Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels

  1. Michaela A Edmond
  2. Andy Hinojo-Perez
  3. Xiaoan Wu
  4. Marta E Perez Rodriguez
  5. Rene Barro-Soria

  • Curated by eLife

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    Evaluation Summary:

    This study investigates the mechanism of voltage-dependent activation of the KCNQ class of potassium channels that regulate neuronal firing, and are mutated in monogenic forms of epilepsy. This study makes an important technical step forward by reporting measurements of voltage-dependent conformational changes of KCNQ2/Kv7.2 channels, measurements which are known to be extremely difficult for this biologically important channel. Understanding these conformational changes allows the authors to investigate models of how voltage-dependent changes are coupled to opening of the channel pore, and also identify diverse mechanisms by which disease-linked mutations of KCNQ2/Kv7.2 may alter channel function.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage-sensing mechanisms that trigger channel gating. Here, we define the parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of eight to nine amino acids in the S4 becomes exposed upon voltage sensing domain activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

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  1. Version published to 10.7554/elife.77030 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    1. The manuscript seems to claim that the study shows that S4 is the voltage sensor and S4 moves in KCNQ2. This has been repeated in Abstract, Introduction and Results. However, by this time S4 movements as a voltage sensor are well accepted mechanisms. The importance of the work is actually that it defines parameters of the VSD movement in KCNQ2 such as the stretch of S4 in and out of the membrane, and the relationship between VSD activation and pore opening. These points should be brought out as the rationale and significance of this work, rather than the well-known S4 function.

    We thank Reviewer# 2 for this important comment that was also brought up by Reviewer# 3. We apologize for over emphasizing that the 4th TM segment is the voltage sensor and that the S4 moves in KCNQ2 channels. This might be the result of the author’s past struggle to convince earlier reviewers that the fluorescence signals at a given position are not an experimental artifact, but S4 moving during channel opening. We are very happy to learn that this is now a well-accepted mechanism.

    In the revised version, we now state:

    Abstract: “Here, we define parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-causing mutations using cysteine accessibility and voltage clamp fluorometry (VCF).”

    Introduction: “Similar to that seen in other Kv channels, the fourth transmembrane segment contains several highly conserved positively charged amino acid residues that move in response to changes in membrane voltages that functions as the voltage sensor(25-28)[…]Although these studies provided insight into S4 rearrangements, they did not define parameters of S4 movement, such as the dynamic relationship between S4 activation and pore opening during voltage-controlled gating of KCNQ2 channels.

    Results: We deleted: “Collectively, these close correlations in time (Figure 3) and voltage dependence (Figure 2C) of fluorescence and current suggest that the environmental changes around labeled F192C at the outer end of S4 rendered fluorescence signals that seem to report on S4 motion associated with the opening and closing of the channel gate.”

    And simply state: “The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43)”

    We also rewrote in its entirety the subsection: “Disease-causing mutations differentially affect S4 and gate domains” (Pages 10-11).

    1. The closeness of fluorescence and current traces and FV and GV curves led to the conclusion that the movement of a single VSD could trigger channel opening. The rationale for connecting the experimental observations to this conclusion needs to be well explained when the conclusion is first made. References that have made similar arguments such as Osteen et al PNAS 2010; Westhoff et al PNAS 2019 should be cited. In addition, as the authors recognized in Discussion, the same observations can also lead to an alternative conclusion such that the movements of four VSDs highly cooperative to all activate and then open the pore. However, this alternative mechanism is not mentioned until at the end of the manuscript, while "the movement of a single VSD opening the pore" is firmly claimed in Abstract and Results. Some justifications need to be provided for this.

    Thank you for this important observation, the wording we used was clumsy. Since we removed the kinetic model (Figure 6 in the original manuscript), we have also deleted any sentences that discuss concerted or independent S4 movement in the Abstract and Result sections. We only discussed that these alternatives, concerted or independent S4 movement, might explain our VCF data which shows that both the steady-state voltage dependence of S4 transitions and the kinetics closely follow those of ionic currents. Both references – Osteen et al PNAS 2010 and Westhoff et al PNAS 2019 have also been added – as recommended by the reviewer and apologize for overlooking these references in the original manuscript.

