Bulky mutations show more attenuation of the niclosamide-potentiation effect. (A) 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. (B) Representative current traces of WT and mutants with and without 10 µM niclosamide. The currents were elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and a holding potential at −60 mV. (C) Niclosamide dose–response curves of the mutants. The currents in A were normalized to the instantaneous tail current of the same patch elicited by a +140 mV test pulse in the presence of 10 µM niclosamide in B, right, *. (D) Comparison of mutational effects on 5 µM niclosamide-induced TMEM16A potentiation at −60 mV. Data was derived from C. Statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparisons test. ****: P < 0.0001, n = 9 for WT and n = 4–5 for the mutations. (E) Comparison of the mutational effects of F601 and R605 on niclosamide-induced potentiation. Statistical analysis was done with Student’s t-test for F601 mutations and one-way ANOVA followed by Tukey’s multiple comparisons test for R605 mutations. *: P < 0.05, **: P < 0.01, ****: P < 0.0001, and ns means no significance, n = 5).