Revealing a hidden conducting state by manipulating the intracellular domains in K_V10.1 exposes the coupling between two gating mechanisms

Reviewer #1 (Public Review):

Gating of Kv10 channels is unique because it involves coupling between non-domain swapped voltage-sensing domains, a domain-swapped cytoplasmic ring assembly formed by the N- and C-termini, and the pore domain. Recent structural data suggests that activation of the voltage sensing domain relieves a steric hindrance to pore opening, but the contribution of the cytoplasmic domain to gating is still not well understood. This aspect is of particular importance because proteins like Calmodulin interact with the cytoplasmic domain to regulate channel activity. The effects of Calmodulin (CaM) in WT and mutant channels with disrupted cytoplasmic gating ring assemblies are contradictory, resulting in inhibition or activation, respectively. The underlying mechanism for these discrepancies is not understood. In the present manuscript, Reham Abdelaziz and collaborators use electrophysiology, biochemistry, and mathematical modeling to explore the mechanistic effects on gating of various mutations and deletions that disrupt inter-subunit interactions at the cytoplasmic gating ring assembly and the consequences for channel modulation by CaM. From the beginning, it becomes challenging for non-experts to grasp the structural basis of the perturbations that are introduced (ΔPASCap and E600R), because no structural data or schematic cartoons are provided to illustrate the rationale for those deletions or their potential mechanistic effects. In addition, the lack of additional structural information or illustrations, and a somewhat confusing discussion of the structural data, make it challenging for a reader to reconcile the experimental data and mathematical model with a particular structural mechanism for gating, limiting the impact of the work.

By expressing mutants in oocytes and recording currents using Two Electrode Voltage-Clamp (TEV), it is found that both ΔPASCap and E600R mutants have biphasic voltage-activation curves, with two clear components contributing to activation and deactivation kinetics. Notably, the first component involving activation occurs at voltages where WT channels are mostly closed. Larger deletions at the N-terminus that further disrupt the cytoplasmic gating ring assembly accentuate the first component by heavily disfavoring the second one. The data can be well described by three components involving a closed state and two open states O1 and O2, in which the second component O2 is the one affected by the mutations and deletions. Based on the structural data, the first component is hypothesized to be associated with voltage sensor activation, whereas the second component is associated with conformational changes at the cytoplasmic ring. Consistent with this interpretation, a deletion construct where the covalent link between the voltage sensor and pore has been severed is shown to primarily affect that first component. Also consistent with the first component involving voltage-sensor activation, it is found that divalent cations that are known to stabilize the voltage sensor in its most deactivated conformations, shift the occupancy of the first component to more depolarizing potentials. Activation towards and closure from the first component is slow, whereas channels close rapidly from O2. A rapid alternating pulse protocol is used to take advantage of the difference in activation and deactivation kinetics between the two open components in the mutants and thus drive an increasing number of channels toward state O1. Currents activated by the alternating protocol reached larger amplitudes than those elicited by a long depolarization to the same voltage. This finding is interpreted as an indication that the first component (O1) has a larger conductance than the second (O2). It is shown that conditioning pulses to very negative voltages results in currents that are larger and activate more slowly than those elicited at the same voltage but starting from less negative conditioning pulses. In voltage-activated curves, the component corresponding to state O1 is shown to be favored by increasingly negative conditioning voltages as compared to less negative ones. This is interpreted as indicating that the first open component O1 is primarily accessed from so-called 'deeply closed' states in which voltage sensors are in their most deactivated position(s). Consistently, a mutation that destabilizes these deactivated states is shown to largely suppress the first component in voltage-activation curves for both ΔPASCap and E600R channels. It is also shown that stimulating calcium entry into the oocytes with ionomycin and thapsigargin, which is assumed to enhance CaM-dependent modulation, results in preferential potentiation of the first component in ΔPASCap and E600R, and this potentiation is attenuated by including an additional mutation that disfavors deeply closed states where voltage sensors are (mostly) deactivated. Together, these results are interpreted as an indication that calcium-CaM preferentially stabilizes O1 in mutant channels, thus favoring activation, whereas in WT channels lacking occupancy of O1, CaM stabilizes closed states and is therefore inhibitory. Moreover, it is found that the potentiation of ΔPASCap and E600R by CaM is more strongly attenuated by mutations in the channel that disrupt interaction with the C-terminal lobe of CaM than mutations affecting interaction with the N-terminal lobe. Finally, a mathematical model is proposed consisting of two layers involving two activation steps for the voltage sensor, and one conformational change in the cytoplasmic gating ring - completion of both sets of conformational changes is required to access state O2, but accessing state O1 only requires completion of the first voltage-sensor activation step in the four subunits. The model qualitatively reproduces most major findings on the mutants.