    1. An explanation is needed for how same the covalent MTS modification of N190C at two voltages resulted in different GV relations (Fig 1E).

    Thank you for pointing out this important point. We have spent a good deal of time since we received the reviews answering this important point that was also raised as a concern by Revewer# 1. To that end, we have included additional data that support the idea that N190C channels are accessible in both the open and closed states. This is now clearly addressed in Recommendations for the Authors, first Specific Suggestions from Reviewer #1. See above Response to the first Specific suggestions from Reviewer# 1 on Pages 2-5.

    In the original submission, we only used the protocols shown old Figure 1. We applied MTSET only at +20-mV for the open state and – 80-mV for the closed state. We used – 100-mV and – 120 mV for the closed state of A193C and S199C, respectively, because compared to the wt channels, these cysteine mutants shifted the GV relationship to negative voltages.

    In the revised version, to further strengthen our conclusions, we have used a new protocol: For each cysteine mutant, we have designed a protocol in which we first apply MTSET at hyperpolarized voltages (closed) before switching to depolarized voltages (open) on the same cell, in a pairwise manner.

    This is now described in the Result subsection “State-dependent external S4 modifications consistent with S4 as voltage sensor”, Pages 6-8 of the revised manuscript and new Figure 1 and Figure 1-figures supplement 3 and 4.

    We also apologize for the lack of clarity in citing reference 40 in the original submission. This reference is deleted in the revised version, in light of our new data on N190C (new Figure 1 and Figure 1-figures supplement 3 and 4), which strengthen our claims that N190C modification occurs in in both states (open and closed).

    1. The model in Fig 6F raises several concerns including vertical transitions having the rates of VSD activation and detailed balance is violated.

    The reviewer raises an important concern in our original Figure 6F (model). Based on the Editors and reviewers comments, we have removed Figure 6 from the original manuscript to eliminate any of potential misunderstanding about the data presented. In future studies, we will gather additional fluorescence and current data using different protocols and dimer constructs to provide a more in depth description of KCNQ2 gating.

    1. Discussion. The argument of no intermediate open state based on K/Rb permeability ratio assumes that the pore properties such as ion selection and permeability of KCNQ2 are the same as that of KCNQ1. The evidence for this assumption is not provided or discussed. On the other hand, some evidence suggests that the VSD of KCNQ2 may activate in two steps. For instance, the time course of VSD activation can be fitted with two exponentials, and the fluorescence increases after a plateau at voltages > 0 mV in FV curves (Fig 2C). How these results affect the conclusion should be discussed.

    We agree with the reviewer that the claim of a lack of an intermediate open state in KCNQ2 channels based on the Rb/K data provided in the original submission assumed that the pore properties of KCNQ2 are the same as those seen in KCNQ1 channels. Since we did not show sufficient experimental evidence to prove this point, we have removed Figure 6 (the model) from the revised manuscript. In the future, we will provide more evidence to build stronger support for the potential existence of intermediate and active open states in KCNQ2 channels. As such, we have removed the model shown in the original manuscript. Future studies will be performed to refine the KCNQ2 model, including the use of mutations that can lock the S4 in the intermediate or activated states in KCNQ2, as has been performed in the KCNQ1 channel by Zaydman et al; PMID: 25535795). These experiments will provide more conclusive results regarding the different S4 states.

    We have now re-analyzed the data and concluded that while the time course of the fluorescence appeared to have multiple exponentials, our fluorescence data lacked sufficient resolution to reliably estimate the first (fast) component. This might be because of the low signal-to-noise ratio of our VCF or/and because the filtering might have limited the tau-on from the optical signal (shown to be 20 ms in Figure 3C of the original submission).

