Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel

Figure 1. Kinetics of Ca2+-dependent activation of the TMEM16F wt-mediated current. (A) Whole-cell recordings obtained from TMEM16F wt-transfected cells with a pipette solution containing 100 μM Ca2+. The development of the current with time was monitored by repeatedly applying a +80 mV step every 10 s, as indicated in the upper traces. (B) Current amplitudes at the end of voltage steps were plotted against the time after membrane breaking for the recordings in (A). (C) The time required to obtain the maximal current activation was plotted against the intracellular Ca2+ concentration (n = 3–8, ** p < 0.01 Dunn–Holland–Wolf test after Kruskal–Wallis test). (D) An inside-out excised membrane patch expressing TMEM16F wt was repeatedly stimulated every 15 s with a solution containing 1 mM Ca2+ as indicated in the upper trace. The holding potential was +60 mV. The red line superimposed to the upper trace is the fit with a double exponential function. (E) Ratios between the peak current values measured at various times after patch excision and the value measured after 10 s were plotted against the time after patch excision for several patches (grey dots and lines). Black circles are the average…

Figure 2. Ca2+ sensitivity of the TMEM16F wt-mediated current. (A) Representative whole-cell currents recorded from TMEM16F wt-expressing cells with a pipette solution containing the indicated intracellular Ca2+ concentrations (x). The voltage protocol is shown at the top of the panel. (B) Average steady-state IV relationships from several cells at the indicated Ca2+ concentrations (n = 4–10). (C) Averages ± sem of the current density at different voltages were plotted against Ca2+ concentration (n = 4–10). The continuous lines are the fit with the Hill equation. (D) An inside-out excised membrane patch expressing TMEM16F wt was kept in 0 Ca2+ and exposed for 1 s to solutions containing different free Ca2+ concentrations (x) at the time indicated in the upper traces. The holding potential was +60 mV, or +100 mV as indicated. (E) Dose–response relations of activation by Ca2+ obtained by normalised currents and fitted to the Hill equation. Current amplitudes were calculated as the average value in the last 50 ms to each Ca2+ exposure and the current amplitude in 0 Ca2+ was subtracted. (F) Comparison of K1/2 obtained in different recording configurations (n = 7–8 for inside out, * p < 0.05).

Figure 3. TMEM16F wt ion permeability ratio PNa/PCl depends on the recording configuration. (A,B) Representative whole-cell voltage-clamp recordings obtained from representative TMEM16F wt-transfected cells with intracellular solution containing 50 μM Ca2+. Voltage protocols are shown at the top of the panels. (B) A cell was first exposed to a control solution containing 140 mM NaCl, then to 14 mM NaCl and returned to 140 mM NaCl. (C) IV relations measured from tail currents of the cell shown on the left with 140 mM NaCl (squares), 14 mM NaCl (circles) and after returning to 140 mM NaCl (triangles). (D,E) Inside-out patches expressing TMEM16F wt were exposed to the indicated NaCl concentrations and the IV relations were determined by voltage ramps from +100 to −80 mV. Only the regions around the reversal potentials are shown. Currents were activated by 100 μM CaCl2 and leakage currents measured in 0 Ca2+ were subtracted. The pipette solution contained 140 mM NaCl (D) or 14 mM NaCl (E). (F) Comparison of PNa/PCl calculated with the Goldman-Hodgkin-Katz equation from reversal potentials measured in different recording conditions (n = 7–19; *** p < 0.001 Tukey test after ANOVA, F = 48.256 p =…

Figure 4. TMEM16F wt ion permeability ratio PNa/PCl in excised inside-out patches depends on ionic strength but not on intracellular Ca2+ concentration. (A) An inside-out patch expressing TMEM16F wt was exposed to the indicated cytoplasmatic NaCl concentrations (in mM) and the IV relations were determined by a voltage ramp from +100 to −80 mV. Only the regions around the reversal potentials are shown. The current was activated by 100 μM CaCl2. Leakage currents measured in 0 Ca2+ were subtracted. (B) Average reversal potentials (Vrev) corrected for liquid junction potentials were plotted versus NaCl concentrations (n = 11–19). The continuous line was calculated according to the Goldman-Hodgkin-Katz equation with PNa/PCl = 3.7. (C) An inside-out patch expressing TMEM16F was exposed to 140 or 14 mM cytoplasmatic NaCl and the IV relation was determined by a voltage ramp from +100 to −80 mV. The current was activated by 13 μM CaCl2. (D) Comparison of PNa/PCl calculated with the Goldman-Hodgkin-Katz equation from reversal potentials measured with the indicated Ca2+ concentration (n = 8–19, p = 0.19 unpaired t-test).