There are several concerns associated with the analysis and interpretations that are provided. First, the conductance-voltage (G-V) relations for the mutants do not seem to saturate, and the absolute open probability is not quantified for any mutant under any condition. This makes it impossible to quantitatively compare the relative amplitudes of the two components because the amplitude of the second component remains undetermined. This makes it challenging to interpret results involving perturbations that affect the relative occupancy of O1 and O2, such as those in Figures 2, 6, and 7, and also raises concerns about the extent to which model parameters can be constrained. This issue is made even more serious by the observation that the currents in both key mutants (ΔPASCap and E600R) are extremely slow and do not appear to reach steady-state over the intervals that are studied. This reduces confidence in the parameters associated with G-V relations, as the shape and position of both components might change significantly if longer pulses were used. This is not addressed or acknowledged in the manuscript. Further, because the mutant channel currents do not saturate at the most positive potentials and time intervals examined, the kinetic characterization based on reaching 80% of the maximum seems inappropriate, because the 100% mark is arbitrary. Further, the kinetics for some of the other examined mutants (e.g. those in Fig. 2A) are not shown, making it difficult to assess the extent to which the data could be affected by having been measured before full equilibration. There are additional aspects associated with gating kinetics that are not appropriately explored. For example, I would expect that the enhanced current amplitudes from Figure 5 are only transient, ultimately reaching a smaller steady-state current magnitude that depends only on the stimulation voltage and is independent of the pre-pulse. The entire time course including the rise-time and decay is not examined experimentally. This raises concern on whether occupancy of state O1 might be overestimated under some experimental conditions if a fraction of the occupancy is only transient. The mathematical model is not utilized to examine some of these slower relaxations - this may be because the model does not reproduce these slow processes, which would represent a serious shortcoming given that the slow kinetics appear to be intrinsic to transitions around state O1. The significance of the results with the Δ2-10.L341Split is unclear. First, structural as well as functional data has established that the coupling of the voltage sensor and pore does not entirely rely on the S4-S5 linker, and thus the Split construct could still retain coupling through other mechanisms, which is consistent with the prominent voltage dependence that is observed. If both state O1 and O2 require voltage sensor activation, it is unclear why the Split construct would affect state O1 primarily, as suggested in the manuscript, as opposed to decreasing occupancy of both open states.

The figure legends and text do not describe which solutions exactly were utilized for each experiment, and the rationale for choosing some solutions over others is not properly explained. The reversal potential for solutions used to measure voltage-activation curves falls right at the spot where occupancy of the first component peaks (e.g. see Figure 1B). Because no zero-current levels are shown on the current traces, it becomes very hard to determine which voltages correspond to each of the currents (see Fig. 1A). It is unclear whether any artifacts could have been introduced to the mutant activation curves at voltages close to the reversal potential. One key assumption that is not well-supported by the data pertains to the difference in single-channel conductance between states O1 and O2 - no analysis or discussion is provided on whether the data could also be well described by an alternative model in which O1 and O2 have the same conductance. No additional experimental evidence is provided related to the difference in conductance, which represents a key aspect of the mathematical model utilized to interpret the data. The CaM experiments are potentially very interesting and could have wide physiological relevance. However, the approach utilized to activate CaM is indirect and could result in additional non-specific effects on the oocytes that could affect the results.

