PONE-D-25-14924Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine.PLOS ONE

Dear Dr. Senning,

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We thank Dr. Sharona Gordon for TRPV1.pcDNA3.1 and Dr. Jason Mclellan for JSM-164 NW10 3C-GFP-HS (GFP). We also thank Dr. Shai Silberberg for a conversation that started the ball rolling on the mysterious M308 residue of TRPV1. This work was supported by start-up funds from the University of Texas at Austin (E.N.S.) and the NSF-MCB grant no. 2129209 (E.N.S.).

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This work was supported by start-up funds from the University of Texas at Austin (E.N.S.) and the NSF-MCB grant no. 2129209 (E.N.S.).

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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: Partly

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

Reviewer #1: Yes

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: No

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: The general quality of the data presented in the manuscript is good, and the study addresses a relevant scientific question. However, the presentation of the data lacks clarity and, at times, appears sloppy. This significantly affects the readability and interpretation of the results. I have several major comments that need to be addressed to improve the manuscript.

Please see the attached review for more details.

Reviewer #2: In the manuscript “Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine.”, by Grace C. Wulffraat, Sanjana Mamathasateesh, Rose Hudson1, Benjamin He, Andrés Jara-Oseguera1, Eric N. Senning. Authors provide new data about isolates residue M308 in the ankyrin repeat domain of TRPV1 channel, which exhibits a large number of missense variants in humans while being conserved across species. Mutagenesis experiments have shown that substitutions in M308 can affect channel function, with larger or more polar residues increasing activity. In addition, the conservation of M308 may be related to its role in redox-dependent modifications, and a splice variant lacking M308 reduces TRPV1 expression on the surface of HEK293 cells. These data suggest that M308 is critical for channel regulation and expression dynamics.

Although the study is of undoubted interest and expands our knowledge about functions of TRPV receptors to increase the level of validity of the material and publication in the journal, it needs some improvements.

In the Materials and Methods section, the missense variant analysis, then electrophysiology. I would put the information about the object of study (Cell Culture) at the beginning. The electrophysiology section goes on to cell culture and it's not clear what kind of culture it is.

All the Materials and Methods sections are quite detailed, but the molecular biology section contains mostly references to literature, it should be expanded to include information on the experimental approaches used in the study.

Capsaicin was diluted in ethanol. Potentially ethanol can have an effect on cells . It is necessary to specify what concentration of solvent was used and whether the same concentration of solvent was added to the control solutions. If not, explain why this can be neglected.

In the experiments with temperature change a modified setup was used, it is necessary to indicate how fast and with what accuracy it was possible to change the temperature in the experiment.

The Materials and Methods section lacks information about the method of staining of the preparation in Fig. 4 E and the microscopic technique with which it was obtained. There is no scale in Fig. 4 E.

There are two large sections in the Materials and Methods are devoted сalcium imaging experiments. But in the Results and Discussion section I found very little information regarding the description of these experiments. It is necessary to clearly describe why they were performed and what the authors wanted to show. Why ionomycin was used, what it allows to reveal, what follows from the obtained data. I did not find in the discussion section any specific conclusions on this block of results.

The same applies to data obtained by electrophysiological methods, and there is no discussion of these data or conclusions from these experiments in the discussion section.

I think it is also good to add to the discussion information about the physiological significance of the obtained results

With all the suggested additions, the paper can certainly be accepted for publication.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review_PlosONE.pdf

Missense variant analysis in the TRPV1 ARD reveals the unexpected functional

significance of a methionine.

PONE-D-25-14924R1

Dear Dr. Senning,

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,

Alexander G Obukhov, Ph.D.

Academic Editor

PLOS ONE

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: In general, the author tried to take into account all the comments of the reviewers. The manuscript can be accepted for publication

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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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