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. 2019 Feb 14;10:51. doi: 10.3389/fphar.2019.00051

FIGURE 4.

FIGURE 4

Niclosamide and related compounds provide potent and nearly full inhibition of TMEM16A chloride currents. Whole cell patch clamp studies using 170 nM free intracellular calcium revealed the average potency of TMEM16A antagonists in blocking the TMEM16A outward current at +100 mV (A), the compounds maximum antagonist activity (B) and concentration-response relationship for inhibition (C,D). Niclosamide, Cpd 2 and Cpd4 showed potent inhibition of the TMEM16A calcium-activated chloride current (A–D) but spared the cAMP-induced CFTR chloride current (E) exhibiting < 20 percent of inhibition (POI) of the forskolin-induced current. (F) Shows representative current–voltage traces from TMEM16A whole cell patch clamp electrophysiology studies using 170 nM free intracellular calcium, where the y-axis is current in nA and the x-axis is membrane potential changes from –100 mV to +100 mV in 20 mV steps from Vhold of 0mV. While benzbromarone inhibited both the outward and inward currents (Figure 3), Cpd 4, 1PBC and niclosamide suppressed the outward current but showed concentration-dependent effects on the inward current ranging from inhibition to stimulation (F). The stimulation was especially noticeable for niclosamide and 1PBC at –100 mV and higher concentrations. In contrast, 1PBC (H) and Cpd 4 (G) were found to provide dose dependent inhibition (not stimulation) at –100 mV when tested by perforated patch clamp electrophysiology, where the ionomycin stim (10 μM) and compounds (from 0.01 to 30 μM) were added at the 0 s time point. The DMSO Vehicle Control (gray, open squares; mean ± SD, n = 8) included on each patch plate shows the rapid calcium-dependent activation and inactivation of TMEM16A described earlier. ∗∗P < 0.01, ∗∗∗P < 0.001; significantly different from benzbromarone (A,B) or niclosamide (B); ns, not significant (unpaired t-test). Panels (G,H), ∗P < 0.05, ∗∗P < 0.01, #P < 0.001, $P < 0.0001 from two-way ANOVA for differences from the Vehicle Control; all other data points showed no significant difference (p > 0.05) compared to Vehicle. Number of replicates: n = 3–11 (A–D), n = 3–4 (E), mean ± SEM. Average of quadruplicate measures (G,H).