The description of the mathematical model that is provided is difficult to follow, and some key aspects are left unclear, such as the precise states from which state O1 can be accessed, and whether there is any direct connectivity between states O1 and O2 - different portions of the text appear to give contradictory information regarding these points. Several rate constants other than those explicitly mentioned to represent voltage sensor activation are also assigned a voltage dependence - the mechanistic basis of that voltage dependence is unclear. Finally, a clear mechanistic explanation for the full range of effects that the ΔPASCap and E600R mutants have on channel function is lacking, as well as a detailed description of how those newly uncovered transitions would influence the activity of the WT channel; this latter point is important when considering whether the findings in the manuscript advance our understanding of the gating mechanism of Kv10 channels in general, or are specific to the particular mutants that are studied. It is unclear, for example, how both the mutation or the deletion at the cytoplasmic gating ring enable conduction by state O1, especially when considering the hypothesis put forward in this study that transition to O1 exclusively involves transitions by the voltage sensor and not the cytoplasmic gating ring. It is also not clearly described whether a non-conducting state with the equivalent state-connectivity as O1 can be accessed in WT channels, or if a state like O1 can only be accessed in the mutant channels. Importantly, if a non-conducting state with the same connectivity to O1 were to be accessed in WT channels, it would be expected that an alternating pulse protocol as in Fig. 4 would result in progressively decreasing currents as the occupancy of the non-conducting state equivalent to O1 is increased. Because this is not the case, it means that mutation and deletion cause additional perturbations on the gating energetics relative to WT, which are not clearly fleshed out.

Reviewer #2 (Public Review):

Summary:
The EAG family of ion channels is associated with many pathological conditions and are considered a target for the treatment of disease such as cancer. In this study, Abdelaziz et. al. examine the role of interaction between PAS domain and CNBHD in voltage-dependent gating of EAG channels. Based on their data, the authors conclude that they have identified a hidden open state that is only accessible in the mutant channels but not in the wild type. This hidden open state O1 can distinguished from the canonical open state O2 because it exhibits very different voltage-dependence. Although it is clear that the kinetics of these two open states are different, I have concerns about whether the data presented in this manuscript rule out alternate explanations. The idea that PAS domain deletions uncover a hidden open state is an extraordinary claim and if established, it has the potential to open a completely new approach to studying early gating transitions of these channels.

Strengths:
1. The study has identified a number of potentially interesting mutants that modulate voltage-dependent gating.
2. The discovery of a hidden open state due to mutations in the cytosolic domains is quite astonishing.

Weaknesses:
1. WT EAG currents are far right shifted compared to previously published data. It is not clear whether it is the recording conditions but at 0 mV very few channels are open. Compare this with recordings reported previously of the same channel hEAG1 by Gail Robertson's lab ( Zhao et. al. (2017) JGP). In that case, most of the channels are open at 0 mV. There must be at least 25 mV shift in voltage-dependence. These differences are unusually large.

2. In most of the mutants, O2 state becomes more prevalent at potentials above +50 mV. At these potentials, endogenous voltage-dependent currents are often observed in xenopus oocytes. The observed differences between the various mutants might simply be a function of the expression level of the channel versus endogenous currents.

3. Voltage-dependence of the kinetics of WT currents appears a bit strange. Why is the voltage-dependence saturated at 0 mV even though very few channels have activated at that point? I cannot imagine any kinetic model that can lead to such unusual voltage-dependence of kinetics.

4. One of the other concerns I have is that in many cases, it is clear that the pulse is too short to measure steady-state voltage-dependence. For instance, the currents in -160 mV and -100 mV in Figure 6A and 6B are not saturated.

