PONE-D-22-29508Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channelsPLOS ONE

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"We thank Shigeki Higashiyama (Ehime University) for the AP-fused constructs; Makoto Arita (Keio University) for the helpful information on the TRP channel and TGF-α shedding response; Kayo Sato, Shigeko Nakano and Ayumi Inoue (Tohoku University) for their assistance in plasmid preparation. A.I. was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grants 21H04791, 21H05113, JPJSBP120213501 and JPJSBP120218801; FOREST Program JPMJFR215T and JST Moonshot Research and Development Program JPMJMS2023 from Japan Science and Technology Agency (JST); Takeda Science Foundation; Uehara Memorial Foundation. M.T. received JSPS KAKENHI 22J10475. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this work, Tatsumi et al. developed a new molecular biology method to assay TRP channels activity exogenously expressed in cell lines, based on the use of a transforming TGF alpha shedding assay. This assay has the advantage of being low cost compared to calcium imaging or patch-clamp tecniques. That said, the information obtained by this method doesnt´t allow to monitorize TRP channel activity since the measurement is based on cummulated activity. Further, it is not useful to evaluate the activity of natively expressed TRP channels. Despite those limitations, this work might help the high-throughput screening of TRP channels ligand and to clarify the relationship between TRP channels and patho-physiologically relevant proteins as ADAM. Overall, the manuscript is well-written, well-structured, and the data support the conclusions.

As a main concern, authors show spontaneous TRPV1 activation (Figure 1H), however the origin of this activity remains unclear in the manuscript, since authors do not discuss this point or mention any reference supporting this. Is this TRPV1 spontaneous activation developed as a consequence of TGF alpha transfection? Has been this spontaneous activity previously reported in calcium imaging or patch-clamp experiments? If so, authors should add the corresponding references to the draft.

Reviewer #2: The present manuscript by Tatsumi and colleagues is an attempt to develop a low-cost and easy-to-access method for evaluating TRP channel activation. This will be useful for researchers, especially those who have limited access to specialized equipment, and may broaden the TRP channel field.

The present manuscript should be accepted in its present form.

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Reviewer #1: No

Reviewer #2: No

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Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels

PONE-D-22-29508R1

Dear Dr. Inoue,

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.

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Kind regards,

Agustín Guerrero-Hernandez

Academic Editor

PLOS ONE

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