In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.TMEM16F is activated by elevated intracellular Ca When ac2+ . This pr2+ . TMEM16F channel , raisingn to Cl- . Surprisectivity  or even n to Na+ . This di2+-activated Cl- channels (CaCC) without lipid scrambling functions   in different Ca2+ concentrations. The shift of permeability ratio does not correlate with alterations of channel open states, but instead is regulated by the change of electrostatic field along the permeation pathway, on which divalent cations such as Ca2+ and Zn2+ have more significant impact than monovalent ions. Depolarization, which facilitates intracellular cation entry into the membrane electric field, promotes the shift of the relative permeability toward a preference for Cl- over cations. Such an electrostatic modulation could reflect a general feature of the mechanism of ionic transportation by TMEM16 proteins, and it suggests that TMEM16F harbors an inherent machinery that allows it to dynamically modulate preference between cations and anions in response to its local environment.In TMEM16F, glutamine 559 (Q559) faces the ionic permeation pathway, and lysine substitution of this pore-lining residue (Q559K) reduces the ratio of Naa+/PCl-) . Previoufication . In this2+ in a dose-dependent manner. The current started to decrease when Ca2+ was higher than ~30 µM, as a result of both desensitization and rundown . Rundown refers to a reduction of current magnitude (induced by 1 mM Ca2+ in this case). To measure the voltage dependence of activation, we recorded with a voltage-family protocol from −40 mV to +160 mV with 10 mV increments followed by holding at 100 mV to obtain ‘tail-currents’, which we used as indicators of steady-state conductance at the range of voltages. Fitting to the sigmoidal conductance-voltage (G-V) relationship yielded V1/2 of 76 ± 11 mV in 15 µM Ca2+ for wild-type TMEM16F, while in 1 mM Ca2+, desensitization strongly reduced the voltage-gating, as was previously reported (rev) of wild-type TMEM16F in 1 mM Ca2+. In our experiments, we measured the Erev from the current recorded with a hyperpolarizing ramp  following holding the excised patch at +80 mV. When we switched the bath  from 150 mM NaCl to 45 mM or 15 mM NaCl , the current recorded from Tmem16f-transfected cells immediately diminished with the hyperpolarizing voltage and was indistinguishable from the background current endogenous to HEK293 cells , significantly different from 0.36 ± 0.13 for wild type  or wild type in 1 mM Ca2+ . Q559K mutant channels in 1 mM Ca2+ had a lower threshold for voltage activation than in 15 µM Ca2+  in TMEM16F also prevents current rundown  (- (versus Na+) when Ca2+ concentration was raised from 15 µM to 1 mM, in both wild type and mutants.We asked whether the phenotype of E current . Thus, w rundown . We meastration) . The abo+, K+, Ca2+) were higher than that to Cl-, while the permeability to some other cations may be lower  from those recorded in 6 and 15 µM Ca2+ for each excised patch. We found that for each recording from either WT or Q559K, the current evoked by 6 µM Ca2+ reversed at a more positive voltage than that by 15 µM Ca2+  for 90–100 s and measured Erev once every 10 s  remained unchanged despite the constant decrease of current magnitude and the increase of outward rectification. We therefore concluded that the reversal potential of Q559K shifted only when Ca2+ concentration was changed, independently of current magnitude, open state, or rectification level. Also, although it was not clear whether the permeation pathway could be partially composed of the headgroups of lipids being scrambled, we inferred that at least PIP2, which was slowly depleted during incubation in 36 µM Ca2+, did not contribute to the shift of ion permeability ratio.To dissociate the correlation between Catization . We mainery 10 s . The Ere2+ sensitivity  works as a hinge to allow the rearrangement of the helical segments during channel activation to generate the ionic permeation pathway . Alaninesitivity . This glsitivity . Cryo-EM TMEM16F . Previousitivity , and we vity  TMEM16F , although it remains possible that TMEM16F might undergo miniscule conformational changes at different Ca2+ concentrations that contribute to the transition of relative permeability.Notably, the steady-state voltage dependence of TMEM16F G615A channel in 15 μM Ca mM Ca2+ . We thentionship . Recordetionship . The cha TMEM16F . This do2+ alters anion accessibility to the TMEM16A pore through electrostatic effect  with elevation of Zn2+ concentration from 10 μM to 1 mM  and tetraethylammonium (TEA) , so intrtrations . Compareectively , consistNa+/PCl- . The PNa mM NaCl , suggest2-SO4. We observed that the wild-type TMEM16F activated by 15 µM Ca2+ in both conditions reversed at ~0 mV  or 68 mM NMDGersisted , which c2-SO4 to the pipette solution , while the bath  contained 150 mM NaCl. With the correction of liquid junction potentials , the currents reversed at ~0 mV when activated by 15 µM Ca2+ in all the conditions , while at 11 ± 2 mV, 19 ± 1 mV and 24 ± 1 mV, respectively, when activated by 1 mM Ca2+  would reduce the channel relative permeability to cations. We performed whole-cell recording with 150 mM NaCl and 15 µM Ca2+ in the pipette, the Erev was 1.4 ± 3.1 mV in 15 mM NaCl, indicative of PNa+/PCl- of 1.0 ± 0.1  or I- shifted Erev to positive or negative values respectively, suggesting the presence of Cl- conduction  and trivalent cations (Gd3+) having stronger effects than monovalent ions (Na+ and NMDG). This suggests that TMEM16F employs a charge-screening mechanism to dynamically alter the preference for permeating ion species: Increase of positive charges, by introduction of basic amino acids or by entry of cations, enhances the channel preference for anions. This mechanism allows TMEM16F to open as a non-selective ion channel at low intracellular Ca2+ level and as a Cl- channel when local Ca2+ concentration increases, indicative of physiological functions that have not been investigated thus far.Taken together, the elevation of intracellular cation level underlies the increased permeability to Cl2+ and depolarization in the gating of TMEM16A. Depolarization facilitates Ca2+ entry into the membrane electric field and thus reducing the EC50 for channel activation by Ca2+  followed by a hyperpolarizing ramp from +80 mV to −80 mV (−2 V/s), to measure the Erev  , leading to a right-shift of Erev, which was opposite to our results. Traces of Q559K currents following different conditioning potentials in 1 mM Ca2+ overlapped at low membrane voltages  . The trachannels , since ivoltages , a phenoages . We then recorded the Erev with hyperpolarizing ramps of variable speeds (0.3 V/s to 3 V/s) following holding the patch at +120 mV, to test whether the channel would ‘forget’ the ion permeability ratio at the conditioning potential (+120 mV) by observing Erevs under hyperpolarization ramps of various rates. We did not see a significant shift of the Erev for both WT and Q559K, although wild-type TMEM16F current became more outwardly rectifying at slower hyperpolarizing ramps (Cl-/PNa+) can be maintained during hyperpolarization, raising the possibility that the Ca2+ ion that modulates the pore electrostatic field dwells at the permeation pathway or a proximal site that allows it to still affect permeation pathway instead of leaving the pore with the ionic flow.The observation that TMEM16F in 15 µM Cang ramps . Thus, t2+-binding to the conserved Ca2+-binding pocket. The elevation of intracellular divalent or trivalent cation concentration brings positive charges into the permeation pathway, which gradually increases the relative permeability of anions. Depolarization promotes the pore attraction to anions by facilitating Ca2+ entry into the membrane electric field, wherein it may remain until the channel is closed.We summarized the mechanism of TMEM16F dynamic ion selectivity based on the experiments shown above and published previously . TMEM16F2+ concentration leads to activation of TMEM16F channel that mediates Ca2+ influx . With membrane potential and ionic strength unchanged, the permeability ratio PNa+/PCl- is only correlated with Ca2+ concentration rather than Ca2+-induced conformational change, suggesting that the ion selectivity machinery is not coupled to channel gating. At low Ca2+ when channels display modest selectivity between Na+ and Cl-, the ion permeability ratio is also altered by monovalent ions and membrane potential, which indicates that the ion selectivity filter is sensitive to electrostatic disturbance and thus might explain the discrepancy of experimental results from different labs. Depolarization, which drives Ca2+ into membrane electric field and facilitates Ca2+-gating, synergistically promotes the permeability alteration towards a preference for Cl-. The ion permeability ratio can be memorized during the recording with a hyperpolarizing ramp, suggesting that Ca2+ retreating from the site that affects the permeation pathway is preceded by the dissociation of Ca2+ from the binding pocket, so that the pore electrostatic field can be maintained until the channel is closed. PIP2 reduces the EC50 for Ca2+-activation but does not affect ion selectivity, thus facilitating TMEM16F opening when the pore is still more attractive to cations in low intracellular Ca2+. Controlling the selectivity between cations and anions by altering the pore potential, referred to as a charge-screening mechanism, is also documented in other ion channels, such as ligand-gated channels  in the Ca2+-binding pocket, or in the permeation pathway, may alter the electrostatic field of the pore. The former resembles what has been proposed for TMEM16A  is outwardly rectifying, while at high intracellular Ca2+ level that allows two Ca2+ ions to occupy the binding-pocket, the TMEM16A channel conductance becomes ‘Ohmic’  than that in low NaCl balanced with mannitol  displayed a dispersed distribution of Erev  might provide a brake to the positive feedback to reduce the influx of cations including Ca2+. In a typical neuronal cell, such a transition towards Cl- selectivity might reduce the entry of Ca2+ into the cell and drive membrane potential close to Cl- equilibrium potential, both of which will reduce the neuronal excitability and prevent Ca2+-overloading. The ‘memory’ of the permeability ratio during repolarization allows TMEM16F to modulate the waveform of action potentials in accordance to local cytoplasmic Ca2+ level. In non-excitable cells, this function might enable TMEM16F to sense cellular Ca2+ level and modulate membrane potential, which affects cellular proliferation and differentiation  were respectively fused with mCherry and subcloned into pcDNA3, as previously reported (CVCL_0045) were aliquoted and preserved in liquid nitrogen. One vial of cells was thawed and plated in 10-mL cell culture flask  once every 3 months. Cell were cultured in Dulbecco's Modified Eagle Medium  containing 10% FBS  and 1% penicillin-streptomycin, at 37°C and with 5% CO2, and they were passaged into a new flask with a 1:10 ratio once every 3 ~ 4 days. They were disposed of if they were not confluent in the flask in 4 days, their morphologies deviated from pictures in instructions (ThermoFisher website), or if the batch had been used for 3 months, whichever coming first, but were not in particular tested for mycoplasma contamination. Cells for electrophysiology recording were plated on 12 mm round coverslip  during the passaging procedure. Transient transfection was performed with Lipofectamine 2000  2 days before recording. The cDNA constructs for wild-type TMEM16F-mCherry, the mutants Q559K-, G615A-, G614A-, I612A-, G614_G615A-, N620A-, and E667Q- mCherry were stably transfected in HEK293 cells as previously reported . For whole-cell recording, the pipette solution contained 150 mM NaCl, 10 mM HEPES, 5 mM HEDTA and 4.1 mM CaCl2, and the final free Ca2+ concentration (15 µM) was confirmed with Oregon Green BAPTA 5N. The osmolality of each solution was adjusted to 290 ~ 310 mOsm/kg.For all electrophysiology recordings, bath solution contained 145 mM NaCl, 10 mM HEPES, 2 mM CaCl2+ < 100 µM) with isotonic mannitol solution , NMDG-MES solution , or NMDG2-SO4 solution . Ca2+ concentration was subsequently confirmed with Oregon Green BAPTA 5N. The 1 mM Ca2+ solutions were made by directly mixing the solution containing 150 mM NaCl, 10 HEPES with the respective isotonic solutions, and Ca2+ was added at the end. Solutions in 2+. Zn2+ and Gd3+ were directly added to solution of 150 mM NaCl, 10 HEPES from 100 mM stocks. The pH values of all the solutions were confirmed to be ~7.3. All the chemicals were purchased from Sigma-Aldrich .Solutions with NaCl lower than 150 mM were made by mixing the above solutions  and plated onto 12 mM coverslip  3 ~ 4 days before recording. For recording, coverslips with cells were transferred to a chamber on a Nikon-TE2000 Inverted Scope  and transfection was confirmed with fluorescent microscopy. Patch borosilicate pipets  were pulled from a Sutter P-97 puller with resistances of 2–3 MΩ for inside-out patch recordings. Solutions were puffed to the excised patch using VC3-8xP pressurized perfusion system . Data were acquired using a Multiclamp 700B amplifier controlled by Clampex 10.2 via Digidata 1440A . All experiments were performed at room temperature (22–24°C).Na+/PCl-). For recordings with 150 mM NaCl pipette solution, the liquid junction potentials for all the other conditions were within ±2 mV, and were not corrected in the figures. For recordings where the pipette solution contained 15 mM NaCl , the liquid junction potentials were corrected by adding 12 mV, 6 mV and 2 mV, respectively, to the measured values, as estimated by Clampex. The data in For measurements of reversal potentials, a 3 M KCl salt bridge was used. For experiments with 150 mM NaCl in the pipette solution, based on prediction by Clampex , the liquid junction potentials for 15 mM NaCl, 45 mM NaCl  were −1.7 mV and −1.0 mV respectively and were only corrected for calculation of permeability ratios , OriginLab , and Graphpad Prism . For the measurement of Ca2+-sensitivity, every trace was fitted to the Hill equation to generate its respective EC50 and H (Hill coefficient). The curves in the figures display the averaged current magnitudes normalized to their respective maximal values (I/Imax %). PNa+/PCl- values were calculated with the simplified equation derived from Goldman-Hodgkin-Katz equation:+]o, [Cl-]o, [Na+]i, and [Cl-]i are extracellular and intracellular Na+ and Cl− concentrations, respectively.All data were analyzed using pClamp10 .Thank you for submitting your work entitled "Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in rev, including potential problems arising from not subtracting background conductances or controlling for potential changes in ion concentrations during your recordings. There were also concerns with your proposed mechanism and whether the changes you see may be physiologically relevant, The reviewers articulate their concerns at considerable length, which we hope you will find helpful in sorting out whether the ion selectivity of TMEM16F is indeed dynamic, and if so what the underlying mechanism and physiological relevance may be.The reviewing editor and reviewers found your manuscript on TMEM16F to be interesting and provocative. However, after careful consideration, the consensus was that there are substantive technical concerns with your measurements of VReviewer #1:This manuscript by the Jan lab investigates the selectivity of the TMEM16F channel/scramblase. Multiple contrasting publications in the literature reported that TMEM16F-mediated ionic currents are anion-, cation- or