Reviewer #3 (Public Review):

Summary:
The present manuscript by Reham Abdelaziz and colleagues addresses the gating of Kv10.1, which belongs to the KCNH gene family and contains other subfamilies such as Kv11 (ERG) and Kv12 (ELK). They all have fundamental physiological roles, from cardiac repolarization to modulation of neuronal excitability and cancer physiology. They have a non-domain swapped architecture at the molecular level; both voltage and Ca-CaM modulate the channel function. They contain an intracellular gating ring formed by a PAS domain (in the N-term) that interacts intimately with the cNBHD (C-term) of the neighbor subunit but also with the cytosolic part of the voltage sensor domain and the C-linker. Mutations in the N- or C- terminus modify the gating dramatically. This complex network of interactions makes the cytosolic section and the PAS domain in particular, an alluring part of the channel to study as responsible for the coupling between the movements of the voltage sensor and the gating ring.

In this paper, Reham Abdelaziz and colleagues address a fundamental question of how in the Kv10.1 channels, the movement of the voltage sensor is coupled to the intracellular gating ring rotation to make the channel conduct ions. The authors perform a series of deletions and mutations in the N-terminal section of the channel (PAS domain) and in the C-terminus (cNHBD) and observe a biphasic behavior on the modified EAG channels that they interpret as two populations of open states, one of them not visible in the WT and only available because of the mutations introduced. While this is a fascinating hypothesis and it fits with the depolarizing range of potentials of the WT channels, there are some issues that, if addressed, will make this work very valuable for biophysicists and molecular physiologists interested in voltage-gated ion channels.

Strengths:
The work presented addresses one of this channel's most fascinating and challenging features in the KCNH family. The authors use adequate mutations and electrophysiological techniques to address the questions they are trying to answer. They help them explore the behavior of the channels and build a Markov model to understand the underlying mechanism.

Weaknesses:
Although very well established, the experimental conditions used in the present manuscript introduce uncertainties, weakening their conclusions and complicating the interpretation of the results. The authors performed most of their functional studies in Cl-based solutions that can become a non-trivial issue when the range of voltages explored extends to very depolarizing potentials such as +120mV. Oocytes endogenously express Ca2+-activated Cl- channels that will rectify Cl- at very depolarizing potentials -due to an increase in the driving force- and contribute dramatically to the current's amplitude observed at the test pulse in the voltage ranges where the authors identify the second open state.

The authors propose a two-layer Markov model with two open states approximating their results. However, the results obtained with the mutants suggest an inactivated state accessible from closed states and a change in the equilibrium between the close/inactivated/open states that could also explain the observed results; therefore, other models could approximate their data.

That said, if the results obtained by the authors get confirmed under different experimental conditions in the absence of Cl-, this present work could be instrumental in understanding the gating mechanisms of the KCNH family.

Author Response

We thank the reviewers and editors for their deep, thoughtful and constructive assessment of our manuscript. We nevertheless would like to reply to the Reviewers reports.

Reviewer #1.

(...) The data can be well described by three components involving a closed state and two open states O1 and O2, in which the second component O2 is the one affected by the mutations and deletions

This statement is not completely clear to us. What we propose is that O1 is not visible in WT, only in the mutants. What would be affected is the access to O1 and the transition between O1 and O2, but not O2 itself.

From the beginning, it becomes challenging for non-experts to grasp the structural basis of the perturbations that are introduced (ΔPASCap and E600R), because no structural data or schematic cartoons are provided to illustrate the rationale for those deletions or their potential mechanistic effects. In addition, the lack of additional structural information or illustrations, and a somewhat confusing discussion of the structural data, make it challenging for a reader to reconcile the experimental data and mathematical model with a particular structural mechanism for gating, limiting the impact of the work.

Thank you very much for pointing this out and our apologies for the missing cartoon. It will be provided in the revised version.

There are several concerns associated with the analysis and interpretations that are provided. First, the conductance-voltage (G-V) relations for the mutants do not seem to saturate, and the absolute open probability is not quantified for any mutant under any condition. This makes it impossible to quantitatively compare the relative amplitudes of the two components because the amplitude of the second component remains undetermined. […] This reduces confidence in the parameters associated with G-V relations, as the shape and position of both components might change significantly if longer pulses were used.

