Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability
Round 1
Reviewer 1 Report
Summary of Research and overall impression
The authors present work whereby they mutated the conserved aspartate of transmembrane helix 4 of PsChR and Chronos into either histidine or cysteine (psChR D139H, psChR D139C, Chronos D179H, Chronos D179C). These new mutants were then compared to their wild types, and also to ChR2 wildtype and its two previously existing aspartate mutants (XXL and XXM), with a focus on light sensitivity, kinetics, and ion permeabilities. To examine the mutations’ effect on calcium permeability, they were also compared to CatCh in a final experiment. The manuscript is fairly comprehensive, covering basic properties and important ion permeabilities, plus the authors have created 4 additional channelrhodopsin mutants for use in the field. For the most part, figures were simple and easy to read, as were tables (with some issues, see later). While the story/rationale behind the choice of mutations is both included and interesting, it may benefit from a slight re-write to improve the clarity and overall flow, and to properly highlight important milestones. The discussion is very concise, and effective in conveying the overall aims and outcomes of the research.
The manuscript requires both major and minor revisions, with major revisions related to manuscript clarity (particularly the abstract and introduction, and certain figures/tables), plus expansions to the materials and methods section.
Abstract
Major issues
1. Though the Chronos mutations are referred to in Background, they are not mentioned in the Results section – consider adding a brief summary of their results.
2. Consider shortening the description of CatCh for clarity in the abstract (can be expanded upon later). Lines 23-24.
Minor issues
1. Typo – PsChR D173H should read PsChR D139H. Line 23.
1.1 Introduction
Major Issues
1. As alluded to previously, the authors may consider rewording the introduction section to improve clarity and flow. For example, nomenclature of mutants should be kept consistent, and as lines 50-55 are currently very detail heavy, consider how this section may be reworded to be more concise while getting across the same point. It may also be unclear to readers outside the field what certain notation means (eg E90R, C128, D156), and a brief explanation may bring further clarity to the introductory “story”. Also, in this section it should be made clearer exactly which mutants were created by the authors, and which previously exist and have been obtained for experimentation.
2. Materials and Methods
Major Issues
1. More detail should be added to section 2.5 Fluorescence Imaging so as to allow replication (filters, magnification, sample preparation etc).
2. The data processing section (2.6) should be expanded upon, for example, detailing which program was used for each type of analysis.
3. In general, experimental details outlined in figure legends may benefit from being included in the main Materials and Methods section (eg. closing time calculation, curve fitting, chemical composition of measurement solutions)
3. Results
3.1
Major Issues
1. The authors state that “expression in the plasma membrane might have been increased even more”, but this comes across as speculation, rather than fact. Consider removing this statement, unless some additional evidence can be provided along with a more quantitative statement on the fluorescence increase. Lines 124-125.
2. The authors state that steady state photocurrents were increased ~30 and ~48 fold, and while this is written in Table 1, it is not clearly apparent from Figure 1 that this is the case. As it is a quite important finding, consider modifying graphs to highlight this result.
Minor Issues
1. Typo – The “hill” in “hill equation” should be capitalised. Line 172.
2. There is extensive use of the “ ~ “ symbol in Table 1. It may improve clarity to omit this, and add a note in the legend to the effect of “Values are presented as approximates”.
3.2
Minor Issues
1. Consider rewording lines 174-175 to be less conversational language.
2. Typo – remove the comma after “Both”. Line 179.
3. Typo – remove comma after “the most Na+ permeable channerhodopsin…”. Line 205.
4. Typo – line 197 states that the PNa+/PH+ of Chronos D173C is ~4.9x10-7, whereas in Table 2, this is stated to be ~5x10-7. The correct value should be in both places.
3.4
Major Issues
1. In Figure 5, only “selected channelrhodopsin variants” are included. This is inconsistent with the previous 4 figures, the authors may wish to include the other variants, space permitting.
Minor Issues
1. Typo – The second C of Catch on line 244 should be capitalised to CatCh. This is also the case in the x axis of Figure 5a.
Author Response
Point 1: Abstract Major issues 1. Though the Chronos mutations are referred to in Background, they are not mentioned in the Results section – consider adding a brief summary of their results.
Response 1: We have added a brief summary in the abstract accordingly.