non-selective. Here, Ye and colleagues show that the selectivity of TMEM16F changes dynamically as a function of divalent cations, ionic strength, and voltage. The authors propose that this shift in selectivity is not due to the ability of the TMEM16F ion pore to adopt conformations with different selectivity properties, but rather to the modulation by divalent cations of the electric field within the pore.These findings are important because they provide a unifying framework to interpret the contrasting and confusing data present in the literature. However, I have three major concerns with regards to the interpretation of the data, and several minor issues that need to be addressed.2+ concentrations, while lipid scrambling requires only elevation of Ca2+, but not depolarization. Thus, it is not clear under what physiological conditions the self-braking mechanism enabled by the dynamic switch in selectivity might play a role. For example, does the membrane potential of platelets and/or apoptotic cells reach and maintain the depolarizations necessary for channel activation? Furthermore, the magnitude of the observed switch is relatively modest; in all conditions the channel is poorly selective between anions and cations, displaying only modest preferences for one over the other. In no cases do the currents approach ideal anion vs cation selectivity. Given the overall poor selectivity, I wonder how much of an effect would be caused by the observed switch in selectivity. Thus, while the phenomenon described here is biophysically interesting, I am not convinced of its physiological relevance. Some quantitation of the effects observed here within the context of a cell would be needed to establish a clear link to physiology.1) TMEM16F is a scramblase, as such its main function is lipid transport to externalize PS to initiate blood coagulation and recognition of apoptotic cells by macrophages. The physiological relevance of TMEM16F-mediated ionic currents is -at the moment- unclear. Further, activation of TMEM16F currents require sustained depolarizations and high Ca2) It is not clear through what type of pore ions move in TMEM16F. It has been proposed that the TMEM16 scramblases form a lipid-lined pore through which ions can diffuse together with the lipids that are being scrambled . If this were the case, then the selectivity of the channel would be affected by multiple factors such as lipid headgroups, voltage and mono- and di-valent ions. On the other hand, the TMEM16A homologue forms a more conventional, protein-delimited, pore. In this case, the observed changes in selectivity could reflect either a charge-screening effect (as the authors propose) or the ability of the channel's pore to adopt slightly different conformations. Further, most of the measurements are carried out using a mutant, Q559K that affects both selectivity and gating of TMEM16F. I understand that the mutant was used because of the removal of run-down. It is unclear whether this construct can serve as a good model for the WT pore. The mutation could case subtle rearrangements in the pore rendering it more sensitive to environmental changes. While the present experiments provide evidence suggesting that the selectivity of TMEM16F changes, they shed little light into the mechanisms underlying such a switch.2+ are naturally smaller than those in 1 mM Ca2+, and therefore these effects are less pronounced leading to the observed differences. Did the authors use different directs of the ramp and/or tail current protocols to measure reversal potentials? Is there an effect on the direction of the ion switch (i.e. going from 150 NaCl to 15 NaCl is the same as going from 15 NaCl to 150 NaCl)? Do they observe a dependence of the magnitude of the effects as a function of the current in the patch?3) A technical concern I have is that the measured currents are relatively large (~1 nA) and the stimulation protocols used are long. Can the authors rule out that the effects they see are due to ion accumulation/depletion effects? The currents in 15 μm Ca2+ concentration”. The reversal potential of the QK/EQ mutant suggest this mutant is less Cl- selective than the QK mutant alone. Thus, at an intermediate Po the selectivity is also intermediate. This is more consistent with a model where the pore can adopt multiple open states with different selectivity. What happens to the currents of the QK mutant alone (or WT) when measured at a Po~0.5? I do not think that the authors' conclusion that "This indicates either that TMEM16F ion selectivity is not correlated with its open state, or that the E667Q mutation circumvents the intermediate open state (if any) and causes the mutant channel to directly enter a fully-open conformation" is warranted by the data.4) Subsection “Q559K channel ion selectivity varies with intracellular Carev depends on divalent ions  then the shift should depend in a graded manner on their concentration. Did the authors test what happens when using intermediate increases between the 15 and 1000 μm concentrations shown?5) Subsection “The switch of ion selectivity correlates with electrostatic change in permeating pathway”. If the shift in V6) Are divalent anions inert? Since the channel is permeable to anions under all conditions , these ions can enter the pore and therefore should have the opposite effect of the divalent cations.7) The inactivation in Figure 4A is slow (tau~30s – 1 min). Why weren't the reversal potentials measured after reaching activation but before inactivation sets in? Is inactivation dependent on voltage? If the patches are held at a membrane voltage closer to reversal is there a similar effect?2+ confusing where they regard the rapid run-down that is PIP2 independent. Are there multiple run-down mechanisms?8) I find the effects of Zn9) Discussion section. The authors state "However, TMEM16F does not have an "Ohmic" state: the channel always requires depolarization to be activated." What does this mean? That the pore is intrinsically rectifying? Ohmic state would refer to conductance, not gating.Reviewer #2:While TMEM16F's dual roles as a calcium-activated phospholipid scramblase and ion channel are well established, its’ ionic selectivity remains the subject of debate. In this work, the authors examine whether the ionic selectivity of TMEM16F can change in response to solution composition and membrane voltage. Using the Q559K mutation to prevent channel rundown, the authors report that TMEM16F switches its preference from cations to anions depending on the intra-cellular calcium concentration and membrane voltage used to activate it, and they propose that this switch results from the calcium ions and membrane potential changing the electrostatic field in the permeation pathway.The authors results are very interesting, but I have several concerns.1) Experimental artifacts: To combat channel rundown and voltage-dependent activation, the authors have resorted to protocols with sizeable resting membrane currents and conductance, conditions that can easily lead to ion accumulation/depletion artifacts : 115-124.).2+], holding voltage). If the reversal potential for each condition is essentially independent of the holding current/conductance, ion accumulation/depletion artifacts are unlikely. Reporting key experimental details  would help address these concerns.I am sure the authors have thought carefully about this issue and probably already have data to exclude this possibility. For example, since the channel undergoes desensitization, they should have recordings in which early ramps have a much larger conductance than later ramps recorded under identical conditions (e.g. , but becomes anion selective at high Ca2+. Also, they show that manipulating the opening state of the channel does not affect this ion-selectivity shift, but manipulating electrostatic field in the permeation pathway of the channel does. Finally, they also found that more positive potential tends to increase the anion permeability. They speculate that the discrepancy of TMEM16F ion selectivity reported in the literature may result from different recording conditions such as holding potentials and ion concentrations.This manuscript explores how Ca2+ concentrations used) is a little bit hand waiving. In the literature, the permeability difference in TMEM16F between Na+ and Cl- is not huge. Even accepting the results in this study as face value, the TMEM16F's PNa/PCl ratios range from 0.5 to 2 . It is therefore strange that the authors would call such a several-fold difference of Na+/Cl- permeability as Na+ selective (or Cl- selective in the other way around). Even worse, the changes of the PNa/PCl permeability ratios reported here may actually be artefacts.While resolving the controversy in the cation/anion selectivity of TMEM16F is important, the authors' conclusion that the controversy results from different recording conditions used in various labs  shift from ~ 0 mV to ~ -15 mV . In Yang et al., 2012, a similar 10-fold change of [NaCl]i in the presence of 0.5 mM Ca2+ generated a right shift of the reversal potential to ~+20 mV . Both experiments are excised inside-out patch recordings, and the solutions are similar (150 mM/15 mM vs 140 mM/14 mM). The [Ca2+]i in Yang et al. was 0.5 mM, which should be closer to 1 mM Ca2+ than to 15 µM Ca2+. Why did the authors obtain a left shift of the reversal potential while in Yang et al., 2012, the reversal potential shifted to the right?In the present study and the Yang et al. paper cited by the authors , there are at least two serious technical problems that could contribute to the controversy. In fact, the result from Yang et al., 2012 and the result in this paper, both from the same lab, already contradict each other. Here is a striking example: in the present study, the authors show that with a high concentration (1 mM) of CaTwo technical issues in both the current study and Yang et al., 2012 are not seriously dealt with.2+  should only be ~ -4 mV (with a 10-fold NaCl gradient). The reversal potential shift in Q559K in 15 µM Ca2+ in the paper is ~ +10 mV . After junction potential correction, this should be ~+20 mV.The first serious technical problem is the junction potential correction. Throughout the whole paper of Yang et al., 2012, there was no description of junction potential correction. In the present study, the authors mention that "the liquid junction potentials for 15 mM NaCl, (and) 45 mM NaCl were -1.7 mV and -1.0 mV respectively.……". I suppose that the authors calculated these values with respect to the 150 mM NaCl solution. If so, these values are severely under-estimated. In fact, using the same calculator (Clamplex), the value of liquid junction potential for 15 mM NaCl (with respect to 150 mM NaCl) is -11 mV . Since the data points in the figures  are not corrected for the junction potential, the reversal potential of ~ -15 mV in 1 mM Ca2+) and ~ +60 mV (in 15 µM Ca2+) . The conductance of the untransfected cell patches includes the leak conductance and the conductance from the endogenous channels/transporters. If we sum the two as the background conductance (abbreviated as gb), and the conductance from TMEM16F (the channel conductance) is denoted as gc, the measured reversal potential (Erev) should be a weighted sum of the reversal potential of gc (abbreviated as Ec) and that of gb (abbreviated as Eb) according to the following equation:The second and even more problematic issue is a lack of background current subtraction. Throughout the whole manuscript , I do not see a description of background current subtraction for the presented I-V curves . In the absence of TMEM16F conductance, the HEK293 cells have a cation-selective endogenous conductance. The authors actually show this fact from the background current measurement where the reversal potentials under 150 mM/15 mM NaCl condition were ~+20 mV (in 1 mM Carev = [gc/(gc+gb)] × Ec + [gb/(gc+gb)] × EbE2+ will reduce the TMEM16F conductance (gc), and therefore, Erev will approach Eb (which is +20 mV or +60 mV). When the gc is much larger than gb (this is the case when TMEM16F is activated by 1 mM Ca2+), the contribution of gb to the measured Erev is less significant . However, when gc is small relative to gb, the measured Erev will be severely contaminated by Eb. The example is no better than the one provided by the authors in Figure 6, where when the holding voltage is more positive (the TMEM16F conductance is larger because of its outward rectification property), the reversal potential is less positive (less contaminated by Eb). This phenomenon is consistent with all the results throughout the paper. Various manipulations in the paper  would not change this trend-the larger the gc, the less positive the measured Erev.Reducing [NaCl] or Ca2+, how large the total current should be so that the measured Erev is good enough to reflect the true TMEM16F reversal potential (Ec)? The equation above indicates that gc needs to be larger than 90% of the total conductance (gc + gb) in order to have a measurement where <10% of Eb  is added to the measured reversal potential. The authors show that the background current from untransfected cell patches at +80 mV is 50-80 pA . If the total current at +80 mV is 500 pA (0.5 nA) in the solution with 15 µM Ca2+ and 15 mM NaCl, as in many of the authors' recording traces, the conductance of TMEM16F at +80 mV is ~ 9-10 fold that of the background current. However, since WT TMEM16F and Q559K mutant are outwardly rectifying, their conductances are quickly reduced by several fold near the reversal potential (the outward rectification is even more severe in low NaCl and low Ca2+ condition). Therefore, the 9-10 fold gc/gb ratio at +80 mV may reduce to 2-3 near Ec (gb is constant at different voltages). Suppose the true value of Ec is 0 mV in the 150 mM/15 mM NaCl condition , and the value of Eb is +60 mV (as shown in the supplementary figure), a gc/gb ratio of 2-3 will give a measured reversal potential of +20 mV – +30 mV. If the total current at +80 mV is less than 0.5 nA, the situation will be even worse. Interestingly, the reversal potentials measured in 150 mM/15 mM NaCl condition in many experiments in this manuscript are in this range.Without subtracting the background current obtained in the absence of Ca2+ from current obtained in the presence of Ca2+. By doing background current subtraction, the authors would eliminate the shift in Erev caused by the background conductance and thus isolate the shift in Erev caused by Ca2+. It is interesting that similar background current contamination of the reversal potential measurement is also shown in TMEM16A and its K584Q mutant in this manuscript . However, it appears that the authors have not noticed this problem. Taking K584Q recordings as an example , the K584Q conductance is bigger in 1 mM Ca2+ than in 0.4 µM Ca2+. Therefore, the reversal potential is more shifted to the right in 0.4 µM Ca2+. Here, the measured reversal potentials (10 fold NaCl gradient) of K584Q in 0.4 µM and 1 mM Ca2+ are both far from the perfect Cl- Nernst potential. It has already been shown  that after background current subtraction, the K584Q mutant, like the wild-type TMEM16A, is perfectly Cl- selective in 20 µM Ca2+. In other words, based on the measured reversal potentials, K584Q in 20 µM Ca2+ in Jeng et al., 2016, is more Cl- selective than the K584Q current activated by 1 mM Ca2+ shown in this paper. The key difference between the two studies is the background current subtraction.The above example therefore emphasizes the importance of subtracting the background current in order to evaluate EC more precisely. However, I am only using the data provided by the authors to make the above estimate. Since the background currents vary from patch to patch, the easy way to minimize the contribution of the background conductance is to subtract the current obtained in the absence of CaWith these technical flaws, the reported reversal potentials in this manuscript are simply not correct. I am not sure if the authors have measured background I-V curve in every recorded patch. If not, I do not think the problem can be corrected within a short time. If these two technical problems are corrected, I anticipate that conclusion of the paper is probably not going to hold.