Niclosamide directly targets TMEM16A from the extracellular side by enhancing its apparent Ca ²⁺ sensitivity. (A) Representative currents were recorded with outside-out patches from HEK293T cells overexpressing TMEM16A. Intracellular solution contains 0.2 µM free Ca²⁺. The current was elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and the holding potential at −60 mV. The patch clamp configuration is shown on the left. (B)G-V relation of the currents recorded in A. The instantaneous tail current was normalized to the tail current elicited by the +140 mV pulse in bath solution (G/G_{+140 mV, bath}). Error bars represent SEM, n = 5. (C) Representative currents were recorded in HEK293T cells overexpressing TMEM16A with inside-out patches. The current was elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and the holding potential at −60 mV. The intracellular solution contains 0.387 µM free Ca²⁺. The patch clamp configuration is shown on the left. (D)G-V relation of the currents recorded in C. The tail current normalization was the same as B. Error bars represent SEM, n = 4. (E and F) Representative current traces of TMEM16A to different Ca²⁺ concentrations as indicated. The experiments were done under an inside-out configuration with a holding potential of −60 mV. The pipette solution contains 5 µM niclosamide for F and vehicle only (DMSO) for E. (G) Representative dose–response curve of TMEM16A to different Ca²⁺ concentrations as indicated. (H) EC₅₀ of the dose–response curves shown in G. Statistical analysis was done with Student’s t-test (**** means P < 0.0001, n = 7).