    As suggested by reviewers # 3, we have removed the kinetics comparison of fluorescence and current in the revised version of Figure 3, and simply state: …” There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B, D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43).”

    As for the last part of the reviewer comments, the apparent increase in fluorescence after a plateau at voltages > 0mV has now also been revised. We have attempted new VCF at voltages more positive than + 40 mV to probe if a putative second fluorescence component after the plateau phase develops or if it is just artifacts of the experimental system. To get reliably fluorescence signals, we need a huge expression of labeled KCNQ2* channels (often producing currents larger than 100uA). It is considerably more difficult to properly clamp these high expressing cells, especially at extreme voltages. This experimental limitation makes it challenging to draw conclusions about the occurrence of a second fluorescent component. It may be possible to perform the cut—open technique coupled with VCF in order to shed light on this issue, but these experiments would require significant upgrade of the set up that we currently do not have this in place.

    Reviewer #3 (Public Review):

    1. I am convinced that the fluorescence signals reflect the voltage sensor conformation in the system. The authors focus quite a lot of attention on demonstrating that the fluorescence signals are not an experimental artifact, which is fine.

    We thank Reviewer# 3 for this observation. We apologize for over emphasizing that the fluorescence signals reflect the voltage sensor conformation in the system. As state above in response to a similar comment from Reviewer #1, this might be the result of the author’s past struggle to convince earlier reviewers that the fluorescence signals at a given position are not an experimental artifact, but S4 moving during channel opening. This has been amended in the revised version.

    However, I feel the authors could be more cautious in terms of describing how the mutations or dye conjugation may alter some of the gating properties. A place where this may be very important is in the description or characterization of activation kinetics as lacking sigmoidicity, which is part of the argument that these channels may open with only a fraction of voltage sensors activated. This may be correct in the modified (dye-conjugated) channel recordings, but many other recordings of unmodified channels (Figure 1) or WT KCNQ2 or 3 channels exhibit some sigmoidicity. I wonder if this difference may arise because the dye labeling may prevent complete VSD deactivation or interfere with gating in some other way. I would also add that this comment isn't meant to diminish the importance of the findings, I just think it would be wise to qualify some of the description of data with these possible caveats.

    We thank the reviewer for this suggestion, which we believe improves the flow and description of data considering all possible limitations. The reviewer is right. The mutation F192C on its own accelerates the kinetics of activation and causes a leftward shift in the GV curve of KCNQ2 channels. Moreover, labeling F192C with either fluorophore further shifts the GV towards negative potentials.

    In the revised version, we have rewritten the Result subsection ‘Tracking S4 movement of KCNQ2 channels using voltage-clamp fluorometry (VCF)’ almost in its entirety. In this subsection, we now bring to the forefront the changes associated with the measurement of gating properties caused by the mutations or dye conjugation that we agree helps with data interpretation. We made a direct comparison of voltage dependence and kinetics between wt, unlabeled KCNQ2-F192C, and labeled-KCNQ2F192C channels (new Figures 2 and Figure 2-figure supplement 1).

    These differences are also discussed on Pages 12-13 of the revised manuscript. See also below response to Recommendations for the authors:

    1. A brief aside on this point is that a lack of sigmoidicity does not necessarily imply a single transition required for opening - it can also arise if there is a rate-limiting step during a sequence of pre-open transitions.

    Thanks -good point-. We will keep this possibility in mind for future studies where the model will be developed.

    1. The generation of a quantitative model is a useful application of the data. It was not clear to me whether there was a benefit to using multiple-exponential components to fit the fluorescence signals and generate a more complex model. This may add complexity where it may not be necessary, as it is not clear whether the fluorescence signals require multiple components for an adequate fit.

    Thank you for your comment. We agree with the reviewer that our model is underdeveloped and needs additional VCF data to better describe KCNQ2 gating. Based on all three reviewers concerns and as suggested by the Reviewing editor in his summary, we removed the kinetic model from this manuscript and will work to refine this model in our future studies.

