Monitoring Substrate-Driven Structural Changes in a CLC Chloride-Proton Antiporter with Double Electron-Electron Resonance Spectroscopy

736-Pos Board B491 Electrophysiological Characterisation of TMEM16A Currents in Esophageal Squamous Cell Carcinoma Cells Mariana Oana Popa, Hedaythul Choudhury, Christopher Rothwell, Larry Alex Gaither, Martin Gosling, Pamela Tranter. Novartis Institutes for Biomedical Research, Horsham, United Kingdom. TMEM16A is a calcium activated chloride channel that has been linked to a number of cancer types including prostate, head and neck and esophageal squamous cell carcinomas (ESCC). The TMEM16A gene has been shown to be amplified and overexpressed in ESCC. To assess the presence of functional TMEM16A channels in human TE-11 cells, an established ESCC cell line, whole-cell current measurements were performed in the presence of different intracellular calcium concentrations using both conventional and planar (QPatch, Sophion) patch clamp technology. Channel characterization was based on the voltage, time and calcium dependence of the currents. Pharmacological validation was also undertaken. In the presence of 338 nM intracellular free Ca2þ, the chloride currents showed time dependent activation and deactivation and a characteristic outward rectifying current-voltage relationship consistent with TMEM16A. These currents were blocked by 100 mM CaCCinh-A01 (95 5 2 % inhibition, n = 17). The CaCCinh-A01 sensitive chloride current density was increased from 20.5 510.7 pA/pF (n = 7) in the absence of intracellular Ca2þ, to 171 5 26 pA/pF (n = 11) in the presence of 1 mM intracellular Ca2þ, indicating these currents are calcium dependent as expected for TMEM16A. The currents were also sensitive to other non-specific calcium activated chloride channels blockers e.g. niflumic acid and DIDS. Treatment of TE-11 cells with shRNAs targeting TMEM16A significantly decreased the chloride conductance in TE-11 cells consistent with them being mediated by TMEM16A. Data generated using conventional patch clamp was in good agreement with that from the QPatch, thus validating the application of the QPatch platform for this type of study. The data provides the first electrophysiological characterisation in esophageal squamous cell carcinoma cells of an endogenous Ca2þactivated chloride current, confirming the presence of functional TMEM16A in these cancer cells.