Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity

Figure 1.. TMEM16F Q559K shifts its reversal potential in response to change of intracellular Ca2+ concentration. (A, B) Representative recordings of TMEM16F wild type (WT) and Q559K in different Ca2+ concentrations. Traces were recorded from transfected HEK293 cells and the inside-out patches were held at +80 mV. The shades illustrate 1 min treatment with 1 mM Ca2+ that catalyzes PIP2 degradation by membrane-tethered phospholipase. (C) Dose-response curves for Ca2+-activation of WT and Q559K before and after 1 mM Ca2+ treatment, respectively. Currents before and after 1 mM Ca2+ were separately fitted to the Hill equation and normalized to their respective maximal amplitudes. (D) Change of current magnitudes of WT and Q559K. The currents were normalized to the maximal magnitudes before 1 mM Ca2+ for each cell. *p<0.05 (one sample t test against hypothetical value ‘1’). (E, F) Representative I-V relationships of WT and Q559K recorded in indicated conditions. The traces were recorded with a hyperpolarizing ramp from +80 mV to −80 mV (−1 V/s) following holding at +80 mV. (G) Scatter plot of reversal potentials (Erev) obtained from traces as in E and F without background correction. p-Values…

Figure 1—figure supplement 1.. Voltage-dependence of TMEM16F WT and Q559K steady-state activation. (A, B, C, E, G, F) Representative traces recorded with a voltage family protocol from −40 mV to +160 mV with 10 mV increments followed by holding at +100 mV, as in (D). The traces recorded at +100 mV are highlighted for comparison. (H) Averaged G-V relationships of the currents recorded in indicated conditions. Tail current (at +100 mV) magnitudes were measured from traces as in A ~ C, E ~ G. For WT and Q559K in 15 µM Ca2+ and Q559K in 1 mM Ca2+. Tail current magnitudes of each recording were fitted to the Hill equation against voltage, and they were subsequently normalized to the respective maximal values. We chose to use Hill equation since it provides a simplified estimation for the kinetics of voltage activation (Yifrach, 2004). Averaged G/Gmax values were calculated at the end for each group. For the other conditions, the tail current magnitudes were directly normalized to those following +160 mV for each recording, and the averaged G/Gmax values for each group were calculated at the end.

Figure 1—figure supplement 2.. Recordings in indicated conditions with a hyperpolarizing ramp protocol. (A,G) Time courses of TMEM16F WT and Q559K activation by 15 µM Ca2+ in 150 mM NaCl. The currents were recorded when the patches were held at +80 mV. (B, E, H, K) Recordings of reversal potential with a hyperpolarizing ramp protocol following constant holding at +80 mV. For the demonstrated traces, the solutions were applied in the order of 150 mM, 45 mM and 15 mM NaCl, and back to 150 mM NaCl (not shown). Ca2+-free solution was applied at the end to confirm the seal. (C, F, I, L) Detail amplification showing tail currents in B, E, H, K recorded at +80 mV following the hyperpolarizing ramp. Note that in F, the tail current is not significantly bigger than endogenous current (gray trace), excluding the feasibility to calculate Erev out of it. (D, J) Current magnitudes activated by 15 µM Ca2+ in 150 mM NaCl were plotted against the Erev values measured with 15 mM NaCl intracellular solution, indicating that there is no correlation.

Figure 1—figure supplement 3.. Additional control experiments for Figure 1. (A ~ D) Representative I-V relationships of currents recorded in indicated conditions. The recording protocol was the same as in Figure 1E,F. Wild-type TMEM16F current in 1 mM Ca2+ is not distinguishable from HEK293 cell endogenous currents. (E) Representative I-V relationships of TMEM16F Q559K current to show the Erev in 1 mM Ca2+. Ca2+-free solution was applied at the end to confirm the seal. (F) One example of TMEM16F Q559K recorded in 1 mM Ca2+ to illustrate the challenge of background subtraction. Note that the trace of the current recorded in Ca2+-free 15 mM NaCl solution (orange solid trace) did not cross with that in 1 mM Ca2+ 15 mM NaCl (blue trace), attributable to the inhibition of endogenous current by 1 mM Ca2+, shown in C. Extrapolation (orange dotted trace) indicates that the current after background subtraction (a) should reverse at a more negative potential than that without subtraction (b), justifying our ‘safer’ step of not performing background subtraction (using b) for comparison involving recordings in 1 mM Ca2+.

Figure 1—figure supplement 4.. Permeability ratio PNa+/PCl- was altered in WT TMEM16F and Q559W in response to change of Ca2+. (A) Representative recordings of WT TMEM16F held at +80 mV with a hyperpolarizing ramp (−1 V/s) once every 5 s in indicated conditions. (B) Representative I-V relationships of currents recorded at labeled time points. (C) Scatter plot of the reversal potentials (Erev) at labeled time points without background subtraction. p-Values were determined with Tukey's multiple comparisons following one-way ANOVA. (D) Representative I-V relationships of Q559W recorded in indicated conditions. Traces were recorded with the same protocol as in Figure 1E,F. (E) I-V relationships of the currents in 15 µM Ca2+ and 1 mM Ca2+ (both in 15 mM NaCl) after background subtraction (subtraction of the current in Ca2+-free 15 mM NaCl solution, orange trace in D). (F) Scatter plot of reversal potentials (Erev) obtained from traces as in D (without background subtraction) and in E (after background subtraction). p-Values were respectively determined with Sidak's multiple comparisons following two-way ANOVA and two-tailed Student's t-test.