  3. eLife

    Evaluation Summary:

    This study investigates the mechanism of voltage-dependent activation of the KCNQ class of potassium channels that regulate neuronal firing, and are mutated in monogenic forms of epilepsy. This study makes an important technical step forward by reporting measurements of voltage-dependent conformational changes of KCNQ2/Kv7.2 channels, measurements which are known to be extremely difficult for this biologically important channel. Understanding these conformational changes allows the authors to investigate models of how voltage-dependent changes are coupled to opening of the channel pore, and also identify diverse mechanisms by which disease-linked mutations of KCNQ2/Kv7.2 may alter channel function.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This manuscript presents foundational studies of voltage sensing of human KCNQ2 channels, a drug target for epilepsy, and how voltage sensing is altered by epilepsy-causing human mutations. The studies probe identify extracellular regions of the KCNQ2 voltage sensor that change conformation upon voltage activation, important information for drug design. The study develops a fluorescence method for measuring a KCNQ2 voltage sensor movement, although it is not clear which subset of voltage sensor movements produce the fluorescence changes. The fluorescence measurements reveal that the KCNQ2 channel operates distinctly from the more thoroughly characterized KCNQ1 channels. The fluorescence measurement techniques were able to establish that a human epilepsy mutation separates voltage sensor movements from pore opening. The study attempts to reconcile the KCNQ2 fluorescence and conductance measurements with a Markov-chain model, but the model is underdeveloped, limiting conclusions that can be drawn from the modeling. Overall the conclusions that a stretch of the S4 becomes exposed upon activation of KCNQ2 channels, that voltage dependence and kinetics of S4 movement and channel opening/closing correlate in wild-type channels , and that different human mutations distinctly alter voltage sensor to pore coupling are justified by the data. This study indeed provides insight into KCNQ2 channel function.

  5. eLife

    Reviewer #2 (Public Review):

    Strengths:
    The study by Edmond and colleagues characterized voltage sensor (VSD) movements in KCNQ2 channels, which is an important component of the M-channel that controls neuronal excitability. Cysteine modification accessibility and voltage clamp fluorometry were used to measure VSD movements and the mechanism of how two epilepsy-associated mutations alter KCNQ2 voltage dependent activation. The authors report that the S4 transmembrane segment moved outward to expose a stretch of residues to the extracellular cysteine modifiers during activation, and the movement of VSD is followed closely by pore opening in kinetics and voltage dependence. A kinetic model is proposed to represent the experimental observations. The VSD movements and mechanism of channel gating were reported in KCNQ1 and other Kv channels. However, this is the first time similar studies are reported in KCNQ2. The optical measurements and chemical modification methods used in this study are known to be extremely difficult in the study of KCNQ2 channels. Therefore, this work establishes a detailed mechanism of voltage sensing in KCNQ2 channels for the first time based on a technical achievement.

    Weaknesses:
    1. The manuscript seems to claim that the study shows that S4 is the voltage sensor and S4 moves in KCNQ2. This has been repeated in Abstract, Introduction and Results. However, by this time S4 movements as a voltage sensor are well accepted mechanisms. The importance of the work is actually that it defines parameters of the VSD movement in KCNQ2 such as the stretch of S4 in and out of the membrane, and the relationship between VSD activation and pore opening. These points should be brought out as the rationale and significance of this work, rather than the well-known S4 function.
    2. The closeness of fluorescence and current traces and FV and GV curves led to the conclusion that the movement of a single VSD could trigger channel opening. The rationale for connecting the experimental observations to this conclusion needs to be well explained when the conclusion is first made. References that have made similar arguments such as Osteen et al PNAS 2010; Westhoff et al PNAS 2019 should be cited. In addition, as the authors recognized in Discussion, the same observations can also lead to an alternative conclusion such that the movements of four VSDs highly cooperative to all activate and then open the pore. However, this alternative mechanism is not mentioned until at the end of the manuscript, while "the movement of a single VSD opening the pore" is firmly claimed in Abstract and Results. Some justifications need to be provided for this.
    3. An explanation is needed for how same the covalent MTS modification of N190C at two voltages resulted in different GV relations (Fig 1E).
    4. The model in Fig 6F raises several concerns including vertical transitions having the rates of VSD activation and detailed balance is violated.
    5. Discussion. The argument of no intermediate open state based on K/Rb permeability ratio assumes that the pore properties such as ion selection and permeability of KCNQ2 are the same as that of KCNQ1. The evidence for this assumption is not provided or discussed. On the other hand, some evidence suggests that the VSD of KCNQ2 may activate in two steps. For instance, the time course of VSD activation can be fitted with two exponentials, and the fluorescence increases after a plateau at voltages > 0 mV in FV curves (Fig 2C). How these results affect the conclusion should be discussed.

