Fig. 3: Niclosamide and 1PBC in the same hydrophobic groove of TMEM16F.

Atomic model of the TM1-TM6 region of (a) Class 1 and (b) Class 2 of the drug-free TMEM16F, (c) niclosamide-bound TMEM16F and (d) 1PBC-bound TMEM16F. In each case, the additional cryo-EM densities found in the area are shown. Below, zoom into the TM1-TM6 groove with the residues shown as sticks and colored by heteroatom and the additional density found within the pocket shown in semitransparent outline. Structures of niclosamide and 1PBC as determined by computational docking using Glide are shown in purple and green, respectively. Bottom panels, differential effects of K370A and F374A mutations on TMEM16F-mediated (e) PS exposure (n = 84 for WT; 35 for F321A; 26 for K370A and 43 for F374A) and (f) Ca²⁺ influx (n = 162 for WT; 76 for F321A; 32 for K370A and 52 for F374A). At least three independent experiments have been performed for each condition, each with distinct cell populations to assess biological rather than technical variability. The mean ± SEM is shown along with the statistical significance determined by unpaired t-test (two-tailed) for each mutant as compared to the wildtype control (F321A: p = 0.0254; K370A: p < 0.0001; F374A: p = 0.0051 in (e) and F321A: p < 0.0001; K370A: p = 0.1003; F374A: p = 0.9862 in (f), ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001).