[Editors’ note: below is the decision letter following the authors’ appeal.]eLife. Your letter of appeal has been considered by Rick Aldrich as Senior Editor and Kenton Swartz as Reviewing editor, and we are prepared to consider a revised submission with no guarantees of acceptance.Thank you for choosing to send your work entitled "Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity" for consideration at The editors and reviewers have read and discussed your rebuttal. It is important to point out that our initial concerns remain, and we are not convinced that your plan for revision will satisfy those concerns. However, we are willing to reconsider a revised manuscript after you have had a chance to do the additional experiments. The following are several points that you should consider in preparing the revised manuscript.rev. The data provided in your rebuttal does not address this issue, as in different ionic concentrations the shape and Vrevof the leak will change. We suggest that you bracket each IV measurement in any solution containing calcium with measurements of the background IV (i.e. 0µM Ca2+) in solutions with identical NaCl concentration so that the voltage dependence of the total and leak currents can be directly compared. We understand that there is no perfect way to identify and subtract leak currents for TMEM16F, but by showing raw and leak corrected data it will be possible to estimate the total and leak conductances at the reversal potential and thereby gauge the accuracy and reliability of the Vrevcalculations. Since this does not appear to have been done in your initial experiments, we would request that they show a set of experiments between the extremes of low and high current density where you do these experiments, perhaps using a couple of different constructs.1) We remain concerned that leak currents could contribute in a non-linear fashion to the observed shifts in Vrevshift in Author Response Image 1D of your rebuttal only partially addresses this concern since the conductance of TMEM16F is nonlinear and you have not reported the TMEM16F and leak conductances at the reversal potential. Furthermore, the data in Author Response Image 1D shows that there is a ~2-fold variability in the Vrevmeasurements (between ~15 and ~35 mV), in a similar range to the changes in Vrevthat lead you to conclude that the ion selectivity of TMEM16F changes. We are not certain how best to resolve this issue, but we suggest that it might be helpful to provide Vrevmeasurements as outlined above for both low and high calcium and to extend the range shown in Figure 1D to somewhat lower current density, in the range of 500 pA. While these might be affected more by background leak currents, they might also serve to validate the conclusions by providing an empirical calibration of the role of the leaks and other errors.2) As you point out, the current in your experiments is typically quite high, in the range of 1-8 nA when activated with calcium at +80mV. Although increasing current density diminishes the contribution of leak currents, currents of this magnitude will cause substantial voltage errors due to series resistance and will also alter ion concentrations (via ion depletion and accumulation). The plots of the current magnitude vs Vrevcan be unambiguously demonstrated, it would be more appropriate to refer to the protein as a poor ion selective channel that slightly changes its ionic preference (rather than a sodium selective channel that becomes chloride selective).3) We suggest that you rephrase how you describe the ion selectivity of TMEM16F. Assuming a clear shift in V4) We think the potential physiological effects of the ion selectivity changes you report are overstated. You argue that the effects might induce a ~13 mV change in the resting potential of a cell where TMEM16F is the main conductance. However, we are unaware of conditions where a cell would be exposed to millimolar intracellular calcium and sustained large depolarizations. Moreover, the presence of other currents would significantly diminish the impact of small changes in the selectivity of TMEM16F, especially since in the -80 to +50 mV range , TMEM16F has a very low Po . Therefore, the physiological effects on a cell's membrane voltage -if any- will be considerably smaller than ~13 mV. We request that the physiological relevance should be removed from the Abstract and significantly toned down in the Discussion. The focus of the manuscript should be on the biophysical mechanism underlying the small shift in selectivity.5) We suggest that you add a discussion about the origin of the discrepancy of the ion selectivity of TMEM16A and TMEM16F.eLife. Your revised article has been favorably evaluated by Richard Aldrich (Senior Editor), Kenton Swartz (Reviewing Editor), and three reviewers.Thank you for resubmitting your work entitled "Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Summary:The authors' extensive revisions resolve many issues with the earlier version. The article now describes the authors' considerable efforts to exclude artifacts that might bias the reversal potential methods, and also provides sufficient detail that their measurements can be meaningfully compared to other published work. The following are suggestions for revising the manuscript:Major comments:1) The term "electrostatic gate" seems out of place. The ordinary meaning of a "gate" is to control a flux in an all-or-none way. It is very odd to read a sentence like “…the "closed state" of the electrostatic gate allows the permeation of cations.”. Throughout the whole paper, there are no results showing that TMEM16F is only conducting cations or only conducting anions. It seems inappropriate to use the concept of "gate" to represent the control by a change of electrostatic potential. Controlling the cation versus anion selectivity by altering the pore potential (through charge residue mutations) is well documented in pentameric ligand-gated channels. The authors may want to reference those papers to strengthen the main conclusion in this paper.2+ and Depolarization: The authors propose that depolarization indirectly modifies permeation by "driving Ca2+ into the membrane electric field". While the conceptual simplicity of this idea is appealing, the authors observe that depolarization still alters the relative permeability even at high (i.e. 1mM) calcium concentrations where the site of action for calcium should be saturated.2) Synergy of Ca [Editors’ note: the author responses to the first round of peer review follow.]We thank the reviewers for their helpful suggestions. We are grateful for the acknowledgement by all the reviewers that our work provides a unifying mechanism for previous discrepancy pertinent to ion selectivity of TMEM16F. Parts of comments by reviewer 1 and 2, and almost all of the comments by reviewer 3 are focusing on technical issues, mainly questioning whether the shift of reversal potential is an artifact resulting from issues involving ion accumulation / depletion, liquid junction potential and background current contamination. Reviewers 1 and 3 also raise the question about the physiological implication of the ion selectivity shift. In addition, reviewers 1 and 2 inspire us to perform a few more practical experiments to support our proposed mechanism. In this appeal letter, we will clarify these main issues followed by point-to-point responses.We will address our responses to the concerns raised by multiple reviewers, including technical issues and physiological implications, and believe these concerns could be cleared. Upon the request by the reviewers, we will perform a few additional experiments to corroborate our mechanistic model.1) Technical issuesrev is an experiment artifact. We apologize for not having explained the methodology clearly in the manuscript. We will add the experimental details and two figures ; In most cases we only included recordings with Ca2+-activated current > 1000 pA. In all cases, we made sure that the Ca2+-activated current (recorded at +80 mV) is >ten-fold larger than endogenous current before switching protocol for testing Erev, and this is consistent with reviewer 3's estimation. In addition, for data in Figure 1 , Figure 2, Figure 3, Figure 4, Figure 5, Figure 6—figure supplement 1, Figure 7, we always recorded the current in 0 Ca2+ solutions at the end to make sure that the seal is good .First, for all the experiments, we started recording in a 150 mM NaCl, Carev and current magnitude, but we did not see such a trend. This analysis also partially answers the question of reviewer 1 and 2 regarding artifacts caused by ion accumulation / depletion.Second, there is no correlation between reversal potential and current magnitude . If the current was contaminated by endogenous current (which is cation-selective), there would be a negative correlation between E2+ mostly permeates cation. (2) The "reverse tail current"  following the reverse ramp is still 6~7-fold the size of the background current, meaning that the current at ~0 mV will be larger than that. The current underlying the reversal potential thus is dominated by TMEM16F current. To be cautious with our conclusions, we did not interpret the reversal potential of WT TMEM16F in 1 mM Ca2+ . (3) Most importantly, the double mutant Q559K_G615A reduces the rectification in both 15 µM and 1 mM Ca2+, using current magnitude at +80 mV as “quality control” will underestimate the effect of background current contamination, because TMEM16F current is outwardly rectifying while background current is linear (as pointed out by reviewer 3). We have thought about this and have made sure that our conclusion is valid: (1) In Figure 1—figure supplement 2C, the current at 0 mV is ~-200 pA, which would not have been "eliminated" even if we performed background subtraction, suggesting that the channel activated by 15 µM Ca1 mM Ca2+. For thirev and current magnitude . In addition, the main conclusion of our study is not affected by any deviation  caused by the ion accumulation or depletion: We used a hyperpolarizing ramp protocol following a holing potential at +80 mV . This will cause Na+ efflux and/or Cl- influx, potentially leading to Na+ accumulation on the extracellular side and Cl- accumulation on the intracellular side (or intracellular Na+ depletion and extracellular Cl- depletion). All of these possibilities should push ENa and ECl to “more positive” values, leading to a positive shift of Erev (as observed in the JGP paper1 cited by reviewer 2). This is opposite to the difference between 15 µM and 1 mM Ca2+, where the Erev in 1 mM Ca2+ is more negative. Also, In Figure 6D, a more depolarized holding potential should exacerbate the accumulation/depletion effect (if there is any), also pushing Erev to more positive values. The data show the opposite.Fourth, reviewer 1 and 2 raise the issue of ion accumulation / depletion. For most recordings, we switched solutions "back and forth" to make sure traces in the same solution reversed at the same voltage despite the change of current magnitude. We also successively recorded multiple traces in the same solution to make sure there was no drift. Between different cells, there was no correlation between E2+ (pink) and by 1 mM Ca2+ (gray) were similar in magnitude . In contrast, since the red trace is more Na+-selective than the blue trace, in 15 mM [NaCl]i the driving force for Na+ efflux at +80 mV is small (only 20 mV), while in 1 mM Ca2+ (the blue trace) the outward current is mainly carried by Cl- influx. Thus, this comparison between the red and blue traces is in fact consistent with our main idea. In addition, among different cells expressing WT or Q559K channels, there is no correlation between current magnitude and reversal potential .Fifth, reviewer 3 states that "the smaller the magnitude, the more cation-selective the current" suggesting that the current is contaminated by endogenous current. We believe this is a misinterpretation of our data. The reviewer might be comparing the magnitudes between the currents in 15 mM NaCl , which seems inappropriate; instead, we should compare the pink trace and the gray trace . For each cell expressing Q559K mutant channels, the current activated by 15 µM Ca2+-activated current > 1 nA, validation with "reversal tail current" and the utilization of a mutant with reduced rectification, we hope the reviewer would be convinced that the measured reversal potentials are accurate. Also, in the point-to-point response to reviewer 3, we will demonstrate that background current correction will actually "expand" the magnitude of Erev shift and cause additional problems, given how Ca2+ affects the background current. We will also demonstrate in our response to reviewer 3 that our correction of liquid junction potential is reliable.Last but not least, reviewer 3 raises the issue of background current contamination and liquid junction potential. Given the above explanation of experiment details, including selection of Ca2) Physiological implication- equilibrium potential is -60 mV and extracellular / intracellular cations are Na+ / K+ respectively, a shift of PNa+/PCl- from 0.5 to 2 can cause the membrane potential shift from -22 mV to -9 mV , big enough to regulate some signaling molecules such as NMDA receptors, voltage-gated Ca2+ and Na+ channels. Given that TMEM16F is proposed to be on the dendritic spines of neurons2, a local membrane potential change can be very consequential.Reviewer 1 and 3 point out that the shift of reversal potential is modest and question whether it is physiologically significant. We believe that the “relatively modest” ion selectivity change is sufficiently large to be relevant for physiological functions. In a hypothetical cell whose Cl3 as well as odorant receptor neuron sensory cilia4. Primary cilia are specialized organelles containing high Ca2+ and being constantly depolarized5, offering a “platform” for the dynamic ion selectivity. The ion transport in primary cilia is still a new area of investigation, and our work is indicative of a new regulatory machinery of primary cilia signaling. Our studies of TMEM16F might provide novel hints to people studying ion transport at subcellular regions, such as dendritic spines and cilia.TMEM16F is one of the several TMEM16 members universally expressed in diverse cell types, including epithelial cells, neurons and immune cells, but its physiological functions have not been fully characterized. It was reported that TMEM16F is localized in the primary cilia of epithelial cells3) Additional experiments as suggested by reviewers2SO4-based pipette solution and record the reversal potential of Q559K with the same protocol. We expect to see a less substantial reversal potential shift as we switch from 15 µM Ca2+ to 1 mM Ca2+, since we predict divalent anions might have effects opposite to those of divalent cations with respect to the alteration of electrostatic field in the permeation pathway.Based on suggestions of reviewer 1, we will test the effect of divalent anion on reversal potential. We will use Na+ selective as the membrane repolarizes during a slow ramp.Based on the suggestions of reviewer 2, we will test TMEM16F's "memory" of factors impacting the reversal potential at a given holding voltage (+80 mV). We will hold the membrane potential at +80 mV for ~5 s followed by hyperpolarizing ramps of different speeds. We expect to see that the recorded reversal potential is shifted to a more positive value as the ramp speed is reduced. This would indicate that TMEM16F ion selectivity is restored to being more NaAlso based on the suggestion of reviewer 2, we will test the reversal potential of Q559K with whole cell configuration.2-SO4 would affect the ion selectivity of TMEM16A.Based on the comment of reviewer 3, we will test whether replacing NaCl with mannitol or NMDGReviewer #1:2+ concentrations, while lipid scrambling requires only elevation of Ca2+, but not depolarization. Thus, it is not clear under what physiological conditions the self-braking mechanism enabled by the dynamic switch in selectivity might play a role. For example, does the membrane potential of platelets and/or apoptotic cells reach and maintain the depolarizations necessary for channel activation? Furthermore, the magnitude of the observed switch is relatively modest; in all conditions the channel is poorly selective between anions and cations, displaying only modest preferences for one over the other. In no cases do the currents approach ideal anion vs cation selectivity. Given the overall poor selectivity, I wonder how much of an effect would be caused by the observed switch in selectivity. Thus, while the phenomenon described here is biophysically interesting, I am not convinced of its physiological relevance. Some quantitation of the effects observed here within the context of a cell would be needed to establish a clear link to physiology.