Fig. 2

Effects of W-scanning on scrambling activity of nhTMEM16. a, b Schematic representation of the scrambling assay for protein-free vesicles (a) and proteoliposomes (b). c–f Representative traces of the dithionite induced fluorescence decay for WT (c) and low- (d), medium- (e) and high-impact (f) tryptophan mutants of nhTMEM16 in the presence (red) and absence (black) of Ca²⁺. Dashed yellow lines indicate fits to Eq. (6). Protein-free traces are shown in green. g, h Quantification of scrambling rate constants α and β for WT and mutant nhTMEM16 in the presence (g) and absence of Ca²⁺ (h). The effects were categorized into three classes as low- (<10-fold reduction, blue shading), medium- (10 to 100-fold reduction, green shading) and high-impact residues (>100-fold reduction, red shading). Individual data points are shown as empty circles. All data is reported as the mean ± S.D. The number of replicates are indicated in Supplementary Table 1. i Mapping of the tested residues on the nhTMEM16 structure according to their impact on scrambling, colors as in g, h