Point 2: Abstract Major issues 2. Consider shortening the description of CatCh for clarity in the abstract (can be expanded upon later). Lines 23-24.
Response 2: Changed accordingly.
Point 3: Abstract Minor issues 1. Typo – PsChR D173H should read PsChR D139H. Line 23.
Response 3: Changed.
Point 4: 1.1 Introduction Major Issues 1. As alluded to previously, the authors may consider rewording the introduction section to improve clarity and flow. For example, nomenclature of mutants should be kept consistent, and as lines 50-55 are currently very detail heavy, consider how this section may be reworded to be more concise while getting across the same point. It may also be unclear to readers outside the field what certain notation means (eg E90R, C128, D156), and a brief explanation may bring further clarity to the introductory “story”. Also, in this section it should be made clearer exactly which mutants were created by the authors, and which previously exist and have been obtained for experimentation.
Response 4: We have reorganized the introduction part and also explained items like E90R, C128, D156.
Point 5: 2. Materials and Methods Major Issues 1. More detail should be added to section 2.5 Fluorescence Imaging so as to allow replication (filters, magnification, sample preparation etc).
Response 5: added.
Point 6: 2. Materials and Methods Major Issues 2. The data processing section (2.6) should be expanded upon, for example, detailing which program was used for each type of analysis.
Response 6: expanded.
Point 7: 2. Materials and Methods Major Issues 3. In general, experimental details outlined in figure legends may benefit from being included in the main Materials and Methods section (eg. closing time calculation, curve fitting, chemical composition of measurement solutions)
Response 7: Some general experimental details have been moved to Materials and Methods section.
Point 8: 3. Results 3.1 Major Issues 1. The authors state that “expression in the plasma membrane might have been increased even more”, but this comes across as speculation, rather than fact. Consider removing this statement, unless some additional evidence can be provided along with a more quantitative statement on the fluorescence increase. Lines 124-125.
Response 8: removed.
Point 9: 3. Results 3.1 Major Issues 2. The authors state that steady state photocurrents were increased ~30 and ~48 fold, and while this is written in Table 1, it is not clearly apparent from Figure 1 that this is the case. As it is a quite important finding, consider modifying graphs to highlight this result.
Response 9: We have changed the format of Figure 1b to show this more obviously.
Point 10: 3. Results 3.1 Minor Issues 1. Typo – The “hill” in “hill equation” should be capitalised. Line 172. 2. There is extensive use of the “ ~ “ symbol in Table 1. It may improve clarity to omit this, and add a note in the legend to the effect of “Values are presented as approximates”. 3.2 Minor Issues 1. Consider rewording lines 174-175 to be less conversational language. 2. Typo – remove the comma after “Both”. Line 179. 3. Typo – remove comma after “the most Na+ permeable channerhodopsin…”. Line 205. 4. Typo – line 197 states that the PNa+/PH+ of Chronos D173C is ~4.9x10-7, whereas in Table 2, this is stated to be ~5x10-7. The correct value should be in both places.
Response 10: All are changed accordingly.
Point 11: 3. Results 3.4 Major Issues 1. In Figure 5, only “selected channelrhodopsin variants” are included. This is inconsistent with the previous 4 figures, the authors may wish to include the other variants, space permitting.
Response 11: For the Ca2+ conductance experiment in Figure 5, we selected several most promising and informative constructs for the comparison. Because this test is difficult, we need to inject many oocytes to get the reliable data. The Chronos D173H and D173C were excluded because of the relative high H+ conductance and smaller current. XXL and PsChR D139C mutations were excluded because of the relative high H+ conductance and much slower kinetics.
Point 12: 3. Results 3.4 Minor Issues 1. Typo – The second C of Catch on line 244 should be capitalised to CatCh. This is also the case in the x axis of Figure 5a.
Response 12: changed accordingly.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Thanks for asking me to review this paper which describes the properties of a number of optogenetic tools, and importantly examines the effect of the DC gate mutants in PsChR.
The work is well presented, but a few grammatical points are made below to try an rephrase the occasional sentence that did not feel right. The data is clear, and fairly interpreted.
My two major concerns
1) that the work has been entirely conducted in Xenopus oocytes at 16 degrees Celsius. Channel Rhodopsins have a thermal sensitivity, and expression levels (which potentially contribute to some of the changes in photocurrent) may depend on the experimental model system used (codon optimised for mouse for example).