[…] 1) TMEM16F is a scramblase, as such its main function is lipid transport to externalize PS to initiate blood coagulation and recognition of apoptotic cells by macrophages. The physiological relevance of TMEM16F-mediated ionic currents is -at the moment- unclear. Further, activation of TMEM16F currents require sustained depolarizations and high Ca6 and preprints7 indicated that monovalent ions (not only Ca2+) are necessary for the execution of these functions. Clarification of TMEM16F ion selectivity will help unravel the mechanism of lipid scrambling, providing answers to questions such as whether scrambling occurs at a particular stage of the ion permeability transition, and whether movement of different ions drives the scrambling of different lipids.We thank the reviewer for raising the question regarding the physiological relevance of our work. It is true that previous studies regarding TMEM16F functions are mostly focused on its function of phospholipid scrambling and vesiculation. However, recently published papers3, which are specialized organelles containing high Ca2+ and being constantly depolarized5 – physiological conditions where TMEM16F ion selectivity shift is very likely. Our studies might suggest implications for researchers studying these subcellular regions. Also, as we discussed in "responses to all reviewers", the “relatively modest” ion selectivity change is sufficiently large to be relevant for physiological functions, such as the regulation of NMDA receptors and voltage-gated ion channels. Given that TMEM16F is proposed to be on the dendritic spines of neurons2, a local membrane potential change can be very consequential.TMEM16F is one of the few TMEM16 members universally expressed in diverse cell types, but its subcellular localization and physiological functions have not been fully characterized. It was reported that TMEM16F is localized in the primary cilia of epithelial cells2) It is not clear through what type of pore ions move in TMEM16F. It has been proposed that the TMEM16 scramblases form a lipid-lined pore through which ions can diffuse together with the lipids that are being scrambled . If this were the case, then the selectivity of the channel would be affected by multiple factors such as lipid headgroups, voltage and mono- and di-valent ions. On the other hand, the TMEM16A homologue forms a more conventional, protein-delimited, pore. In this case, the observed changes in selectivity could reflect either a charge-screening effect (as the authors propose) or the ability of the channel's pore to adopt slightly different conformations. Further, most of the measurements are carried out using a mutant, Q559K, that affects both selectivity and gating of TMEM16F. I understand that the mutant was used because of the removal of run-down. It is unclear whether this construct can serve as a good model for the WT pore. The mutation could case subtle rearrangements in the pore rendering it more sensitive to environmental changes. While the present experiments provide evidence suggesting that the selectivity of TMEM16F changes, they shed little light into the mechanisms underlying such a switch.8. A recent paper also proposes that mutation of Q559 establishes the feasibility to study ion transportation of TMEM16F9. Although WT is not feasible for mechanistic research about the impact of high internal Ca2+ concentration on ion selectivity, we showed that the critical phenotypes as found in Q559K mutant channel could all be recapitulated in WT, including the shift of Erev in accordance to ionic strength , voltage  and Ca2+ level . Several studies of the TMEM16 family of proteins regulated in multiple ways have exemplified the strategy of using mutants to “stabilize" some dynamic features , as long as critical phenotypes inferred from the mutant studies can be recapitulated in WT.We thank the reviewer for the question regarding the structure of TMEM16F. Previous studies have suggested that the Q559K mutant is deficient in scrambling, suggesting that it is a mutant stabilized in an open channel conformation with scrambling defect2+ are naturally smaller than those in 1 mM Ca2+, and therefore these effects are less pronounced leading to the observed differences. Did the authors use different directs of the ramp and/or tail current protocols to measure reversal potentials? Is there an effect on the direction of the ion switch (i.e. going from 150 NaCl to 15 NaCl is the same as going from 15 NaCl to 150 NaCl)? Do they observe a dependence of the magnitude of the effects as a function of the current in the patch?3) A technical concern I have is that the measured currents are relatively large (~1 nA) and the stimulation protocols used are long. Can the authors rule out that the effects they see are due to ion accumulation/depletion effects? The currents in 15 μm Ca+ efflux and/or Cl- influx, supposedly leading to Na+ accumulation on the extracellular side of the channel and Cl- accumulation on the intracellular side (or intracellular Na+ depletion and extracellular Cl- depletion). All of these should push ENa and ECl to “more positive” values, leading to a positive shift of Erev. This is opposite to the shift we observed between 15 µM and 1 mM Ca2+, where the Erev in 1 mM Ca2+ is more negative. Also, In Figure 6D, a more depolarized holding potential should exacerbate the accumulation/depletion effect (if there is any), also pushing Erev to more positive values. The data show the opposite.We thank the reviewer for the helpful suggestion. The effects we observe are unlikely due to ion accumulation/depletion. We used a hyperpolarizing ramp following holding the membrane potential at +80 mV . This will cause Narev with current magnitude . We will perform additional experiments to look into the effect of varying the ramp speed.We did not use a different ramp protocol because we predict the measured values will surely be different due to our hypothesis that membrane potential will drive ion movement into/away from the electric field. But for every experiment, we did switch solutions “back and forth” to make sure the results are reproduceable. Also, there is no correlation between E2+ concentration”. The reversal potential of the QK/EQ mutant suggest this mutant is less Cl- selective than the QK mutant alone. Thus, at an intermediate Po the selectivity is also intermediate. This is more consistent with a model where the pore can adopt multiple open states with different selectivity. What happens to the currents of the QK mutant alone (or WT) when measured at a Po~0.5? I do not think that the authors' conclusion that "This indicates either that TMEM16F ion selectivity is not correlated with its open state, or that the E667Q mutation circumvents the intermediate open state (if any) and causes the mutant channel to directly enter a fully-open conformation" is warranted by the data.4) Subsection “Q559K channel ion selectivity varies with intracellular Ca2+ in the binding pocket and ion entry into the permeation pathway, which has been shown in a recent paper for TMEM16A10. What we intended to indicate with this mutant is: Q559K channels in 15 µM Ca2+ or 1 mM Ca2+ are close to "full activation", and if the shift was due to the activation state, the "half-activation" mutant (i.e. Q559K_E667Q in 1 mM Ca2+)_should have more "positive" Erev – which is not the case. We will take the advice by adding the data in 6 µM Ca2+ and revising the text to clarify the point.We thank the reviewer for making this point. . The "less negative shift" of Q559K_E667Q compared to Q559K can be attributed to the "cross talk" of the electrostatic field effect of Carev depends on divalent ions  then the shift should depend in a graded manner on their concentration. Did the authors test what happens when using intermediate increases between the 15 and 1000 μm concentrations shown?5) Subsection “The switch of ion selectivity correlates with electrostatic change in permeating pathway”. If the shift in V2+. We will mention this point in the presentation of Figure 3.We thank the reviewer for this question. Yes, Figure 3 shows our results in ~40 µM Ca6) Are divalent anions inert? Since the channel is permeable to anions under all conditions , these ions can enter the pore and therefore should have the opposite effect of the divalent cations.42-, so we are not sure if this will work for TMEM16F Q559K. But nonetheless, we can try testing the reversal potential with SO42- in the solution.This is a very interesting question. However, TMEM16A is not permeable to divalent ions such as SO7) The inactivation in Figure 4A is slow (tau~30s – 1 min). Why weren't the reversal potentials measured after reaching activation but before inactivation sets in? Is inactivation dependent on voltage? If the patches are held at a membrane voltage closer to reversal is there a similar effect?2+ is "slow", the change of its rectification is relatively fast . Combining these changes together, we cannot draw convincing conclusions from these data. But interestingly, we noticed that if the patch was exposed to 1 mM Ca2+ in 15 mM NaCl, the change of rectification is slower, enabling us to obtain data as in Figure 1_figure supplment 4. We speculate that the inactivation indeed depends on the difference between voltage and Erev, and more specifically, the amount of permeating cation. But this is beyond the scope of this manuscript.We thank the reviewer for the insight. We did this experiment. Although the rundown of TMEM16F WT current magnitude in 1 mM Ca2+ confusing where they regard the rapid run-down that is PIP2 independent. Are there multiple run-down mechanisms?8) I find the effects of Zn2+-induced rundown was reversed in ~1 minute. We thus believe Zn2+-induced rundown was not caused by degradation of PIP2, which is irreversible, although we are not sure whether Zn2+ can sequester PIP2 in a reversible manner. We will re-write the sentence to make it clear.We thank the reviewer for mentioning this point. From Figure 4—figure supplement 1, the Zn9) Discussion section. The authors state "However, TMEM16F does not have an "Ohmic" state: the channel always requires depolarization to be activated." What does this mean? That the pore is intrinsically rectifying? Ohmic state would refer to conductance, not gating.2+ concentration (>a few µM) stably binds to two Ca2+ ions so that its conductance is "Ohmic". For TMEM16F, the data indicate that it is always outwardly rectifying regardless of Ca2+ concentration, but we don't know whether the pore is "intrinsically rectifying" or Ca2+ ions are not stably bound without depolarization. We will rewrite the sentence to clarify this point.We apologize for the confusing statement. TMEM16A in high CaReviewer #2:[…] The authors results are very interesting, but I have several concerns.1) Experimental artifacts: To combat channel rundown and voltage-dependent activation, the authors have resorted to protocols with sizeable resting membrane currents and conductance, conditions that can easily lead to ion accumulation/depletion artifacts : 115-124.).2+], holding voltage). If the reversal potential for each condition is essentially independent of the holding current/conductance, ion accumulation/depletion artifacts are unlikely. Reporting key experimental details  would help address these concerns.I am sure the authors have thought carefully about this issue and probably already have data to exclude this possibility. For example, since the channel undergoes desensitization, they should have recordings in which early ramps have a much larger conductance than later ramps recorded under identical conditions .For most recordings, we switched solutions "back and forth" to make sure traces in the same solution reversed at the same voltage despite the change of current magnitude. We also successively recorded multiple traces in the same solution to make sure there was no drift. Between different cells, there was no correlation between EIt is important to clarify that the main conclusion of our study is not affected by any deviation  caused by the ion accumulation or depletion:+ efflux and/or Cl- influx, potentially leading to Na+ accumulation on the extracellular side and Cl- accumulation on the intracellular side (or intracellular Na+ depletion and extracellular Cl- depletion). All of these possibilities should push ENa and ECl to “more positive” values, leading to a positive shift of Erev (as observed in the cited JGP paper1). This is opposite to the difference between 15 µM and 1 mM Ca2+, where the Erev in 1 mM Ca2+ is more negative. Also, In Figure 6D, a more depolarized holding potential should exacerbate the accumulation/depletion effect (if there is any), also pushing Erev to more positive values. The data show the opposite.We used a hyperpolarizing ramp from the holing potential at +80 mV . This will cause Na2) Proposed mechanisms for holding voltage changing ionic selectivity:2+  ions driven into the permeation pathway by the electric fields/driving force during the holding/activation voltage somehow modify permeation during the subsequent voltage ramp step. I have two concerns with this idea. Firstly, the authors have previously reported that TMEM16F is quite permeable to calcium. Larger depolarizations will drive more calcium out through the channel which could lower the calcium concentration on the intra-cellular side of the permeation pathway, rather than increasing it. Secondly, for the permeation pathway to act as a channel, ions must be able to enter and leave it on the microsecond timescale and thus one would expect rapid turnover of all permeating ions during the voltage ramp. For the channel to "remember" the holding voltage, it seems like voltage would either have to induce a conformational change or modulate the binding of ions to specific sites that turnover slowly enough to remain present during the subsequent ramp.The authors seem to be proposing that Ca2+ permeation may potentially reduce Ca2+ concentration at the intracellular interface of the pore, it is not fast enough