Figure 6. The TMEM16F Channel Pore and Functional Tests of Pore-Lining Residues.

(A–C) The solvent-accessible (mesh) pore of Ca²⁺-bound TMEM16F in PIP₂-supplemented nanodiscs (orange for class 1; blue for class 2; A and B) or digitonin (green; C).

(D) Pore radius along the z axis (green for TMEM16F in digitonin; orange and blue for class 1 and class 2 of TMEM16F in PIP₂ supplemented nanodiscs, respectively).

(E–G) Live imaging of TMEM16F-dependent Ca²⁺ influx (E), PS exposure (F), and GPMV generation (G). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca²⁺ influx via Fluo-8 fluorescence. Time-lapse imaging of individual cells viewed with 603 magnification was performed to monitor PS exposure via pSIVA fluorescence. Data are represented as mean ± SEM.

(H–J) Scattered dot plots of time of onset of TMEM16F-dependent Ca²⁺ influx (H), PS exposure (I), and GPMV generation (J). Time of onset could not be determined for those time courses with a linear rather than sigmoidal rise. Data are represented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Statistical significance of all mutants as compared to TMEM16F WT is determined by one-way ANOVA followed by Holm-Šídák multiple comparisons test.

(K) Normalized currents fit to the Hill equation. The Hill coefficient is 2, and the EC₅₀ values of E529A and E604 (24.0 ± 2.3 μM and 20.1 ± 2.2 μM, respectively) are significantly increased compared to the EC₅₀ of wild-type control (7.4 ± 1.9 mM; p < 0.0001; corrected Dunnett test following one-way ANOVA). EC₅₀ of K530A (6.5 ± 2.9 μM) is not significantly different from WT value (p = 0.99).

See also Figures S2, S3, and S6, Table S2, and Videos S1 and S2.