The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC50s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents, but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC50s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.
Keywords: ANO1; QPatch; TMEM16A; drug development; electrophysiology; small molecule.