While not wishing to question the quality of the data, or the long standing role the Xenopus oocyte model has played in ion channel physiology, I suspect that many groups who may consider using these tools may feel better about doing that if some comparative analyses were presented from a mammalian cell line at body temperature.
If this is not technically feasible, or is perhaps part of an extended application of this project that will be part of a larger submission in due course, perhaps the authors would agree to observe these present limitations? Attempts to transduce or transfect mammalian cells inevitably produce variable transgene levels which may erode some of the apparent differences seen, mentioning this caveat may help set the expectations of those that may follow this papers lead.
2) I was interested to see the experiment performed with BAPTA, and also its interpretation in Figure 5.
A recent series of experiments with the fluorescent BAPTA derivatives
(N.A. Smith, B.T. Kress, Y. Lu, D. Chandler-Militello, A. Benraiss, M. Nedergaard
Fluorescent Ca(2+) indicators directly inhibit the Na,K-ATPase and disrupt cellular functions Sci. Signal., 11 (2018))
has revealed some rather unpleasant truths about this group of compounds, which are further reviewed and consolidated in this review (https://doi.org/10.1016/j.ceca.2018.04.005 by Bootman and colleagues in Cell Calcium, 2018, 73, p82-87) where the Ca2+ independent misdemeanours of BAPTA are summarised in Table 1. Some of these appear relevant to optogenetics.
Therefore I wonder whether it would be prudent to repeat some of the analyses in figure 5 in the presence of dibromo-BAPTA. I recognise this is a departure from what has historically been considered a reasonable standard, but I am not sure that standard should remain unchallenged in light of the recent findings described by others.
Minor comments
While I accept the main purpose of this paper is to examine the stable photocurrent these channels produce it appears unlikely that depolarising tools will be used with 5, 30 or 100sec stimulation protocols in practice. I would be grateful if the authors would consider evaluating short burst protocols of stimulation.
There are some additional minor comments pertaining to presentation
Line 42 – red shifted "activation" spectrum rather than "activity"
GtACR1 – phylus for GT - present for all other ChR's described (Guillardia theta)
Line 106 "were ranging" to “ranged”
Line 172 hill should be Hill
Line 180 "outside pH" should be "external" or "extracellular pH"
Line 261 "down" can be deleted
Line 261 "a much increased change" to "an increased change"
References some of the titles are in bold, some are not,
Author Response
Response to Reviewer 2 Comments
Point 1: that the work has been entirely conducted in Xenopus oocytes at 16 degrees Celsius.Channel Rhodopsins have a thermal sensitivity, and expression levels (which potentially contribute to some of the changes in photocurrent) may depend on the experimental model system used (codon optimised for mouse for example).
While not wishing to question the quality of the data, or the long standing role the Xenopus oocyte model has played in ion channel physiology, I suspect that many groups who may consider using these tools may feel better about doing that if some comparative analyses were presented from a mammalian cell line at body temperature.
If this is not technically feasible, or is perhaps part of an extended application of this project that will be part of a larger submission in due course, perhaps the authors would agree to observe these present limitations? Attempts to transduce or transfect mammalian cells inevitably produce variable transgene levels which may erode some of the apparent differences seen, mentioning this caveat may help set the expectations of those that may follow this papers lead.
Response 1: We were measuring at room temperature (about 20 degrees), which is also suitable for C. elegans and Drosophila. We agree that it would be meaningful to test these new tools in mammalian cell line at body temperature. But we are not working with any mammalian cells by ourselves. Our collaborators working in the mammalian cell field could follow this up in the future. We also think these new mutants have good possibility to work well in mammalian cells as their wild type ChR2, Chronos and PsChR had already been tested in mammalian cells in previous publications.
Point 2: I was interested to see the experiment performed with BAPTA, and also its interpretation in Figure 5.