We agree that the endpoint of activation is ill-defined in the cases where a steady-state is not reached. This does indeed hamper quantitative statements about the relative amplitude of the two components. However, while the overall shape does change, its position (voltage dependence) would not be affected by this shortcoming. The data therefore supports the claim of the “existence of mutant-specific O1 and its equal voltage dependence across mutants.”

Further, because the mutant channel currents do not saturate at the most positive potentials and time intervals examined, the kinetic characterization based on reaching 80% of the maximum seems inappropriate, because the 100% mark is arbitrary.

We agree that the assessment of kinetics by a t80% is not ideal. We originally refrained from exponential fits because they introduce other issues when used for processes that are not truly exponential (as is the case here). To address the concerns, we will add time constants from these fits in the revised version. Please note that in Figure 3, we do provide time constants, and they support the statement made.

Further, the kinetics for some of the other examined mutants (e.g. those in Fig. 2A) are not shown, making it difficult to assess the extent to which the data could be affected by having been measured before full equilibration.

This seems to be a misunderstanding. ∆2-10 kinetics is shown in Fig. 2c. ∆-eag is shown in Fig. 3. We will make sure to state this explicitly in the revised version.

For example, I would expect that the enhanced current amplitudes from Figure 5 are only transient, ultimately reaching a smaller steady-state current magnitude that depends only on the stimulation voltage and is independent of the pre-pulse. The entire time course including the rise-time and decay is not examined experimentally. This raises concern on whether occupancy of state O1 might be overestimated under some experimental conditions if a fraction of the occupancy is only transient. The mathematical model is not utilized to examine some of these slower relaxations - this may be because the model does not reproduce these slow processes, which would represent a serious shortcoming given that the slow kinetics appear to be intrinsic to transitions around state O1.

Thank you for thinking so deeply about the problem. We identified the same questions and did explore them using the model (Figure 8 c). Your intuition is confirmed there, the slow kinetics leads to a decrease of O1 occupancy after a transient accumulation. We intend to study this experimentally as well in the revised version.

The significance of the results with the Δ2-10.L341Split is unclear. First, structural as well as functional data has established that the coupling of the voltage sensor and pore does not entirely rely on the S4-S5 linker, and thus the Split construct could still retain coupling through other mechanisms, which is consistent with the prominent voltage dependence that is observed. If both state O1 and O2 require voltage sensor activation, it is unclear why the Split construct would affect state O1 primarily, as suggested in the manuscript, as opposed to decreasing occupancy of both open states.

Thank you for pointing out the unclear nature of our arguments. We rephrase in the following and will do so in the revised document: If, in non-split mutants, the upward transition of S4 allows entry to O1, it is reasonable to assume that the movement is not transmitted the same way in the split and the transition into O1 is less probable. The observation that, in the split, entry into O1 requires higher depolarization and appears to be less likely, suggests that downstream of S4 (beyond position 342), there is a mechanism to convey S4 motion to the gate of the mutants.

The figure legends and text do not describe which solutions exactly were utilized for each experiment, [...] Because no zero-current levels are shown on the current traces, it becomes very hard to determine which voltages correspond to each of the currents (see Fig. 1A).

Will be corrected.

… the rationale for choosing some solutions over others is not properly explained. […] The reversal potential for solutions used to measure voltage-activation curves falls right at the spot where occupancy of the first component peaks (e.g. see Figure 1B). […] It is unclear whether any artifacts could have been introduced to the mutant activation curves at voltages close to the reversal potential.

The high potassium extracellular solution was chosen to obtain tail currents of sufficient size, warranting precise determination of the reversal potential for every individual experiment. In this way, we ensured that there were no artifacts introduced to the activation curves. Tail currents were used when closing was reasonably fast (∆PASCapL322H and E600RL322H), but otherwise, we used the amplitude at the end of the pulse to get the reversal potential.