Figure 1—figure supplement 5.. I- permeability of TMEM16F WT and Q559K. (A, B) Representative I-V relationships of currents recorded in indicated conditions showing the shift of reversal potentials when the bath (intracellular) solution was switched from 150 mM NaCl to 150 mM NaI. (C) Scatter plot of the changes of reversal potentials (ΔErev) when solution was switched from 150 mM NaCl to 150 mM NaI. WT currents in 1 mM Ca2+ were not included because they were not distinguishable from the currents endogenous to HEK293 cells. Because the interpretation methods for bi-ionic condition were not applicable here, we did NOT perform statistics and instead showed the values directly.

Figure 2.. TMEM16F Q559K_E667Q channel is more permeable to Cl- than to Na+ in 1 mM Ca2+despite being half activated. (A, B) Representative I-V relationships of WT and Q559K currents recorded in indicated conditions. The recording protocol was the same as in Figure 1E,F. Ca2+-free 15 mM NaCl solution was applied at the end for background subtraction. (C) Scatter plot of the changes of reversal potentials (ΔErev) when solution was switched to 15 mM NaCl, obtained from traces as in A and B. p-Values were determined with Fisher's LSD test after two-way ANOVA. (D) Representative recordings of TMEM16F Q559K_E667Q in different Ca2+ concentrations. The recording protocol was the same as in Figure 1A. (E) Dose-response curve for Ca2+-activation of Q559K_E667Q. The gray dotted line represents the curve for Q559K after 1 mM Ca2+, replotted from Figure 1C. (F) Representative I-V relationships of Q559K_E667Q recorded in indicated conditions. The recording protocol was the same as in Figure 1E,F. The insert shows the scatter plot of reversal potentials (Erev) obtained from traces as in F.

Figure 3.. Q559K Erev only shifts with the change of Ca2+ concentration. (A) Representative recording of TMEM16F Q559K held at +80 mV with a hyperpolarizing ramp (−1 V/s) once every 10 s in indicated conditions. (B) Summary of the averaged Erev with background subtraction (subtraction of the current in Ca2+-free 15 mM NaCl solution, orange trace in A, except for the current in 1 mM Ca2+) in indicated conditions. For all the traces recorded in 15 mM NaCl, Erev only shifts when Ca2+ concentration is changed. (C, D, E) Representative I-V relationships of Q559K at arrowed points in A, showing that Erev shifts when Ca2+ is increased from 15 µM to 36 µM, persists in 36 µM Ca2+ despite the constant rundown, and shifts when Ca2+ is increased from 36 µM to 1 mM.

Figure 4.. The change of ion permeability ratio is preserved despite TM6 conformational stabilization. (A, B, C) Representative traces of G615A and Q559K_G615A recorded with a voltage family protocol as in Figure 1—figure supplement 1. The currents recorded at +100 mV are highlighted for comparison. (D) Averaged G-V relationships of G615A and Q559K_G615A currents. The method for data analysis was the same as that for WT in 15 µM Ca2+. The two traces for Q559K were replotted from Figure 1—figure supplement 1H. (D) Representative I-V relationships of Q559K_G615A recorded in indicated conditions. (F) Scatter plot of reversal potentials (Erev) obtained from traces as in E without background correction. p-Values were determined with Sidak's multiple comparisons following two-way ANOVA.

Figure 4—figure supplement 1.. TM6 functions in TMEM16F gating. (A) Alignment of TM6 sequences of TMEM16A and TMEM16F. The numbering for TMEM16A represents the isoform as used by Lam and Dutzler (2018). (B, D, E, F). Representative traces of TMEM16F mutants recorded in 15 µM Ca2+ with a voltage family protocol as in Figure 1—figure supplement 1. Currents recorded at +100 mV are highlighted for comparison. (C, G) Averaged G-V relationships of indicated TMEM16F mutants. The method for data analysis was the same as that for WT in 15 µM Ca2+. The traces for WT and G615A were replotted from Figure 1—figure supplement 1H and Figure 4D.

Figure 4—figure supplement 2.. Recordings in indicated conditions with a hyperpolarizing ramp protocol. (A) Time courses of TMEM16F Q559K_G615A activation by 15 µM Ca2+ in 150 mM NaCl. The currents were held at +80 mV. (B) Recordings of reversal potentials with a hyperpolarizing ramp protocol following constant holding at +80 mV. The solutions were applied in the order of 150 mM, 15 mM NaCl with 15 µM Ca2+, 150 mM NaCl, 15 mM NaCl with 1 mM Ca2+. Ca2+-free solution was applied at the end to confirm the seal. (C) Detail amplification showing tail currents in B recorded at +80 mV following the hyperpolarizing ramp.

Figure 5.. The change of ion permeability ratio is preserved when current is activated by Zn2+ or Gd3+. (A, B) Representative I-V relationships of WT and Q559K recorded in indicated Zn2+-containing solutions. The currents were recorded with the same protocol as in Figure 1E,F. (C) Scatter plot of reversal potentials (Erev) obtained from traces as in A and B. p-Values were determined with Sidak's multiple comparisons following two-way ANOVA. (D, E) Representative I-V relationships of WT TMEM16F recorded in indicated Gd3+-containing solutions. Note that the current in 1 mM Gd3+ 15 mM NaCl is still distinguishable from background current. (F) Scatter plot of reversal potentials (Erev) obtained from traces as in E and F. p-Values were determined with Sidak's multiple comparisons following two-way ANOVA.