A recent series of experiments with the fluorescent BAPTA derivatives
(N.A. Smith, B.T. Kress, Y. Lu, D. Chandler-Militello, A. Benraiss, M. Nedergaard
Fluorescent Ca(2+) indicators directly inhibit the Na,K-ATPase and disrupt cellular functions Sci. Signal., 11 (2018))
has revealed some rather unpleasant truths about this group of compounds, which are further reviewed and consolidated in this review (https://doi.org/10.1016/j.ceca.2018.04.005 by Bootman and colleagues in Cell Calcium, 2018, 73, p82-87) where the Ca2+ independent misdemeanours of BAPTA are summarised in Table 1. Some of these appear relevant to optogenetics.
Therefore I wonder whether it would be prudent to repeat some of the analyses in figure 5 in the presence of dibromo-BAPTA. I recognise this is a departure from what has historically been considered a reasonable standard, but I am not sure that standard should remain unchallenged in light of the recent findings described by others.
Response 2: We agree that different BAPTA derivatives might have different influences in the cell. In our case, we do not study the Xenopus oocyte physiology but only use BAPTA to block the endogenous calcium activated Cl- channel current. As we could see from the kinetics in Figure 5A that the Cl- current (which shows a slower off kinetics) was well blocked. Then we could compare only the photocurrent of our channelrhodopsins reliably.
Point 3: Minor comments While I accept the main purpose of this paper is to examine the stable photocurrent these channels produce it appears unlikely that depolarising tools will be used with 5, 30 or 100sec stimulation protocols in practice. I would be grateful if the authors would consider evaluating short burst protocols of stimulation.
Response 3: In this study, our new tools all showed slower kinetics and increased light sensitivity. They might not be ideal for ultra-fast multiple stimulation as indicated for XXL and XXM in our previous publications by Dawydow et al. 2014 PNAS and Scholz et al. 2017 eLife. But they are useful for experiments which require low light and longtime stimulation. However, we are also working to make new tools with high calcium conductance and fast kinetics to meet different application purposes. This could be a follow up work together with the above-mentioned parts in Point 1 once we succeeded.
Point 4: There are some additional minor comments pertaining to presentation
Line 42 – red shifted "activation" spectrum rather than "activity"
GtACR1 – phylus for GT - present for all other ChR's described (Guillardia theta)
Line 106 "were ranging" to “ranged”
Line 172 hill should be Hill
Line 180 "outside pH" should be "external" or "extracellular pH"
Line 261 "down" can be deleted
Line 261 "a much increased change" to "an increased change"
References some of the titles are in bold, some are not,
Response 4: Many thanks for the careful proofreading. All of them are changed accordingly.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Many thanks for correcting the typo's and making some adjustments to the article overall. I believe they have improved the final product.
The comments to the earlier review are acknowledged. Since additional experiments were not undertaken, I would have preferred to see a "limitations of this study" paragraph in the discussion covering the three points highlighted previously. I believe these help those new to the field, and in an era of increasing scrutiny on the reproducibility in science establish a boundary about what has not been achieved in the present work.
Specifically those limitations are
1) this has not been tested in a mammalian system
2) the kinetics of the current channel appear unfavourable for ultra-fast stimulation
3) one of the chemicals used in the validation of ion channel selectivity may be altering the ion currents in more ways than the explanation presented in the rebuttal suggests.
Clearly this is a matter of opinion, and I will leave it for the authors and editors to determine its inclusion.
I wish the team success in their continued efforts to improve the optogenetic toolset.
Author Response
Point 1: Many thanks for correcting the typo's and making some adjustments to the article overall. I believe they have improved the final product.
The comments to the earlier review are acknowledged. Since additional experiments were not undertaken, I would have preferred to see a "limitations of this study" paragraph in the discussion covering the three points highlighted previously. I believe these help those new to the field, and in an era of increasing scrutiny on the reproducibility in science establish a boundary about what has not been achieved in the present work.
Specifically those limitations are
1) this has not been tested in a mammalian system
2) the kinetics of the current channel appear unfavourable for ultra-fast stimulation
3) one of the chemicals used in the validation of ion channel selectivity may be altering the ion currents in more ways than the explanation presented in the rebuttal suggests.
Clearly this is a matter of opinion, and I will leave it for the authors and editors to determine its inclusion.
I wish the team success in their continued efforts to improve the optogenetic toolset.
Response 1: Many thanks for the comment again. We believe that this comment could help to improve the clarity of our publication. We have added these 3 points in line 265-266, line 270-271 and line 274-278 separately. New reference about BAPTA was added in line 276.