One key assumption that is not well-supported by the data pertains to the difference in single-channel conductance between states O1 and O2 - no analysis or discussion is provided on whether the data could also be well described by an alternative model in which O1 and O2 have the same conductance. No additional experimental evidence is provided related to the difference in conductance, which represents a key aspect of the mathematical model utilized to interpret the data.

We agree that the relative conductance of O1 and O2 is a key point. Our proposal mainly stems from the data presented in Fig. 4 and the amplitudes of the two components of the tail at potentials where both states are visible. We also agree that whole cell currents represent a product of occupancy and conductance and that only single channel recordings can produce unambiguous proof for the higher conductance of O1. We have embarked on a series of experiments directly addressing this in the mutants that will be reported in the revised version. Still, we did explore this issue with the model. Following the path of the least number of assumptions, we initially tested models with equal conductance for both states. None of these models was able to reproduce the shape of the tails and the prepulse-dependent increase.

The CaM experiments are potentially very interesting and could have wide physiological relevance. However, the approach utilized to activate CaM is indirect and could result in additional non-specific effects on the oocytes that could affect the results.

Thank you for the appreciative comments about the relevance of our results. We are aware of the potential side effects of the use of thapsigargin and ionomycin, but we still used this approach as an established method to raise intracellular Ca2+. This said, we would like to point out that the effects of Ca2+ increase on channel behavior do revert with a time course that mirrors the estimated time course of Ca2+ itself (supplement 1 to figure 7), suggesting that we are monitoring a Ca2+-dependent event.

The description of the mathematical model that is provided is difficult to follow, and some key aspects are left unclear, such as the precise states from which state O1 can be accessed, and whether there is any direct connectivity between states O1 and O2 - different portions of the text appear to give contradictory information regarding these points.

This seems to be a misunderstanding: supplement 1 to figure 8 graphically details the model’s layout and explicitly shows the connections to the two open states. It also shows that these are not connected. We will make sure that the text is more clearly stating this fact. We did explore models with one open state connected to more than one other state (loops) and found that none of these models can reproduce the large range of depolarizations for with conductance is reduced as compared to lower and higher depolarization (Figure 1).

Several rate constants other than those explicitly mentioned to represent voltage sensor activation are also assigned a voltage dependence - the mechanistic basis of that voltage dependence is unclear.

Some fundamental properties we observed in the mutants can be explained with constant, voltage-independent rate constants into and out of both open states. Specifically, it was possible to achieve behavior very close to that displayed in Figure 8c with constant η, θ, ε, and ζ. We then attempted to also reproduce the strong prepulse-dependence (Figure 6A and B) and found that we needed additional degrees of freedom to incorporate both behaviors with one parameter set. We could either add more states, and thereby rates, or introduce voltage dependence to η and θ. With already 32 states and 10 rates, we decided to adopt the less complex model variant. We agree that this probably reduced the interpretability of the model. As a rule, a transition with a voltage-dependence of the functional form of Eq.1 corresponds to the kinetic properties of two or three transitions, where one is voltage-independent (setting the maximal rate) and one has the classical exponential shape expected from truly molecular transitions.

We also agree that, conceptually, the transitions between the two layers – tentatively associated with a transition in the ring structure– should be voltage-independent. Interestingly, their voltage dependence is very similar to the voltage dependence of the early activation, i.e. centered at -100 and -120mV, similar to β. We therefore attempted to replace the voltage dependence of κ and λ with a state-dependence. To this end, we introduced a parameter that modified κ and λ depending on the state’s position along the α-β axis. While it seemed possible to include all desired features in a model with state-dependent κ and λ, it proved extremely difficult to tune the parameters. Eventually, we reverted to purely voltage-dependent and not state-dependent transition rates κ and λ. Nevertheless, we believe that their voltage dependence could be replaced by some form of state-dependence, i.e. by rates κ and λ that change systematically from the left-hand side of the scheme to its right-hand side.