to deplete Ca2+. This can be inferred from our data in a previous publication13: holding the membrane patch at a more depolarized membrane potential increases Ca2+ sensitivity of TMEM16F. If local intracellular Ca2+ depletion took place at a faster rate than Ca2+ entry into the membrane electric field, greater depolarization would lead to an increase of the EC50 for Ca2+ activation, which is opposite to what we found in our previous study. We will make this point clearly in the revised manuscript.We thank the reviewer for the question. First, although the Ca14-16. This structural feature is compatible with the possibility that ions can enter the permeation pathway and "resident at specific sites" without "leaving the pore", but still allow other permeating ions to move "beside them", reminiscent of schemes included in early editions of Bertil Hille’s book.The second question concerns the structural basis for TMEM16F ion selectivity shift. The model proposed by the reviewer is based on a "conventional" selective filter exemplified by that in potassium channels, in which a few permeating ions "line up" and move along the pore, though multiple ions may still reside in a wide vestibule below the selectivity filter, as evident from the ability of magnesium and polyamines to cause inward rectification of Kir channels. At this point we do not have a TMEM16F open-channel structure, but we might infer from the structure of TMEM16A. Instead of a conventional selective filter similar to that in voltage-gated ion channels, the TMEM16A permeation pathway has amino acids that are critical for ion selectivity distributed along the pore; with a few narrow constrictions, the permeation pathway has a wide vestibuleReviewer #3:2+ concentrations used) is a little bit hand waiving. In the literature, the permeability difference in TMEM16F between Na+ and Cl- is not huge. Even accepting the results in this study as face value, the TMEM16F's[…] While resolving the controversy in the cation/anion selectivity of TMEM16F is important, the authors' conclusion that the controversy results from different recording conditions used in various labs . Even worse, the changes of theratios range from 0.5 to 2 . It is therefore strange that the authors would call such a several-fold difference of Na PNa/PClpermeability ratios reported here may actually be artefacts.Na/PCl in previously published literature as well as the shift in our study "is not huge". In fact, the moderate permeability ratio is nonetheless physiologically important. In a hypothetical cell whose Cl- equilibrium potential is -60 mV and extracellular / intracellular cations are Na+ / K+ respectively, a shift of PNa/PCl from 0.5 to 2 can cause the membrane potential shift from -22 mV to -9 mV , big enough to regulate some signaling molecules such as NMDA receptors, voltage-gated Ca2+ and Na+ channels. Given that TMEM16F is proposed to be on the dendritic spines of neurons2 and primary cilia of epithelial cells3, a local membrane potential change can be very consequential.The reviewer believes that the discrepancy regarding P2+, when [NaCl]i is changed from 150 mM to 15 mM, the reversal potentials (in both Q559K and wild-type TMEM16F) shift from ~ 0 mV to ~ -15 mV . In Yang et al., 2012, a similar 10-fold change of [NaCl]i in the presence of 0.5 mM Ca2+ generated a right shift of the reversal potential to ~+20 mV . Both experiments are excised inside-out patch recordings, and the solutions are similar (150 mM/15 mM vs 140 mM/14 mM). The [Ca2+]i in Yang et al. was 0.5 mM, which should be closer to 1 mM Ca2+ than to 15 µM Ca2+. Why did the authors obtain a left shift of the reversal potential while in Yang et al., 2012, the reversal potential shifted to the right?In the present study and the Yang et al. paper cited by the authors , there are at least two serious technical problems that could contribute to the controversy. In fact, the result from Yang et al., 2012 and the result in this paper, both from the same lab, already contradict each other. Here is a striking example: in the present study, the authors show that with a high concentration (1 mM) of Ca18, "A voltage ramp from −80 to +100 mV with a rate (dV/dt) = 0.36 V/s was used to determine the reversal potentials for the wild-type mTMEM16A and mTMEM16F channels" – this is a slow depolarizing ramp. By contrast, in the current manuscript, "The traces were recorded with a hyperpolarizing ramp from +80 mV to -80 mV (-1 V/s) following holding at +80 mV" – a fast reverse ramp. The protocol used in our manuscript reflects the channel permeability when it is at a more depolarized membrane potential. Inspired by review 2, we will add one experiment to show that TMEM16F can briefly "memorize" the ion selectivity . Also, please note that in Figure 6, in hundreds of μM Ca2+, the “conditioning membrane potential” impacts significantly on the measurements of reversal potential.We thank the reviewer for the scrutiny. Actually, this question has already been answered in this manuscript: membrane potential matters. In the Yang et al. paperGiven that TMEM16F (and most of the mutants) is outwardly rectifying, in the experiments shown in this manuscript, we mostly performed reversal ramps to obtain "more-opened" current and minimize the contamination by endogenous current. Due to the different experiment protocols used in the two studies, it is understandable that the measurements are different.Two technical issues in both the current study and Yang et al., 2012, are not seriously dealt with.2+  should only be ~ -4 mV (with a 10-fold NaCl gradient). The reversal potential shift in Q559K in 15 µM Ca2+ in the paper is ~ +10 mV . After junction potential correction, this should be ~+20 mV.The first serious technical problem is the junction potential correction. Throughout the whole paper of Yang et al., 2012, there was no description of junction potential correction. In the present study, the authors mention that "the liquid junction potentials for 15 mM NaCl, (and) 45 mM NaCl were -1.7 mV and -1.0 mV respectively.……". I suppose that the authors calculated these values with respect to the 150 mM NaCl solution. If so, these values are severely under-estimated. In fact, using the same calculator (Clamplex), the value of liquid junction potential for 15 mM NaCl (with respect to 150 mM NaCl) is -11 mV . Since the data points in the figures  are not corrected for the junction potential, the reversal potential of ~ -15 mV in 1 mM CaThe reviewer might have neglected that we formed membrane seal in 150 mM NaCl-based solutions and used a 3 M KCl agar bridge as a reference electrode  in both papers. The reviewer might have obtained the result (-11 mV) with the following parameters:With our experimental protocol including forming giga-ohm seal in 150 mM NaCl and using a salt bridge during solution exchange, the correct setting should be:18.This significantly reduces the liquid junction potential. With this setup involving a salt bridge, it is acceptable for Yang et al. not to correct it2+) and ~ +60 mV (in 15 µM Ca2+) . The conductance of the untransfected cell patches includes the leak conductance and the conductance from the endogenous channels/transporters. If we sum the two as the background conductance (abbreviated as gb), and the conductance from TMEM16F (the channel conductance) is denoted as gc, the measured reversal potential (Erev) should be a weighted sum of the reversal potential of gc (abbreviated as Ec) and that of gb (abbreviated as Eb) according to the following equation:The second and even more problematic issue is a lack of background current subtraction. Throughout the whole manuscript , I do not see a description of background current subtraction for the presented I-V curves . In the absence of TMEM16F conductance, the HEK293 cells have a cation-selective endogenous conductance. The authors actually show this fact from the background current measurement where the reversal potentials under 150 mM/15 mM NaCl condition were ~+20 mV (in 1 mM Carev= [gc/(gc+gb)] × Ec + [gb/(gc+gb)] × EbE2+, which implicates higher Na+permeability, is not affected as much as the reversal potential in 1 mM Ca2+, which implicates higher Cl- permeability, whose real value should be even more “negative” . This is shown in rev is upheld.This equation, derived from the chord conductance equation, assumes that the conductance (gc and gb) is constant at all voltages, which is not always applicable here. Nonetheless, the double mutant, Q559K_G615A, with reduced outward rectification , is closBut nonetheless, we agree that it is necessary to reduce the contamination by gb, which we were very careful with. We also agree that currents with strong outward rectification are more susceptible to background contamination – we have explained how we performed "quality control" in the "response to all reviewers", and we will reinstate these points in the following response.2+ will reduce the TMEM16F conductance (gc), and therefore, Erev will approach Eb (which is +20 mV or +60 mV). When the gc is much larger than gb (this is the case when TMEM16F is activated by 1 mM Ca2+), the contribution of gb to the measured Erev is less significant . However, when gc is small relative to gb, the measured Erev will be severely contaminated by Eb. The example is no better than the one provided by the authors in Figure 6, where when the holding voltage is more positive (the TMEM16F conductance is larger because of its outward rectification property), the reversal potential is less positive (less contaminated by Eb). This phenomenon is consistent with all the results throughout the paper. Various manipulations in the paper  would not change this trend-the larger the gc, the less positive the measured Erev.Reducing [NaCl] or Ca2+ will reduce the TMEM16F conductance (gc)". Please note that Q559K current activated by 15 µM Ca2+ and by 1 mM Ca2+ are similar in magnitude . Although exposure to >30 µM Ca2+ leads to current desensitization due to loss of PIP2,  this is not an issue since we performed all the experiments in 15 µM Ca2+ before patch exposure to high Ca2+ in experiments throughout the whole manuscript (We formed the excised patch in 0 Ca2+ and carefully prevented the patch from exposure to Ca2+ > 20 µM13). The reviewer might have misinterpreted Figure 1F: the current activated by 15 µM Ca2+ in 15 mM NaCl at +80 mV “looks” small, but that is because the driving force of cation efflux is smaller , while there is no evidence showing that the conductance is small. In 1 mM Ca2+, the current at +80 mV is not reduced simply because now the outward current is mainly carried by Cl- influx. This does not disprove our hypothesis, but instead, is consistent with our conclusion.We respectfully disagree with the statement that "Reducing [NaCl] or Carev is legitimate  as an example. Figure 6D  shows that holding at different conditioning voltages changes the reversal potential recorded with the hyperpolarizing ramp protocol. The traces were obtained from the same patch, so the contaminating gc is universal for the four traces. In whatever way the hypothetical background current is drawn, the shift of Eshown in .2+, how large the total current should be so that the measured Erev is good enough to reflect the true TMEM16F reversal potential (Ec)? The equation above indicates that gc needs to be larger than 90% of the total conductance (gc + gb) in order to have a measurement where <10% of Eb  is added to the measured reversal potential.Without subtracting the background current obtained in the absence of Carev shift”. This can be illustrated in the Author response images 4 and 5.We thank the reviewer for the friendly reminder. We have explained our detailed methods in the “responses to all reviewers” that we made sure gb is <10% as the reviewer points out. Nonetheless, performing background correction will expand the range of “E2+ and 15 mM NaCl, as in many of the authors' recording traces, the conductance of TMEM16F at +80 mV is ~ 9-10 fold that of the background current. However, since WT TMEM16F and Q559K mutant are outwardly rectifying, their conductances are quickly reduced by several fold near the reversal potential (the outward rectification is even more severe in low NaCl and low Ca2+ condition). Therefore, the 9-10 fold gc/gb ratio at +80 mV may reduce to 2-3 near Ec (gb is constant at different voltages). Suppose the true value of Ec is 0 mV in the 150 mM/15 mM NaCl condition , and the value of Eb is +60 mV (as shown in the supplementary figure), a gc/gb ratio of 2-3 will give a measured reversal potential of +20 mV – +30 mV. If the total current at +80 mV is less than 0.5 nA, the situation will be even worse. Interestingly, the reversal potentials measured in 150 mM/15 mM NaCl condition in many experiments in this manuscript are in this range.The authors show that the background current from untransfected cell patches at +80 mV is 50-80 pA . If the total current at +80 mV is 500 pA (0.5 nA) in the solution with 15 µM CaWe thank the reviewers for pointing this out. We explained our detailed methods in responses to all reviewers, but we would like to summarize here:2+-activated current > 1000 pA, which practically were ~ 2000 pA , 20 fold the size of the background current (<100 pA) . The "0.5 nA" mentioned by the reviewer might refer to the current in 15 mM NaCl , which might actually reflect a reduction of driving force for cation efflux, rather than a reduction of conductance.· We included the recordings with Ca2+ .· We did take rectification into consideration and this consideration did not change the conclusion. We made sure the tail current recorded at + 80 mV after completion of the reversal ramp is still ~5 fold of the background current . Also for this reason, we did not analyze WT current activated by 1 mM Ca2+ at 0 mV is ~-200 pA, and it would not have been eliminated by background subtraction.· In some of the recordings , the current in 15 mM NaCl 15 µM Ca· Q559K_G615A circumvents the concern of outward rectification.2+ from current obtained in the presence of Ca2+. By doing background current subtraction, the authors would eliminate the shift in Erev caused by the background conductance and thus isolate the shift in Erev caused by Ca2+.The above example therefore emphasizes the importance of subtracting the background current in order to evaluate EC more precisely. However, I am only using the data provided by the authors to make the above estimate. Since the background currents vary from patch to patch, the easy way to minimize the contribution of the background conductance is to subtract the current obtained in the absence of Ca2+-activated component. But this is based on the assumption that Ca2+ does not have effects on background current, which is NOT the case. Judging from Figure 1—figure supplement 3, we noticed that there is a cation selective current endogenous to HEK cells, and this current is smaller in 1 mM Ca2+, suggesting that Ca2+ "blocks" the background cation current. We have observed this result frequently when we recorded from untransfected cells  in 1 mM Cashown in . Thus, w2+ than in 0.4 µM Ca2+. Therefore, the reversal potential is more shifted to the right in 0.4 µM Ca2+. Here, the measured reversal potentials (10 fold NaCl gradient) of K584Q in 0.4 µM and 1 mM Ca2+ are both far from the perfect Cl- Nernst potential. It has already been shown  that after background current subtraction, the K584Q mutant, like the wild-type TMEM16A, is perfectly Cl- selective in 20 µM Ca2+. In other words, based on the measured reversal potentials, K584Q in 20 µM Ca2+ in Jeng et al., 2016, is more Cl- selective than the K584Q current activated by 1 mM Ca2+ shown in this paper. The key difference between the two studies is the background current subtraction.It is interesting that similar background current contamination of the reversal potential measurement is also shown in TMEM16A and its K584Q mutant in this manuscript . However, it appears that the authors have not noticed this problem. Taking K584Q recordings as an example , the K584Q conductance is bigger in 1 mM Ca- over Na+ also has discrepancy among different groups, and several groups report that TMEM16A is moderately selective for Cl- over Na+, not perfectly meeting the Cl- Nernst potential11,19. In our recording of WT TMEM16A activated by 1 µM Ca2+ or in 1 mM Ca2+, the reversal potential is only shifted by ~15 mV in 45 mM NaCl , different from the value reported by Jeng et al. (by ~30 mV). TMEM16A in 1 mM Ca2+ is linear and 60 times bigger than endogenous current, so background current subtraction will not account for the discrepancy. Jeng et al. had a very insightful discussion in their JGP paper:We thank the reviewer for this comment. The data regarding whether TMEM16A is strictly or moderately selective for Cl4 versus sucrose to replace intracellular NaCl , and/or analyses  may explain the discrepancy.