  6. eLife

    Reviewer #3 (Public Review):

    This study by Edmond and colleagues implements methods to characterize voltage sensor movements in neuronal KCNQ channels, test ideas about how these motions are linked to channel gating, and investigate how different epilepsy-linked mutations might alter channel function. The paper is an important step forward in terms of methodological development for measuring voltage sensor conformation in KCNQ2 channels using fluorescence spectroscopy - this has been very difficult and the progress here will help many other groups. The authors also succeed in demonstrating that the relationship between VSD conformation and gating may be altered differentially by different epilepsy-linked mutations. An aspect of the paper that could be improved is in the description of the details underlying generation of a kinetic model that describes both VSD movements (measured by fluorescence signals) and channel gating.

    Strengths:

    1. This manuscript provides a comprehensive set of observations of conformational changes that underlie voltage sensing in KCNQ2/Kv7.2 channels. The identification and reporting of an approach to measure KCNQ2/Kv7.2 conformation by VCF is a significant step forward and has been a major challenge to many groups studying biophysics of these channels.

    2. The study provides concrete evidence for distinct mechanisms in which disease-linked mechanisms can alter KCNQ2/Kv7.2 function (for example, direct disruption of S4 movement in R198Q versus uncoupling of the pore from the voltage sensor in R214W). Ongoing detailed characterization of mutations may allow categorization into different subtypes based on mechanism, which may be relevant to pharmacotherapy. Mutations with certain properties may be sensitive to Kv7 activating drugs, whereas others may not.

    Weaknesses:
    1. I am convinced that the fluorescence signals reflect the voltage sensor conformation in the system. The authors focus quite a lot of attention on demonstrating that the fluorescence signals are not an experimental artifact, which is fine. However, I feel the authors could be more cautious in terms of describing how the mutations or dye conjugation may alter some of the gating properties. A place where this may be very important is in the description or characterization of activation kinetics as lacking sigmoidicity, which is part of the argument that these channels may open with only a fraction of voltage sensors activated. This may be correct in the modified (dye-conjugated) channel recordings, but many other recordings of unmodified channels (Figure 1) or WT KCNQ2 or 3 channels exhibit some sigmoidicity. I wonder if this difference may arise because the dye labeling may prevent complete VSD deactivation or interfere with gating in some other way. I would also add that this comment isn't meant to diminish the importance of the findings, I just think it would be wise to qualify some of the description of data with these possible caveats.

    2. A brief aside on this point is that a lack of sigmoidicity does not necessarily imply a single transition required for opening - it can also arise if there is a rate-limiting step during a sequence of pre-open transitions.

    3. The generation of a quantitative model is a useful application of the data. It was not clear to me whether there was a benefit to using multiple-exponential components to fit the fluorescence signals and generate a more complex model. This may add complexity where it may not be necessary, as it is not clear whether the fluorescence signals require multiple components for an adequate fit.

  7. Version published to 10.1101/2022.01.19.476944v1 on bioRxiv