Figure 1.
Niclosamide activates TMEM16A exogenously expressed in HEK293T cells. (A) Representative whole-cell current recorded in HEK293T cells overexpressing TMEM16A “a” splicing isoform. Intracellular solution contains 0.5 µM free Ca2+. The current was elicited by a ramp protocol from −100 to +100 mV with the holding potential at −60 mV. The patch clamp configuration and voltage protocol are shown on the left. (B) Representative current traces elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and the holding potential at −60 mV. (C)G-V relationship of TMEM16A current recorded in B. Error bars represent SEM, n = 7. The tail currents were normalized to the tail current at +140 mV in the bath solution. (D) Quantification of TMEM16A current rectification (the peak current ratio between +100 mV and −100 mV). Statistical analysis was done with one-way ANOVA followed by Tukey’s multiple comparisons test. (****: P < 0.0001, n = 7). (E) Representative current traces in response to different niclosamide concentrations as indicated. The current was elicited by a ramp protocol the same as in A. (F) Niclosamide dose–response curve at −60 mV. The data were fitted to the Hill equation with EC50 of 1.27 µM. Error bars represent SEM, n = 5. (G) Representative whole-cell current recorded in HEK293T cells overexpressing TMEM16A. Intracellular solution contains 500 nM free Ca2+. The current was elicited by a ramp protocol the same as A. Three time points (a, b, and c) were picked representing basal, after niclosamide, and niclosamide + Ani9, respectively. (H) Representative current traces at three different points as shown in G. (I) Statistical analysis of peak current at three different points as shown in G. Statistical analysis was done with one-way ANOVA followed by Tukey’s multiple comparisons test. (**: P < 0.01, ***: P < 0.001, n = 5). (J) Summary of niclosamide-induced intracellular Ca2+ change in the presence of 5 mM EGTA + 5 mM BAPTA in the extracellular solution. The error bar represents the SEM of 32 cells from three biological replicates done on different days.

Niclosamide activates TMEM16A exogenously expressed in HEK293T cells. (A) Representative whole-cell current recorded in HEK293T cells overexpressing TMEM16A “a” splicing isoform. Intracellular solution contains 0.5 µM free Ca2+. The current was elicited by a ramp protocol from −100 to +100 mV with the holding potential at −60 mV. The patch clamp configuration and voltage protocol are shown on the left. (B) Representative current traces elicited by a voltage-step protocol from −100 to +140 mV with 20-mV increments and the holding potential at −60 mV. (C)G-V relationship of TMEM16A current recorded in B. Error bars represent SEM, n = 7. The tail currents were normalized to the tail current at +140 mV in the bath solution. (D) Quantification of TMEM16A current rectification (the peak current ratio between +100 mV and −100 mV). Statistical analysis was done with one-way ANOVA followed by Tukey’s multiple comparisons test. (****: P < 0.0001, n = 7). (E) Representative current traces in response to different niclosamide concentrations as indicated. The current was elicited by a ramp protocol the same as in A. (F) Niclosamide dose–response curve at −60 mV. The data were fitted to the Hill equation with EC50 of 1.27 µM. Error bars represent SEM, n = 5. (G) Representative whole-cell current recorded in HEK293T cells overexpressing TMEM16A. Intracellular solution contains 500 nM free Ca2+. The current was elicited by a ramp protocol the same as A. Three time points (a, b, and c) were picked representing basal, after niclosamide, and niclosamide + Ani9, respectively. (H) Representative current traces at three different points as shown in G. (I) Statistical analysis of peak current at three different points as shown in G. Statistical analysis was done with one-way ANOVA followed by Tukey’s multiple comparisons test. (**: P < 0.01, ***: P < 0.001, n = 5). (J) Summary of niclosamide-induced intracellular Ca2+ change in the presence of 5 mM EGTA + 5 mM BAPTA in the extracellular solution. The error bar represents the SEM of 32 cells from three biological replicates done on different days.

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