Finally, a clear mechanistic explanation for the full range of effects that the ΔPASCap and E600R mutants have on channel function is lacking, as well as a detailed description of how those newly uncovered transitions would influence the activity of the WT channel.

We agree. Ultimate mechanistic explanations will have to await data from protein structures of intermediate states and in particular the mutant-specific open state.

…as well as a detailed description of how those newly uncovered transitions would influence the activity of the WT channel; this latter point is important when considering whether the findings in the manuscript advance our understanding of the gating mechanism of Kv10 channels in general, or are specific to the particular mutants that are studied.

We still do not know if the transitions to O1 are identical in the mutants and WT, although our data opens the path to dissecting the interplay of intracellular domains and voltage sensor. We think that the results are relevant for KCNH channels in general because we have made visible otherwise invisible states.

It is unclear, for example, how both the mutation or the deletion at the cytoplasmic gating ring enable conduction by state O1, especially when considering the hypothesis put forward in this study that transition to O1 exclusively involves transitions by the voltage sensor and not the cytoplasmic gating ring.

The transition to O1 is in our model made possible by a displacement of the voltage sensor. In our view, when this occurs with a properly folded and positioned intracellular ring, permeation (access to O1) is precluded. It is precisely the distortion in the intracellular ring induced by mutation or deletion what allows access to O1.

It is also not clearly described whether a non-conducting state with the equivalent state-connectivity as O1 can be accessed in WT channels, or if a state like O1 can only be accessed in the mutant channels. Importantly, if a non-conducting state with the same connectivity to O1 were to be accessed in WT channels, it would be expected that an alternating pulse protocol as in Fig. 4 would result in progressively decreasing currents as the occupancy of the non-conducting state equivalent to O1 is increased. Because this is not the case, it means that mutation and deletion cause additional perturbations on the gating energetics relative to WT, which are not clearly fleshed out.

Thank you for highlighting this important question. Following the arguments in the answer to the previous comment, our experiments cannot provide proof for the existence or accessibility of O1 in WT channels. We favor the interpretation that it is not accessible, because, as you point out, this is supported by the outcome of the alternating pulse on WT (figure 4A) and the paradoxical effect of CaM activation. However, this interpretation hinges on the hypothesis that the kinetics of entry into and departure from O1 would be the same in WT channels, as it is in the mutants. Because transitions into a non-conducting O1 would be only indirectly observable in the WT channel, this assumption would be extremely difficult to test.

Reviewer #2.

WT EAG currents are far right shifted compared to previously published data. It is not clear whether it is the recording conditions but at 0 mV very few channels are open. Compare this with recordings reported previously of the same channel hEAG1 by Gail Robertson's lab (Zhao et. al. (2017) JGP). In that case, most of the channels are open at 0 mV. There must be at least 25 mV shift in voltage-dependence. These differences are unusually large.

G-V curves presented in the literature show a large variability. Depending on the conditions, reported V1/2 values in Xenopus oocytes range from -43 mV (Schönherr et al., 2002 DOI: 10.1016/s0014-5793(02)02365-7) to +16 mV (Lörinczi et al, 2015 DOI: 10.1038/ncomms7672) through +4.1 mV (Lörinczi et al., 2016 DOI: 10.1074/jbc.M116.733576), or +10 mV (in the IUPHAR database). The results in the current manuscript are not significantly different from our previously published results on WT channels. In the report the reviewer is referring to, one source of the difference could be that Zhao et al. had no independent information about the reversal potential. In our experiments, we used solutions with high [K]ext. This places the reversal potential in a voltage range within measurable eag currents and thus allows direct determination of the reversal potential, together with the slow kinetics of the tails and the negative shift in the activation. We would argue that this makes the G-V curves less prone to assumptions, albeit for the price of large error bars around the reversal potential. Additionally, the presence of Mg2+ in the extracellular solutions can change the apparent V1/2 depending on the stimulation protocol.

In most of the mutants, O2 state becomes more prevalent at potentials above +50 mV. At these potentials, endogenous voltage-dependent currents are often observed in xenopus oocytes. The observed differences between the various mutants might simply be a function of the expression level of the channel versus endogenous currents.

