open probability or peak whole-cell current. The current high-throughput screening assays for KCNQ2/3 openers measure changes of channel activity at sub-peak conductances and the output measure is a combination of effects on V(1/2) shift and peak current. Here, we describe a medium-throughput electrophysiological assay for screening KCNQ2/3 openers using the QPatch HT platform. We employed a double-pulse protocol that measures the shift in V(1/2) and the change in current amplitude at peak conductance voltage. Retigabine along with novel KCNQ2/3 openers were evaluated in this assay. Three classes of KCNQ2/3 openers were identified based on the hyperpolarizing shift in V(1/2) and the change in peak current. All three classes of compounds caused a hyperpolarizing shift in V(1/2), but they were differentiated by their respective effects on peak current amplitude (increase, decrease, or only modestly affecting peak current amplitude). KCNQ2/3 blockers were also identified with this assay. These compounds blocked currents without affecting voltage-dependent activation. In summary, we have developed a medium-throughput assay that can reliably detect changes in the biophysical properties of the KCNQ2/3 channel, V(1/2), and peak current amplitude, and therefore may serve as a reliable assay to evaluate KCNQ2/3 openers and blockers.