Using Inducible Expression Vector Technology To Create Stable Cell Lines Expressing KCNQ2/3, KCNQ4, And KCNQ3/5 Currents Suitable For Automated Electrophysiology Platforms

Abstract

The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive expression vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ inducible vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.