Because we were aware of the potential issue of endogenous chloride currents in oocytes, we included data recorded in chloride-free solutions. Those show comparable results, and thus we conclude that endogenous currents are not the origin of the differences between mutants. We will clarify which solutions were used in the figure legends of the revised version and also include the argument against sizable endogenous current contributions in the revision. In a separate line of experiments, we expressed some of the mutants in HEK cells. Despite small current amplitudes, we were able to replicate the findings of two components, providing oocyte-independent evidence for the existence of a second open state.

Voltage-dependence of the kinetics of WT currents appears a bit strange. Why is the voltage-dependence saturated at 0 mV even though very few channels have activated at that point? I cannot imagine any kinetic model that can lead to such unusual voltage-dependence of kinetics.

The fact that voltage dependence of open probability and voltage dependence of activation time constant do not align reflects the multi-state nature of the underlying gating scheme. More than one of several sequential transitions limit the overall kinetics. In this case, the apparent kinetics can reflect a different “bottleneck” transition at different voltage ranges.

One of the other concerns I have is that in many cases, it is clear that the pulse is too short to measure steady-state voltage-dependence. For instance, the currents in -160 mV and -100 mV in Figure 6A and 6B are not saturated.

While we agree that steady-state curves can simplify quantitative evaluation – especially the normalization applied in the I/Imax curves in figure 6 – the conclusion of two components is independent of the absolute amplitude under steady state. The fact that in the raw current traces in Figure 6A, after a -160V prepulse, the same current amplitude is reached for two depolarizations (60 and 90 mV) but not for the intermediate depolarization, can only be explained by an I-V curve that has a minimum. Therefore, the raw data directly support the evidence of finding two components, even if the subsequent analysis is affected by insufficient test pulse durations.

Reviewer #3

Although very well established, the experimental conditions used in the present manuscript introduce uncertainties, weakening their conclusions and complicating the interpretation of the results. The authors performed most of their functional studies in Cl-based solutions that can become a non-trivial issue when the range of voltages explored extends to very depolarizing potentials such as +120mV. Oocytes endogenously express Ca2+-activated Cl- channels that will rectify Cl- at very depolarizing potentials -due to an increase in the driving force- and contribute dramatically to the current's amplitude observed at the test pulse in the voltage ranges where the authors identify the second open state.

As stated above, because we were aware of the potential issue of endogenous chloride currents in oocytes, we performed many of the experiments in chloride-free solutions. We conclude that endogenous currents are not the origin of the differences between mutants because the results were comparable regardless of the presence of chloride. We will clarify which solutions were used in the figure legends of the revised version and also include the argument against sizable endogenous current contributions in the revision. In a separate line of experiments, we expressed some of the mutants in HEK cells. Despite small current amplitudes, we were able to replicate the findings of two components, providing oocyte-independent evidence for the existence of a second open state.

The authors propose a two-layer Markov model with two open states approximating their results. However, the results obtained with the mutants suggest an inactivated state accessible from closed states and a change in the equilibrium between the close/inactivated/open states that could also explain the observed results; therefore, other models could approximate their data.

In the process of model development, we tested a large number of configurations. Those included models with a single open state which we connected to two closed (or inactivated) states that were not directly connected to each other and populated at different voltage ranges. In doing so, we attempted to allow access to the single open state from different regions of the “state-space”, reflecting the two voltage ranges of high conductance. However, in our hands, such a “loop” in the state-space inadvertently leads to a weak separation of the two states and a weak effect of prepulse potentials. The underlying reason is that given the short activation and deactivation time constants, a single open state in a loop provides an effective short-cut, linking otherwise separated parts of the state-space. To achieve the clear separation of the two component’s voltage dependence, two open states that are not connected to each other were essential. As we wrote in response to other comments above, the ultimate proof of two different open states cannot come from modeling, but from single channel measurements.