· We suspect that differences in experimental methods, for example, using NMDG-SO4 versus sucrose to replace intracellular NaCl". Notice that it is consistent with the idea conveyed in this manuscript: the intracellular ionic strength matters. NMDG might be still able to enter the electric field even though it cannot go through the channel, and it could account for the high anion permeability as recorded by Jeng et al.Given the above example, it is more likely that the "key difference" is "using NMDG-SO2+ inhibits the cation-selective current endogenous to HEK cells , background subtraction might cause new issues: The minor cation permeation (<1/10) might have been interpreted as background , and thus the corrected PNa/PCl will be smaller than the actual value. "The key difference between the two studies"  is in fact more than "background current subtraction". These discrepancies can be attributed to:For K584Q, although the current is small, it is nonetheless >10 times bigger than the background current. Since Ca2+ concentration. The lowest Ca2+ concentration in Jeng et al. is 20 µM, while we tested 300 nM Ca2+. Note that 400 nM intracellular Ca2+ is a critical point where most TMEM16A channels are bound to 1 Ca2+ ion11, while in 20 µM Ca2+, TMEM16A binds to 2 Ca2+ ions. The electrostatic field along the TMEM16A permeation pathway is significantly different between the one-Ca2+-bound state and two-Ca2+-bound state10. Thus, in the experiments by Jeng et al., the permeation pathway might have been strongly influenced by the positive charges from two Ca2+ ions (they used 20 µM), overriding the effect of the mutation.· Ca2+, a feature mirroring the slower rundown of TMEM16F Q559K. The mechanism and consequence of rundown have not been studied. Thus, before we figure these out, we were careful about the experimental conditions such as forming the excised patch in 0 Ca2+ toprevent PIP2 degradation catalyzed by exposure to >20 µM Ca2+. Our methodology might "protect" some phenotypes that require PIP2. We infer that Chen lab did not make sure that the measurement of Erev was performed without any prior exposure of the patch to >20 µM Ca2+ (a condition where PIP2 starts to degrade), judging from a recent paper by Chen lab using 900 µM Ca2+ as a reference condition to measure TMEM16F gating and ion selectivity9. It is thus not surprising that they got results different from our results and from a recently deposited preprint12.· Experimental details. Our unpublished data suggest that TMEM16A K584Q quickly runs down in 1 mM CaWith these technical flaws, the reported reversal potentials in this manuscript are simply not correct. I am not sure if the authors have measured background I-V curve in every recorded patch. If not, I do not think the problem can be corrected within a short time. If these two technical problems are corrected, I anticipate that conclusion of the paper is probably not going to hold.2+ to illustrate the point that background subtraction might in fact bring about new issues.We have demonstrated that we had thought about the two technical issues raised by reviewer 3 as we performed experiments. We reduced the liquid junction potential by using a 3 M KCl salt bridge. We have provided experimental details and performed extrapolation to prove that our conclusion is valid regardless of background subtraction, and we also provide recordings of the background current in the presence or absence of Ca[Editors' note: below is the authors’ response to the decision letter following their appeal.]The editors and reviewers have read and discussed your rebuttal. It is important to point out that our initial concerns remain, and we are not convinced that your plan for revision will satisfy those concerns. However, we are willing to reconsider a revised manuscript after you have had a chance to do the additional experiments.Overall revisions:We have performed extensive experiments and added the following figures to our manuscript:2+, with background (recorded in Ca2+-free 15 mM NaCl solution) subtraction.Figure 2: TMEM16F wild type and Q559K ion permeability ratios measured in 6 µM and 15 µM Ca2+, with background (recorded in Ca2+-free 15 mM NaCl solution) subtraction.Figure 3: TMEM16F Q559K ion permeability ratios during current rundown/desensitization in 36 µM Ca2+, with background (recorded in Ca2+-free 15 mM NaCl solution) subtraction.Revised Figure 1—figure supplement 4: TMEM16F Q559W ion permeability ratios in 15 µM and 1 mM Ca3+.Revised Figure 5: TMEM16F wild type ion permeability ratios in 10 µM and 1 mM Gd2-SO4.Figure 6—figure supplement 1: TMEM16F wild type and Q559K recorded in 15 mM NaCl balanced with NMDG-MES or NMDGFigure 6—figure supplement 2: TMEM16F wild type recorded with 15 mM NaCl in the pipette solution and 150 mM NaCl in the bath.- increases when intracellular Ca2+ is elevated. We hope these additional experiments and explanations will clear the concerns regarding technical issues.We have also added Figure 1—figure supplement 2, Figure 1—figure supplement 3, Figure 4—figure supplement 2 to illustrate the experimental details and how we performed "quality control". All the results support the notion that TMEM16F permeability to ClInspired by the editors, reviewer 1 and reviewer 2, we have performed experiments , renamed our mechanism as a “dual-gating model” for the purpose of a comparison with that of TMEM16A. We did not draw a definite conclusion from these experiments, but we hope these results will trigger discussions and new ideas among readers after the paper is published.2+ and voltage, and the addition experiments about channel activation by Zn2+ and Gd3+, the function of TM6 in gating) would be available to researchers working on TMEM16 and other channels with structural similarities (such as TMC and OSCA).We also took the editors’ suggestion to reduce the discussion of physiological implication and focus more on the characterization of channel biophysical properties in the text. In the revised manuscript, we briefly discussed the physiological implication of the ion selectivity shifting in the discussion. As was mentioned in our correspondence with editors regarding the case of BK channel, we believed that a clear understanding of the channel biophysical properties will prompt the elucidation of important physiological functions of the channel. Thus, we wish these results  in solutions with identical NaCl concentration so that the voltage dependence of the total and leak currents can be directly compared. We understand that there is no perfect way to identify and subtract leak currents for TMEM16F, but by showing raw and leak corrected data it will be possible to estimate the total and leak conductances at the reversal potential and thereby gauge the accuracy and reliability of the Vrevcalculations. Since this does not appear to have been done in your initial experiments, we would request that they show a set of experiments between the extremes of low and high current density where you do these experiments, perhaps using a couple of different constructs.1) We remain concerned that leak currents could contribute in a non-linear fashion to the observed shifts in V2+, Figure 2 and 3) and for the new construct, Q559W . We also applied this strategy to our experiments where 15 mM NaCl was balanced with salts (NMDG-MES and NMDG2-SO4) instead of mannitol . We would like to thank the editors for this suggestion, which enabled us to further firm up the conclusions drawn from Figure 3, in which for each patch all the currents recorded in 36 µM Ca2+ crossed with the background current at the same point, suggesting Erev did not shift with rundown or rectification state.We thank the editors for this practical requirement. We adopted the suggested experimental procedures when we performed new experiments for WT and Q559K  As you point out, the current in your experiments is typically quite high, in the range of 1-8 nA when activated with calcium at +80mV. Although increasing current density diminishes the contribution of leak currents, currents of this magnitude will cause substantial voltage errors due to series resistance and will also alter ion concentrations (via ion depletion and accumulation). The plots of the current magnitude vs V in Author Response Image 1D revmeasurements (between ~15 and ~35 mV), in a similar range to the changes in Vrevthat lead you to conclude that the ion selectivity of TMEM16F changes. We are not certain how best to resolve this issue, but we suggest that it might be helpful to provide Vrevmeasurements as outlined above for both low and high calcium and to extend the range shown in Figure 1D to somewhat lower current density, in the range of 500 pA. While these might be affected more by background leak currents, they might also serve to validate the conclusions by providing an empirical calibration of the role of the leaks and other errors.shows that there is a ~2-fold variability in the V2+ after background subtraction reversed at a more positive potential than current in 1 mM Ca2+ without background subtraction, confirming that the permeability ratio transition should be reliable. But there are variations across different samples even for the same batch of cells, recorded on the same day. Thus, "for each experiment in the following study, we performed paired-comparison within the same patch to avoid ambiguities arising from large variations across different recordings."We thank the editors for the suggestion. The current magnitude of Q559W happened to be around ~500 pA, which we thus utilized to perform the measurement as requested. Regardless of background subtraction, the shift was legitimate . Please note that the current in 15 µM Carevcan be unambiguously demonstrated, it would be more appropriate to refer to the protein as a poor ion selective channel that slightly changes its ionic preference (rather than a sodium selective channel that becomes chloride selective).3) We suggest that you rephrase how you describe the ion selectivity of TMEM16F. Assuming a clear shift in VWe have rephrased the statements as suggested. We now used "change of permeability ratio" in most parts of data interpretation. We only use "ion selectivity" when we generally describe channel biophysics.4) We think the potential physiological effects of the ion selectivity changes you report are overstated. You argue that the effects might induce a ~13 mV change in the resting potential of a cell where TMEM16F is the main conductance. However, we are unaware of conditions where a cell would be exposed to millimolar intracellular calcium and sustained large depolarizations. Moreover, the presence of other currents would significantly diminish the impact of small changes in the selectivity of TMEM16F, especially since in the -80 to +50 mV range , TMEM16F has a very low Po . Therefore, the physiological effects on a cell's membrane voltage -if any- will be considerably smaller than ~13 mV. We request that the physiological relevance should be removed from the Abstract and significantly toned down in the Discussion. The focus of the manuscript should be on the biophysical mechanism underlying the small shift in selectivity.We thank the editors for the insight and have revised our manuscript as requested. Now the main text is focused on the biophysical mechanism, while the physiological implication was only briefly discussed at the end.5) We suggest that you add a discussion about the origin of the discrepancy of the ion selectivity of TMEM16A and TMEM16F.We thank the editors for the suggestion. We have added the discussion of the comparison between TMEM16A and TMEM16F, including but not limited to the aspect of ion selectivity.Reviewer #1:2+ concentrations, while lipid scrambling requires only elevation of Ca2+, but not depolarization. Thus, it is not clear under what physiological conditions the self-braking mechanism enabled by the dynamic switch in selectivity might play a role. For example, does the membrane potential of platelets and/or apoptotic cells reach and maintain the depolarizations necessary for channel activation? Furthermore, the magnitude of the observed switch is relatively modest; in all conditions the channel is poorly selective between anions and cations, displaying only modest preferences for one over the other. In no cases do the currents approach ideal anion vs cation selectivity. Given the overall poor selectivity, I wonder how much of an effect would be caused by the observed switch in selectivity. Thus, while the phenomenon described here is biophysically interesting, I am not convinced of its physiological relevance. Some quantitation of the effects observed here within the context of a cell would be needed to establish a clear link to physiology.