Figure 5. TMEM16F wt ion permeability depends on the recording configuration. (A) Representative whole-cell voltage-clamp recordings obtained from TMEM16F wt-transfected cells with intracellular solution containing 100 μM CaCl2. Each cell was exposed to a control solution containing 140 mM NaCl (black traces) or other salts as indicated (brown traces). On the right, IV relations measured from tail currents of the cells on the left with 140 mM NaCl (black squares) or 140 mM of the indicated salt (brown circles). (B) An inside-out patch expressing TMEM16F wt was exposed to bath solutions containing 140 mM NaCl (black traces) or the indicated salts (blue traces). The grey traces represent the wash out with NaCl. IV relations were determined by voltage ramps from +100 to −80 mV. Only the regions around the reversal potentials are shown. Currents were activated by 1 mM CaCl2 and leakage currents measured in 0 Ca2+ were subtracted. (C) Comparison of the shift of reversal potentials obtained in different recording configurations for the indicated ionic solutions (*** p < 0.001, for NaMeS: p = 9.7 × 10−12 and for NaSCN: p = 8 × 10−6, unpaired t-test).

Figure 6. Q559K ion permeability ratio PNa/PCl depends on the recording configuration. (A,B) Representative whole-cell voltage-clamp recordings obtained from TMEM16F Q559K mutant-transfected cells with intracellular solution containing 300 μM Ca2+. Voltage protocols are shown at the top of the panels. (B) A cell was exposed to a control solution containing 140 mM NaCl, then to 14 mM NaCl, followed by wash out with 140 mM NaCl. (C) IV relations measured from tail currents of the cell shown on the left with 140 mM NaCl (squares), 14 mM NaCl (circles) and after wash out (triangles). (D,E) Inside-out patches expressing TMEM16F were exposed to the indicated NaCl concentrations, and the IV relations were determined by voltage ramps from +100 to −80 mV. Only the regions around the reversal potentials are shown. Currents were activated by 1 mM CaCl2 and leakage currents measured in 0 Ca2+ were subtracted. The pipette solution contained 140 mM NaCl (C) or 14 mM NaCl (D). (F) Comparison of PNa/PCl in different recording conditions for Q559K and wt (n = 5–19; *** p < 0.001, * p < 0.05 Tukey test after ANOVA, F = 18.271, p = 8.36 × 10−6; ### p < 0.001, unpaired t-test).

Figure 7. Ion permeability of the TMEM16F wt, D703R and Y563A mutants. (A) Inside-out excised membrane patches expressing TMEM16F wt or the indicated mutant were stimulated with a solution containing 1 mM Ca2+ at the time indicated in the upper traces. The holding potential was +60 mV. Patches were exposed to 140 mM NaCl and (B) 14 mM NaCl, (C) 140 mM NMDG-Cl, (D) 140 mM NaMeS or (E) 140 mM NaSCN. IV relations were determined by voltage ramps from +100 to −80 mV. Only the regions around reversal potentials are shown. Leakage currents measured in 0 Ca2+ were subtracted. (F) Comparison of PNa/PCl for TMEM16F wt and the indicated mutants (n = 3–19, *** p < 0.001, Dunnett test after ANOVA F = 19.303, p = 7.26 × 10−6). (G) Comparison of the shift of reversal potentials after the replacement of 140 mM NaCl with 140 mM NMDG-Cl, NaMeS or NaSCN for TMEM16F wt and the indicated mutants (for NMDG-Cl: *** p < 0.001, Dunnett test after ANOVA F = 30.569, p = 7.79 × 10−6; for NaMeS: *** p < 0.001, Dunnett test after ANOVA F = 26.3, p = 3.86 × 10−7; for NaSCN: ANOVA, F = 1.499, p = 0.25).