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PONE-D-24-28479Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl boratePLOS ONE

Dear Dr. % Korkhov,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Dr. Korkhov:

Thank you for submitting your manuscript to PLOSone. Your manuscript was evaluated for two experts in the field, with significant issues about data presentation and interpretation that requires your attention.

Best regards

Eliseo Eugenin

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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

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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: Yes

Reviewer #2: Yes

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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: From my recollection gap junctional communication is always necessary for normal tissue function whereas as the hemichannel activity is already shown by numerous groups it could be rather deleterious. In addition, there are known small molecules that do not block gap junction channels and block Cx hemichannels providing a great tissue protection in pathological conditions. These issues should be clarified in the introduction and discussion section as well. Otherwise, the manuscript data is very interesting.

Please see the following references. doi.org/10.1038/s41467-019-14063-8; doi.org/10.1073/pnas.2213162119; doi: 10.3389/fncel.2023.1163436.

During inflammation there is reduction in gap junctional communication and increase in hemichannel activity and hemichannel blockers have been shown to prevent or reduced cell death or tissue dysfunction. Clearly, what is needed to test the role of reduced gap junctional communication are molecules that increase the gap junction channel activity without affecting hemichannels.

Reviewer #2: In this study, Lavriha and colleagues set out to explore the molecular basis of inhibition of Cx32-based channels by MFQ and 2APB. They employed a range of techniques, including dye uptake assays, FRAP analysis, Cryo-EM samples preparation and analysis, immunohistochemistry, tryptophan fluorescence quenching-based binding assays and molecular modeling. Additionally, they expressed and analyzed different mutant constructs for Cx32. Their findings revealed that 2APB binds to "site A," near the N-terminal gating helix of Cx32 GJC, restricting access to the channel pore. In contrast, MFQ binds to a different "site M," which is deeply embedded within the pore. Overall, most of the experiments were well-executed, and the chosen methodologies aligned with the primary research questions.

Concerns:

1. Why did the authors choose 2-APB as an inhibitor of connexin-based channels? Its use is less common compared to other well-known hemichannel and GJC blockers (only 18 results in PubMed using the keywords "2-APB" and "connexin"). In fact, 2-APB is better known as an inhibitor of both IP₃ receptors and TRP channels.

2. Line 65. The authors should cite: PMID: 32612499 and PMID: 33769363

3. Why did the authors choose SR101 instead of other commonly used dyes to assess hemichannel activity (e.g., ethidium, YOPRO, DAPI, etc.)? What evidence supports the ability of this dye to permeate Cx32-based channels?

4. How do the authors rule out the contribution of Panx1 hemichannels to SR101 uptake? Previous studies have shown that HEK293 cells endogenously express Panx1 (PMID: 17036048)

5. Fig. 1. The authors should show representative images for SR101 uptake and FRAP analysis.

6. Does the MFQ or 2-APB absorb at or near the excitation or emission wavelengths of tryptophan at the concentrations required for the titration study? If so, is the inner filter effect significant?

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Reviewer #1: No

Reviewer #2: No

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Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate

PONE-D-24-28479R1

Dear Dr. Korkhov,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Eliseo A Eugenin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Dr. Korkhov:

Thank you for submit your manuscript to PLOSone. Thank you for answer the reviewers

Best Regards

Eliseo Eugenin

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Yes

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

Reviewer #2: Yes

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4. 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 #2: Yes

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5. 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 #2: Yes

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6. 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 #2: The authors have thoroughly addressed all of my major concerns by conducting additional data analyses and enhancing the clarity of the text.

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7. 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 #2: No

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