PONE-D-20-03301

Cobalt Ion Interaction with TMEM16A Calcium-Activated Chloride Channel: Inhibition and Potentiation

PLOS ONE

Dear Dr. Tsung-Yu Chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR: Please correct your manuscript according to the criticism of the reviewers.

We would appreciate receiving your revised manuscript by Apr 18 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Eugene A. Permyakov, Ph.D., Dr.Sci.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The submitted manuscript presents an in-dept investigation on the ion conductivity changes in a membrane Ca-activated chloride channel upon competitive binding of Co2+ ions. This protein is a transmembrane homodimer, each subunit having two high affinity binding sites for calcium. Correspondingly, the interpretation of just the current recordings upon the displacement of one type of metal by another type of metal is challenging. The authors have accumulated a respectable volume of current recordings in diverse conditions (including 10 mutant proteins). It would have definitely helped if the stability constants for cobalt ions were measured for all possible binding sites (and in the mutants). However, the complexity of lipid environment adds a whole extra layer of uncertainty. So, it is understandable that the data, as presented, do not make a completed picture.

Long story short, there are a number of questionable parts in the authors’ treatment of data, plus the English of the article is very heterogeneous. All that let me only to recommend a major revision of the manuscript. I have compiled the specific comments below.

Major comments

1. Ln. 101, 104: “…alter the affinity of Ca2+ in activating the channel…” — the affinity and channel activation should be discussed separately.

2. Ln. 169: “…the concentration of NaCl in the intracellular solution was reduced according to the extra [Cl-] from the added [CoCl2].” — such adjustments would have altered the ionic strength of the solution. Few words should be added on the possible impact on the observations.

3. “Data analysis” section is very poorly written, hard to read (compared e.g. to the Discussion, which is good).

4. Ln. 209: “was fit to a single-exponential function” — What kind of function is this? Apparently, it is valid only for the chosen concentration of EGTA?

5. The selection of a Langmuir-type equation for the data analysis is highly questionable. Why not employ standard competitive binding models?

6. Ln. 321-323, 340-341: “the correlation of Co2+ inhibition with the sidechain hydrophobicity is weak (Fig 7 A & B, middle panel). Inhibition appears to correlate with the molecular volume of the amino acid placed at position 589…” — I don’t see any correlations in these cases.

7. Fig. 8: The inverse correlation between the mutant residue volume and the current, could it be interpreted as a mechanical obstruction of the channel?

Minor and technical comments

1. Throughout (e.g. ln. 32, “…charged sidechain carboxylates to coordinate…”): ‘carboxylate’ is an anion of a carboxylic acid; the group is carboxyl (or ‘ionized carboxyl’ if one prefers).

2. Ln. 41: “…mutating a pore residue…” — in the abstract it’s preferable to be specific about which residue was mutated.

3. Ln. 64: “…conduction of ionic currents across lipid membranes. The physiological roles of the current conduction…” — it’s either ‘ion current’ or ‘ionic conduction’.

4. Ln. 68: “…are dimeric proteins consisting of two identical subunits…” — a proper term is ‘homodimeric’.

5. Ln. 96: “…each protein subunit consists of a set of Ca2+-binding sites…” — it doesn’t consist, but has exactly two binding sites, as follows from the published structures.

6. Ln. 106 : “…the binding alters the rectification of the Cl- flux through the channel pore by reducing the negative charge from these Ca2+-coordinating residues…” — not clear, should be rephrased.

7. Ln. 210: Fig 5C is cited not in sequence (the previous one was Fig 2B).

8. Ln. 514: What is CLC-0?

9. Ln. 521: Should be “vestibule”.

Reviewer #2: TMEM16A is a Ca2+-activated Cl- channel that plays key roles in diverse physiological processes including transepithelial Cl- transport. TMEM16A functional studies have been instrumental in deciphering the structure-function of the channel. For example, studies have shown that plotting Ca2+ activation of TMEM16A currents requires a Hill coefficient greater than 1, thereby consistent with multiple Ca2+ binding to activate TMEM16A currents. Moreover, functional studies demonstrated that the channel has two independent Cl- conducting pores.

Manuscript synopsis: Here, the authors explored TMEM16A gating using the divalent Co2+. Nguyen et al. report that Co2+ is a competitive inhibitor for the Ca2+ activation of TMEM16A. They also found that at high concentrations, Co2+ can potentiate Ca2+-evoked Cl- currents. These results were supported by inside-out patch experiments using intracellular Ca2+ application to activate TMEM16A, and application Co2+ to inhibit or potentiate these Ca2+-evoked Cl- currents. By varying concentrations of Ca2+ and Co2+ that were applied, the authors further characterize how Ca2+ and Co2+ regulate TMEM16A conducted Cl- currents. Additional experiments include mutations of the pore residue Y589 which were used to describe the relationship of Co2+ inhibition to amino hydrophobicity, volume, as well as the deactivation kinetics of the TMEM16A channel.

The experiments presented in this manuscript clearly demonstrate inhibition and potentiation Co2+ on TMEM16A Ca2+-evoked Cl- currents. Additionally, these experiments showing the effect of Co2+ on TMEM16A Ca2+-evoked Cl- current are well described and easy to understand. However, this is not as true for the Y589, whose interpretation in the manuscript somewhat confusing. The data included in this manuscript are interesting and should be published. However, improving the writing – specifically motivating the experiments presented in figures 6-10 and discussing the interpretation of these data - will create a more impactful manuscript.

Major points:

1) The motivation for the experiments depicted in figures 6-10 is not easily accessible to the reader. Even though understanding the characteristics of TMEM16A itself is important for the field, the specific angle for why we need to learn more about the biphasic dose response of TMEM16A is not stated. Moreover, reasons why the Y589 mutant was chosen for these experiments, or why this residue was targeted for multiple mutations, even why the amino acids used for these mutations were chosen should be clarified. Perhaps addressing how these data fit with current understanding of TMEM16A gating, or will direct future experimental directions will help the reader.

2) The manuscript alludes to the importance of amino acid carboxylates (line 97) in coordinating Ca2+ but does not address this concept in the context of the Co2+ findings and the Y589 mutants. The importance of these Y589 experiments in addressing the biphasic dose-response is also not clear. Perhaps further discussion on how these mutants address the main question could help.

3) Lines 71-84 and Figure 1 highlight TMEM16F and its structural properties. It is not clear how this information adds to or sets up the motivation for the question being addressed in this manuscript.

Minor points:

1) Lines 42-43 refer to Ca2+ as potentiating TMEM16A and not activating. Are the data suggesting that Ca2+ plays two roles?

2) Correlations described in Figures 7 and 8 are not apparent expect for �off. Are there patterns or features of the selected amino acids that should be described to help with the interpretation? The data are difficult to appreciate as described currently. Additionally, how these data relate to the overall question is quite unclear.

3) The manuscript is written as though it is a follow up to another study in a way that makes the current form of this one difficult to grasp and appreciate. The data are interesting and present new information about TMEM16A, and providing more rationale for the experiments conducted will enhance the value of the data.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Cobalt Ion Interaction with TMEM16A Calcium-Activated Chloride Channel: Inhibition and Potentiation

PONE-D-20-03301R1

Dear Dr. Tsung-Yu Chen,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Eugene A. Permyakov, Ph.D., Dr.Sci.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: