Fig. 3: Mechanistic double-mutant cycle analysis of residues of the gate region.

a Conformational rearrangement of the inner pore entrance upon Ca²⁺ binding. Cα-trace of indicated transmembrane helices of the Ca²⁺-free (PDB: 5OYG) and the Ca²⁺-bound conformation of TMEM16A (PDB: 5OYB) are shown. Sidechains of residues of the gate are displayed as sticks, sphere indicates position of the gating hinge. b Reduced mechanism depicting gating transitions at saturating Ca²⁺ concentrations. c Single-channel current (i) and open probability (Po) estimated from non-stationary noise analysis at saturating Ca²⁺ concentrations. Circles correspond to individual data points. Bars refer to median and interquartile range of data. d–f Equilibrium constants for mutants in the gate region for transitions d, L₃₂, e, L_21, and f, L₁₀. Bars indicate the best-fit values of the averaged data shown in Supplementary Fig. 7d (WT, n = 7; I550A, n = 6; I551A, n = 7; I641A, n = 7; I550A/I641A, n = 7; I551A/I641A, n = 6). Errors are 95% confidence intervals. g Coupling energy (G_coupling) and h independent energetic contribution (G_indep) of the indicated residues. Bars indicate quantities calculated using g, Eqs. 31–32, 35 and h, Eq. 34 from the best-fit values shown in d–f. Errors correspond to standard errors. Asterisks indicate significant deviation from zero in a two-sided one-sample t-test (g I550/I641: ***p = 2e−8, ***p = 3e−7, and ***p = 4e−4; I551/I641: ***p = 1e−13 and ***p = 0.001; h I550: ***p = 2e−15, ***p = 6e−8, and ***p = 0.005; I551: ***p~0, **p = 0.006, and ***p = 2e−13).