[…] 1) TMEM16F is a scramblase, as such its main function is lipid transport to externalize PS to initiate blood coagulation and recognition of apoptotic cells by macrophages. The physiological relevance of TMEM16F-mediated ionic currents is -at the moment- unclear. Further, activation of TMEM16F currents require sustained depolarizations and high CaWe thank the reviewer for raising the question regarding the physiological relevance of our work. It is true that previous studies regarding TMEM16F functions are mostly focused on its function in phospholipid scrambling and vesiculation. But one possible reason that hampers the study of its ion channel functions is the ambiguity of its ion selectivity; the conflicting results might have prevented us from understanding the channel functions thoroughly. Thus, our characterization of its biophysical properties might help unravel the physiological significance involving channel function of TMEM16F. We have included a brief discussion in this regard in the manuscript.2) It is not clear through what type of pore ions move in TMEM16F. It has been proposed that the TMEM16 scramblases form a lipid-lined pore through which ions can diffuse together with the lipids that are being scrambled . If this were the case, then the selectivity of the channel would be affected by multiple factors such as lipid headgroups, voltage and mono- and di-valent ions. On the other hand, the TMEM16A homologue forms a more conventional, protein-delimited, pore. In this case, the observed changes in selectivity could reflect either a charge-screening effect (as the authors propose) or the ability of the channel's pore to adopt slightly different conformations. Further, most of the measurements are carried out using a mutant, Q559K, that affects both selectivity and gating of TMEM16F. I understand that the mutant was used because of the removal of run-down. It is unclear whether this construct can serve as a good model for the WT pore. The mutation could case subtle rearrangements in the pore rendering it more sensitive to environmental changes. While the present experiments provide evidence suggesting that the selectivity of TMEM16F changes, they shed little light into the mechanisms underlying such a switch.We thank the reviewer for the question concerning the structural basis of TMEM16F and the representativeness of the Q559K mutant.3+ to activate TMEM16F for reversal potential measurements. Gd3+, a trivalent ion, increases Cl- accessibility to the permeation pathway likely due to its stronger influence of the electrostatic field, so that wild-type TMEM16F in 1 mM Gd3+ maintains conductivity to Cl- at low membrane potentials. (2) Alternating the pipette solution  and the bath (intracellular solution) so that in 1 mM Ca2+ the current reversed at a positive membrane potential. In addition, we confirmed that the critical phenotypes as found in Q559K mutant channel could all be recapitulated in WT, including the shift of Erev in accordance to ionic strength , to voltage , and "transiently" to Ca2+ level . A recently published paper has also proposed using Q559 mutants to study the ionic transportation mechanism of TMEM16F9.In the revised manuscript, we added a few experiments to show that the shift of permeability ratio also happens to wild-type TMEM16F, including: (1) Using Gd2 does not play a significant role. TMEM16F current activated by Gd3+ - an ion that has not been reported to trigger scrambling – also shows a shifting permeability ratio. In addition, cells heterologously expressing Q559K proteins do not display scrambling activities8, although purified Q559K shows scrambling activities in liposomes2. Taken together, we believe the shifting of permeability ratio is independent of scrambling. However, we could not exclude the possibility that there could be "slightly different conformations" on top of the “charge screening effect”, so we rephrased our statement as "although it remains possible that TMEM16F might undergo miniscule conformational change at different Ca2+ concentrations that contributes to the selectivity transition". In fact, our discussion regarding the relationship between ion selectivity and scrambling function also includes this point.To consider whether the lipid headgroups affect the ion selectivity, we performed an additional experiment as in Figure3, showing that PIP2+ are naturally smaller than those in 1 mM Ca2+, and therefore these effects are less pronounced leading to the observed differences. Did the authors use different directs of the ramp and/or tail current protocols to measure reversal potentials? Is there an effect on the direction of the ion switch (i.e. going from 150 NaCl to 15 NaCl is the same as going from 15 NaCl to 150 NaCl)? Do they observe a dependence of the magnitude of the effects as a function of the current in the patch?3) A technical concern I have is that the measured currents are relatively large (~1 nA) and the stimulation protocols used are long. Can the authors rule out that the effects they see are due to ion accumulation/depletion effects? The currents in 15 μm Ca+ efflux and/or Cl- influx, supposedly leading to Na+ accumulation on the extracellular side of the channel and Cl- accumulation on the intracellular side (or intracellular Na+ depletion and extracellular Cl- depletion). All of these should push ENa and ECl to “more positive” values, leading to a positive shift of Erev. This is opposite to the shift we observed between 15 µM and 1 mM Ca2+,where the Erev in 1 mM Ca2+ is more negative. Also, In Figure 6D, a more depolarized holding potential should exacerbate the accumulation/depletion effect (if there is any), also pushing Erev to more positive values. The data show the opposite.We thank the reviewer for the helpful suggestion. The effects we observe are unlikely due to ion accumulation/depletion. We used a hyperpolarizing ramp following holing the membrane potential at +80 mV . This will cause NaWe thank the reviewer for the helpful suggestion. We have answered this question in our responses to all reviewers: the effects we observe are unlikely due to ion accumulation/depletion, which actually can result in an opposite shift from what has been observed in our study.rev with current magnitude .We did not use a different protocol because we predict the measured values will surely be different due to our hypothesis that membrane potential will drive ion movement into/away from the electric field. There is no correlation between E2+ concentration”. The reversal potential of the QK/EQ mutant suggest this mutant is less Cl- selective than the QK mutant alone. Thus, at an intermediate Po the selectivity is also intermediate. This is more consistent with a model where the pore can adopt multiple open states with different selectivity. What happens to the currents of the QK mutant alone (or WT) when measured at a Po~0.5? I do not think that the authors' conclusion that "This indicates either that TMEM16F ion selectivity is not correlated with its open state, or that the E667Q mutation circumvents the intermediate open state (if any) and causes the mutant channel to directly enter a fully-open conformation" is warranted by the data.4) Subsection “Q559K channel ion selectivity varies with intracellular Carev values of WT TMEM16F and Q559K in 6 µM Ca2+  and they were more positive than those in 15 µM Ca2+ respectively . What we intended to express is: If the transition to being more permeable to Cl- (versus Na+) reflects different activation states, the "half-activation" mutant (Q559K_E667Q) should have more "positive" Erev  - which is not the case. We have revised the text to clarify the point. The "less negative shift" of Q559K_E667Q compared to Q559K can be attributed to the "cross talk" of the electrostatic field resulting from Ca2+-binding to the binding pocket and ion entry into the permeation pathway, to which a similar result has been shown in a recent paper for TMEM16A.10We thank the reviewer for making this point. We measured the Erev depends on divalent ions  then the shift should depend in a graded manner on their concentration. Did the authors test what happens when using intermediate increases between the 15 and 1000 μm concentrations shown?5) Subsection “The switch of ion selectivity correlates with electrostatic change in permeating pathway”. If the shift in VWe thank the reviewer for this question. We revised Figure 2 and added a new Figure 3 to show there is a "dose-dependent" shift.6) Are divalent anions inert? Since the channel is permeable to anions under all conditions , these ions can enter the pore and therefore should have the opposite effect of the divalent cations.4, which did not show different impact, likely reflecting their inaccessibility to the permeation pathway.This is a very interesting question. As part of the new experiments included in the revised manuscript, we compared NMDG-MES and NMDG-SO7) The inactivation in Figure 4A is slow (tau~30s – 1 min). Why weren't the reversal potentials measured after reaching activation but before inactivation sets in? Is inactivation dependent on voltage? If the patches are held at a membrane voltage closer to reversal is there a similar effect?2+ is "slow", the change of its rectification is relatively fast . Combining these changes together, we cannot draw convincing conclusions from these data. But interestingly, we noticed that if the patch was exposed to 1 mM Ca2+ in 15 mM NaCl, the change of rectification was slower, enabling us to obtain data as shown in Figure 1—figure supplement 4. We speculate that the inactivation indeed depends on the difference between voltage and Erev, and more specifically, the amount of permeating cation.We thank the reviewer for the insight. Although the rundown of TMEM16F WT current magnitude in 1 mM Ca2+ confusing where they regard the rapid run-down that is PIP2 independent. Are there multiple run-down mechanisms?8) I find the effects of Zn2+-induced rundown was reversed in ~1 minute. We thus believe Zn2+-induced rundown was not caused by degradation of PIP2, which is irreversible, although we are not sure whether Zn2+ can shield the charges of PIP2 headgroups in a reversible manner. We have added this statement in the manuscript.We thank the reviewer for mentioning this point. From Figure 5—figure supplement 1, the Zn9) Discussion section. The authors state "However, TMEM16F does not have an "Ohmic" state: the channel always requires depolarization to be activated." What does this mean? That the pore is intrinsically rectifying? Ohmic state would refer to conductance, not gating.2+ while TMEM16F is outward rectifying at all tested Ca2+ concentrations, which could be owing to the "intrinsically rectifying" (i.e. non-Ohmic) conductance and/or the voltage-dependent gating. The former is shown with an "instantaneous rectification index" 12, and our steady-state activation curve  shows the latter. We have re-written this part in the discussion.We apologize for the confusing statement. We intended to express that TMEM16A conductance is Ohmic at high CaReviewer #2:[…] 1) Experimental artifacts: To combat channel rundown and voltage-dependent activation, the authors have resorted to protocols with sizeable resting membrane currents and conductance, conditions that can easily lead to ion accumulation/depletion artifacts : 115-124.).2+], holding voltage). If the reversal potential for each condition is essentially independent of the holding current/conductance, ion accumulation/depletion artifacts are unlikely. Reporting key experimental details  would help address these concerns.I am sure the authors have thought carefully about this issue and probably already have data to exclude this possibility. For example, since the channel undergoes desensitization, they should have recordings in which early ramps have a much larger conductance than later ramps recorded under identical conditions  Proposed mechanisms for holding voltage changing ionic selectivity:2+  ions driven into the permeation pathway by the electric fields/driving force during the holding/activation voltage somehow modify permeation during the subsequent voltage ramp step. I have two concerns with this idea. Firstly, the authors have previously reported that TMEM16F is quite permeable to calcium. Larger depolarizations will drive more calcium out through the channel which could lower the calcium concentration on the intra-cellular side of the permeation pathway, rather than increasing it. Secondly, for the permeation pathway to act as a channel, ions must be able to enter and leave it on the microsecond timescale and thus one would expect rapid turnover of all permeating ions during the voltage ramp. For the channel to "remember" the holding voltage, it seems like voltage would either have to induce a conformational change or modulate the binding of ions to specific sites that turnover slowly enough to remain present during the subsequent ramp.The authors seem to be proposing that Ca2+ permeation may potentially reduce Ca2+ concentration at the intracellular interface of the pore, it is not fast enough to deplete Ca2+. This can be inferred from our data in a previous publication13: holding the excised patch at a more depolarized membrane potential increases Ca2+ sensitivity of TMEM16F. If local intracellular Ca2+ depletion took place at a faster rate than Ca2+ entry into the membrane electric field, greater depolarization would lead to an increase of the EC50 for Ca2+ activation, which is opposite to what we found in our previous study. We have added this sentence into our manuscript: "The reduction of EC50 of Ca2+ activation by depolarization is also observed in TMEM16F, although TMEM16F permeates Ca2+, suggesting Ca2+ efflux is not fast enough to deplete Ca2+ in the electric field at the intracellular side."We thank the reviewer for the question. First, although the Ca10,14-16. This structural feature is compatible with the possibility that ions can enter the permeation pathway and remain as "resident at specific sites" without "leaving the pore", but still allow other permeating ions to move "beside them", reminiscent of schemes included in early editions of Bertil Hille’s book. We have considered this mechanism as an "electrostatic gating" mechanism instead of calling it a "selectivity filter", and we have added extensive discussion in the manuscript.The second question concerns the structure basis for TMEM16F ion selectivity shift. The model proposed by the reviewer is based on a "conventional" channel selectivity filter exemplified by that in potassium channels, in which a few permeating ions "line up" and move along the pore. At this point we do not have a TMEM16F open-channel structure, but we might infer from the structure of TMEM16A and the possibly closed TMEM16F. Instead of a conventional selectivity filter similar to that in voltage-gated ion channels, the TMEM16 permeation pathway has amino acids that are critical for ion selectivity distributed along the pore; with a few relatively narrow constrictions, the permeation pathway has a wide vestibuleReviewer #3:2+ concentrations used) is a little bit hand waiving. In the literature, the permeability difference in TMEM16F between Na+ and Cl- is not huge. Even accepting the results in this study as face value, the TMEM16F's[…] While resolving the controversy in the cation/anion selectivity of TMEM16F is important, the authors' conclusion that the controversy results from different recording conditions used in various labs . Even worse, the changes of theratios range from 0.5 to 2 . It is therefore strange that the authors would call such a several-fold difference of Na PNa/PClpermeability ratios reported here may actually be artefacts.