Mutations of a putative niclosamide binding site attenuate the niclosamide-potentiation effect. (A) Computational prediction of a putative niclosamide binding site in TMEM16A extracellular side (PBD accession no. 5OYB). The zoom-in view of potential niclosamide interacting residues is shown on the right. (B) Representative whole-cell current traces of WT and mutant TMEM16A channels were elicited by a gap-free protocol with a holding potential of −60 mV in response to different concentrations of niclosamide. The intracellular free Ca²⁺ was 0.5 µM. (C) Representative current traces of WT and mutants with and without 10 µM niclosamide. The current from the same patches in B were elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and a holding potential at −60 mV. (D) Niclosamide dose–response curves of the mutants. The currents in B were normalized to the instantaneous tail current of the same patch elicited by a +140 mV prepulse in the presence of 10 µM niclosamide in C, right, *. (E) Comparison of mutational effects on 5 µM niclosamide-induced TMEM16A potentiation at −60 mV. Data was derived from D. Statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparisons test. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, n = 9 for WT and n = 4–5 for the mutations.