Automated Patch Clamp of KCNQ2/3 Channels

Methods for expression and recording in CHO cells were previously described in detail (Vanoye et al., 2022 (link)). Human KCNQ2 cDNA (GenBank accession number NM_172108) in pIRES2-EGFP (BD Biosciences-Clontech, Mountain View, CA, USA) was used as template for in vitro mutagenesis. A stable line expressing human KCNQ3 (GenBank accession number NM_004519) was generated as described and maintained under dual selection with Zeocin (100 μg/ml) and hygromycin B (600 μg/ml). Plasmids with WT KCNQ2 or G256W cDNA were introduced into the KCNQ3-expressing CHO cell stable line by electroporation (Maxcyte STX; MaxCyte Inc., Gaithersburg, MD, USA). Automated patch clamp recording was performed using the Syncropatch 768 PE platform using PatchController384 V.1.3.0 software (Nanion Technologies, Munich, Germany). Pulse protocols were performed before and after addition of ezogabine (10 μM, Sigma-Aldrich) and, subsequently, XE-991 (25 μM, Abcam, Cambridge, MA; or TOCRIS, Minneapolis, MN). Currents reported are XE-991-sensitive currents, calculated by subtraction.