In vitro activity: ICA-069673 (ICA73) is a selective KCNQ2/Q3 potassium channel activator with IC50 of 0.69 μM. KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K(+) channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy, suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. ICA069673 acts on a binding site in the voltage-sensing domain that is distinct from the putative retigabine (a KCNQ voltage-gated potassium channel opener that was recently approved as an add-on therapeutic for patients with drug-resistant epilepsy) site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity.

Kinase Assay: Mutant channels were derived from human KCNQ2 or KCNQ3 genes (originally in pTLN vector; gifts from Dr M. Taglialatela, University of Molise, Campobasso, Italy; and Dr T. Jentsch, Leibniz‐institut fuer Molekulare Pharmakologie, Berlin, Germany), expressed in pcDNA3.1(–) plasmid (Invitrogen, Carlsbad, CA, USA). KCNQ3* channels refer to KCNQ3[A315T], carrying a point mutation that allows homomeric expression of KCNQ3. Chimeras between KCNQ2 and KCNQ3 were constructed using an overlapping PCR method. Flanking primers were used to amplify respective segments of KCNQ2 and KCNQ3. PCR approaches were then used to sequentially combine overlapping fragments until all necessary components segments of the chimera were incorporated. Break points for the chimeras were generated. For Q2+Q3[S1–S2] channels, KCNQ2 residues 89–148 were replaced by KCNQ3 residues 117–178. For Q2+Q3[S3–S4], KCNQ2 residues 153–207 were replaced by KCNQ3 residues 183–236. For Q2+Q3[S5–S6] channels, KCNQ2 residues 239–324 were replaced by KCNQ3 residues 268–363. For Q2+Q3[S1] channels, KCNQ2 residues 89–115 were replaced by KCNQ3 residues 117–145. For Q2+Q3[S2] channels, KCNQ2 residues 115–149 were replaced by KCNQ3 residues 145–178. Point mutants in KCNQ2 and KCNQ3 were constructed using a two‐step overlapping PCR method. All constructs were subcloned into pcDNA3.1(–) using NheI and EcoRI (KCNQ2) or NheI and XhoI (KCNQ3) restriction enzymes and verified by Sanger sequencing approaches (Genewiz, South Plainfield, NJ, USA; or Applied Genomics Core, University of Alberta, Edmonton, AB, Canada).

Cell Assay: HEK293 cells were cultured in 50 ml polystyrene tissue culture flasks (Falcon; Corning Inc., Tewksbury, MA, USA) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were grown in an incubator at 5% CO2 and 37°C. Cells were plated into six‐well plates and co‐ ransfected with plasmids encoding the channel of interest and green fluorescent protein using jetPRIME DNA transfection reagent (Polyplus, New York, NY, USA). After 24 h of incubation with transfection reagent, cells were split onto sterile glass coverslips at low density to allow recordings from individual cells, and electrophysiological experiments were conducted 1 day later.