+(or Cl-) permeable” instead of “selective”, and to reduce the discussion of physiological implication. Since the expression and localization of the channel is not known, we could not assert that such a change in channel permeability is “not huge”. In a hypothetical cell with Cl- equilibrium potential of -60 mV and cation “equilibrium potential” (under the assumption that PK = PNa) of 0 mV, a shift of PNa/PCl from 0.5 to 2 can alter the membrane potential from -22 mV to -9 mV , big enough to regulate some signaling molecules. It has also been reported that TMEM16F is localized in primary cilia, a special cellular compartment with elevated Ca2+ level and greater depolarization, providing a condition for TMEM16F to function dynamically. We believe our research can help unravel more physiological functions of TMEM16F.We have taken the editors’ suggestion to refer to the channel as being “more Na2+, when [NaCl]i is changed from 150 mM to 15 mM, the reversal potentials (in both Q559K and wild-type TMEM16F) shift from ~ 0 mV to ~ -15 mV . In Yang et al., 2012, a similar 10-fold change of [NaCl]i in the presence of 0.5 mM Ca2+ generated a right shift of the reversal potential to ~+20 mV . Both experiments are excised inside-out patch recordings, and the solutions are similar (150 mM/15 mM vs 140 mM/14 mM). The [Ca2+]i in Yang et al. was 0.5 mM, which should be closer to 1 mM Ca2+ than to 15 µM Ca2+. Why did the authors obtain a left shift of the reversal potential while in Yang et al., 2012, the reversal potential shifted to the right?In the present study and the Yang et al. paper cited by the authors , there are at least two serious technical problems that could contribute to the controversy. In fact, the result from Yang et al., 2012 and the result in this paper, both from the same lab, already contradict each other. Here is a striking example: in the present study, the authors show that with a high concentration (1 mM) of CaWe thank the reviewer for the scrutiny. Actually, this question has already been answered in this manuscript: membrane potential matters. In the Yang et al paper, "A voltage ramp from −80 to +100 mV with a rate (dV/dt) = 0.36 V/s was used to determine the reversal potentials for the wild-type mTMEM16A and mTMEM16F channels" - this is a slow depolarizing ramp. By contrast, in the current manuscript, "The traces were recorded with a hyperpolarizing ramp from +80 mV to -80 mV (-1 V/s) following holding at +80 mV" – a fast hyperpolarizing ramp. The protocol used in our manuscript reflects the channel permeability when it is at a more depolarized membrane potential. Inspired by reviewer 2, we now show in the revised Figure 7 that TMEM16F can briefly "memorize" the ion selectivity at the preceding holding potential.Given that TMEM16F (and most of the mutants) is outwardly rectifying, in the experiments shown in this manuscript, we mostly performed hyperpolarizing ramps to obtain "more-opened" current to minimize the contamination by endogenous current. Due to the different experimental protocols used in the two studies, it is understandable that the measurements yielded different results.Two technical issues in both the current study and Yang et al., 2012, are not seriously dealt with.2+  should only be ~ -4 mV (with a 10-fold NaCl gradient). The reversal potential shift in Q559K in 15 µM Ca2+ in the paper is ~ +10 mV . After junction potential correction, this should be ~+20 mV.The first serious technical problem is the junction potential correction. Throughout the whole paper of Yang et al., 2012, there was no description of junction potential correction. In the present study, the authors mention that "the liquid junction potentials for 15 mM NaCl, (and) 45 mM NaCl were -1.7 mV and -1.0 mV respectively.……". I suppose that the authors calculated these values with respect to the 150 mM NaCl solution. If so, these values are severely under-estimated. In fact, using the same calculator (Clamplex), the value of liquid junction potential for 15 mM NaCl (with respect to 150 mM NaCl) is -11 mV . Since the data points in the figures  are not corrected for the junction potential, the reversal potential of ~ -15 mV in 1 mM CaThe reviewer might have neglected the fact that we formed the membrane seal with the patch electrode in 150 mM NaCl-based bath solutions and used a 3 M KCl agar bridge as a reference electrode  in both papers. The reviewer might have obtained the liquid junction potential (-11 mV) with the following parameters:The -11 mV actually reflects the offset of the commanding voltage for the liquid junction potential before sealing .With our methods of forming giga-ohm seal in 150 mM NaCl and using a salt bridge at solution exchange, the correct setting should be:This significantly reduces the liquid junction potential.2-SO4 , where the liquid junction potentials are both smaller than 2 mV. Despite the potential complication resulting from NMDG permeation, the channel still displayed a shift in permeability ratio.We have also included the experiments where the osmolarity of 15 mM NaCl solution was balanced with NMDG-MES and NMDG9. Assuming that the authors of that paper did not use the salt bridge and that during the solution exchange there was an interface between the new and old baths, the “10 mV” should be subtracted from rather than added to the measured value . If they had performed the correction correctly, they would have obtained the result showing that TMEM16F (recorded in 1 mM Ca2+) had reversed at a negative potential, corresponding to a channel more permeable to Cl-.Last but not least, we respectfully point out that we do not agree with reviewer’s correction by “adding 10 mV to the measured value”, which coincidently is the method used in a recent paper2+) and ~ +60 mV (in 15 µM Ca2+) . The conductance of the untransfected cell patches includes the leak conductance and the conductance from the endogenous channels/transporters. If we sum the two as the background conductance (abbreviated as gb), and the conductance from TMEM16F (the channel conductance) is denoted as gc, the measured reversal potential (Erev) should be a weighted sum of the reversal potential of gc (abbreviated as Ec) and that of gb (abbreviated as Eb) according to the following equation:The second and even more problematic issue is a lack of background current subtraction. Throughout the whole manuscript , I do not see a description of background current subtraction for the presented I-V curves . In the absence of TMEM16F conductance, the HEK293 cells have a cation-selective endogenous conductance. The authors actually show this fact from the background current measurement where the reversal potentials under 150 mM/15 mM NaCl condition were ~+20 mV (in 1 mM Carev= [gc/(gc+gb)] × Ec + [gb/(gc+gb)] × EbE2+, 15 mM NaCl, see responses to all reviewers) recorded from the same patch in the revised manuscript. The shift of Erev could be observed regardless of background subtraction. We also have revised our manuscript with more detailed description about our methods of data acquisition and quality control. We believe our conclusion is now convincing given the additional controls and explanations. In the following parts, we will also list the supplementary points that we have addressed.We thank the reviewer for expressing the concern of background contamination with considerable length (starting from here). We have included the new data together with the background current (the current recorded in 0 Ca2+ will reduce the TMEM16F conductance (gc), and therefore, Erev will approach Eb (which is +20 mV or +60 mV). When the gc is much larger than gb (this is the case when TMEM16F is activated by 1 mM Ca2+), the contribution of gb to the measured Erev is less significant . However, when gc is small relative to gb, the measured Erev will be severely contaminated by Eb. The example is no better than the one provided by the authors in Figure 6, where when the holding voltage is more positive (the TMEM16F conductance is larger because of its outward rectification property), the reversal potential is less positive (less contaminated by Eb). This phenomenon is consistent with all the results throughout the paper. Various manipulations in the paper  would not change this trend-the larger the gc, the less positive the measured Erev.Reducing [NaCl] or Ca2+ will reduce the TMEM16F conductance (gc)". Please note that the Q559K currents activated by 15 µM Ca2+ or by 1 mM Ca2+ are similar in magnitude . The reviewer might have misinterpreted Figure 1F: the current activated by 15 µM in 15 mM NaCl at +80 mV “looks” small, but that is because the driving force of cation efflux is smaller, while there is no evidence showing that the conductance is small. In 1 mM Ca2+, the current at +80 mV is not reduced because now the outward current is mainly carried by Cl- influx. This does not disprove our hypothesis, but instead, is consistent with our conclusion.We respectfully disagree with the statement that "Reducing [NaCl] or Ca2+, how large the total current should be so that the measured Erev is good enough to reflect the true TMEM16F reversal potential (Ec)? The equation above indicates that gc needs to be larger than 90% of the total conductance (gc + gb) in order to have a measurement where <10% of Eb  is added to the measured reversal potential.Without subtracting the background current obtained in the absence of Ca3+,Figure 5), performing background subtraction will expand the shifting range of the Erev when Ca2+ level changes.The reviewer’s equation, derived from the chord conductance equation, should only be applied if the conductance is constant at all voltages. But nonetheless, we made sure that the data such as those in Figure1, for which we did not perform background correction, met the criteria that gc is 9 times larger than gb . We would also like to remind the reviewer that according to this equation, given that gb is a cation current, we can extrapolate that for a linear current . Therefore, the 9-10 fold gc/gb ratio at +80 mV may reduce to 2-3 near Ec (gb is constant at different voltages). Suppose the true value of Ec is 0 mV in the 150 mM/15 mM NaCl condition , and the value of Eb is +60 mV (as shown in the supplementary figure), a gc/gb ratio of 2-3 will give a measured reversal potential of +20 mV – +30 mV. If the total current at +80 mV is less than 0.5 nA, the situation will be even worse. Interestingly, the reversal potentials measured in 150 mM/15 mM NaCl condition in many experiments in this manuscript are in this range.The authors show that the background current from untransfected cell patches at +80 mV is 50-80 pA . If the total current at +80 mV is 500 pA (0.5 nA) in the solution with 15 µM Ca2+ would have reversed at an even more negative potential if the background current could be adequately removed, thus expanding the shifting magnitude . That was why we chose to “take a safer step by not performing background subtraction”. But nonetheless, for the new data included in the revised manuscript, we have demonstrated that our conclusion is upheld even with background subtraction.Given that TMEM16F current is outwardly rectifying, the background contamination would cause a stronger deviation at a lower membrane potential. Thus, TMEM16F recorded in 1 mM Ca2+ from current obtained in the presence of Ca2+. By doing background current subtraction, the authors would eliminate the shift in Erev caused by the background conductance and thus isolate the shift in Erev caused by Ca2+.The above example therefore emphasizes the importance of subtracting the background current in order to evaluate EC more precisely. However, I am only using the data provided by the authors to make the above estimate. Since the background currents vary from patch to patch, the easy way to minimize the contribution of the background conductance is to subtract the current obtained in the absence of Ca2+.We thank the reviewer for the suggestion. We have added the requested experiments with background subtraction into our revised manuscript. We also have discussed the disadvantage of background subtraction, namely expanding the shifting range in comparisons involving recordings in 1 mM Ca2+ than in 0.4 µM Ca2+. Therefore, the reversal potential is more shifted to the right in 0.4 µM Ca2+. Here, the measured reversal potentials (10 fold NaCl gradient) of K584Q in 0.4 µM and 1 mM Ca2+ are both far from the perfect Cl- Nernst potential. It has already been shown  that after background current subtraction, the K584Q mutant, like the wild-type TMEM16A, is perfectly Cl- selective in 20 µM Ca2+. In other words, based on the measured reversal potentials, K584Q in 20 µM Ca2+ in Jeng et al., 2016, is more Cl- selective than the K584Q current activated by 1 mM Ca2+ shown in this paper. The key difference between the two studies is the background current subtraction.It is interesting that similar background current contamination of the reversal potential measurement is also shown in TMEM16A and its K584Q mutant in this manuscript . However, it appears that the authors have not noticed this problem. Taking K584Q recordings as an example , the K584Q conductance is bigger in 1 mM Carev of TMEM16A recorded in low NaCl balanced with salts (NMDG-MES) is more negative than that in low NaCl with mannitol, with the former corresponding to a channel more selective for Cl- (versus Na+). This was also reported in a paper in the same issue as the one cited by the reviewer, where the authors reported that TMEM16A recorded in low NaCl balanced with NMDG2-SO4 is more selective for Cl- than that in low NaCl balanced with sucrose16. In other words, “the key difference” is the recording condition.We thank the reviewer for the security. We have performed extensive experiments  to show that EWith these technical flaws, the reported reversal potentials in this manuscript are simply not correct. I am not sure if the authors have measured background I-V curve in every recorded patch. If not, I do not think the problem can be corrected within a short time. If these two technical problems are corrected, I anticipate that conclusion of the paper is probably not going to hold.rev still holds. We would like to take the opportunity to friendly express our thoughts to reviewer 3: In scientific research, it happens that different groups might come out with distinct results, under which circumstances we should carefully compare the experimental details to figure out the inherent reasons. Simply distrusting and arbitrarily “anticipating” will not expand our knowledge and are thus not beneficial.We have articulated why we initially did not perform background subtraction. We also included in the revised manuscript experiments with background subtraction, in which we demonstrated that the shift of E1) The term "electrostatic gate" seems out of place. The ordinary meaning of a "gate" is to control a flux in an all-or-none way. It is very odd to read a sentence like “…the "closed state" of the electrostatic gate allows the permeation of cations.”. Throughout the whole paper, there are no results showing that TMEM16F is only conducting cations or only conducting anions. It seems inappropriate to use the concept of "gate" to represent the control by a change of electrostatic potential. Controlling the cation versus anion selectivity by altering the pore potential (through charge residue mutations) is well documented in pentameric ligand-gated channels. The authors may want to reference those papers to strengthen the main conclusion in this paper.We thank the reviewers for the critiques. We now have changed all those statements to "modulation/alteration of electrostatic fields" throughout the text. We also have followed the great suggestion to add the reference to other ion channels which have comparable machineries: "Controlling the selectivity between cations and anions by altering the pore potential, referred to as a charge-screening mechanism, is also documented in other ion channels, such as ligand-gated channels and bacterial porins, through charged residues lining the pore and/or through permeating ions within the pore".2+ and Depolarization: The authors propose that depolarization indirectly modifies permeation by "driving Ca2+ into the membrane electric field" (p16). While the conceptual simplicity of this idea is appealing, the authors observe that depolarization still alters the relative permeability even at high (i.e. 1mM) calcium concentrations where the site of action for calcium should be saturated.2) Synergy of Ca2+ "saturates" the relative permeability of Cl-. A comparison against the trace recorded in 1 mM Gd3+  may indicate that the relative Cl- permeability as in Figure 7E (Q559K) can be even higher, although it is technically hard to test it by further increasing Ca2+ concentration without introducing other ion species in the solution (the NaCl concentration should be maintained at ~15 mM). We now avoid using the word "saturated", and only make the point that the current is now more carried by Cl- than by Na+.We thank the reviewer for the insight. We actually are not sure whether 1 mM CaReferences1 Yu, Y., Kuan, A. S. and Chen, T. Y. Calcium-calmodulin does not alter the anion permeability of the mouse TMEM16A calcium-activated chloride channel. J Gen Physiol 144, 115-124, doi:10.1085/jgp.201411179 (2014).Drosophila. Cell Rep 24, 2273-2286, doi:10.1016/j.celrep.2018.07.095 (2018).2 Sapar, M. L. et al. Phosphatidylserine Externalization Results from and Causes Neurite Degeneration in 3 Forschbach, V. et al. 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