KCNQ channels are proven anti-epileptic drug targets.We took advantage of two strategies to identify novel KCNQ activators.First,a library including 800,000 compounds was screened by a traditional high throughput screening and a series of novel scaffolds were identified.Second,we performed a computer-aid virtual screening targeting the gating charge pathway of KCNQ2 channels.We provided evidence that the gating charge pathway of the voltage-gated KCNQ2 potassium channel can accommodate various small molecule ligands.Combining mutagenesis,molecular simulation and electrophysiological recording,a binding model for the probe activator,ztz240,in the gating charge pathway was defined.This information was used to establish a docking-based virtual screening assay targeting the defined ligand binding pocket.Nine activators with five new chemotypes were identified,and in vivo experiments showed that three of the ligands binding to the gating charge pathway exhibit significant antiepilepsy activity.Identification of various novel activators by virtual screening targeting the pocket supports the presence of a ligand binding site in the gating charge pathway.This work provides new insight into the gating charge pathway of KCNQ2 channel and demonstrates that the gating charge pathways of Kv channels can serve as therapeutic targets.