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This manuscript by Lee et al. presents an extensive structural and functional investigation of the cold- and menthol-activated ion channel TRPM8, providing a series of cryo-EM structures captured in multiple functional states across diverse ligand and temperature conditions. By integrating these structural analyses with mutagenesis and electrophysiology, the authors propose a detailed activation mechanism and delineate distinct conformational pathways underlying cold and menthol activation, including the identification of a putative “coldspot.” In addition, the authors argue that a recently reported “semi-swapped” TRPM8 architecture from another preprint (doi: 10.1101/2025.06.06.658377) is incompatible with the structural and functional requirements for pore dilation and S6-mediated gating, based on the evidence presented here.

The work is ambitious in scope, technically impressive, and addresses long-standing mechanistic questions in the TRPM8 field. Below is a list of comments that may improve clarity and deepen understanding of the manuscript.

Major points

1. The I846V mutant was used to obtain the pre-open and open conformations. However, the rationale for how I846V increases menthol-mediated potency and stabilizes these states is not sufficiently discussed. I would encourage the authors to address the role of this mutated residue in their activation model more in the discussion.

2. The authors propose that Val976 acts as the S6 gate residue, in contrast to the conclusions of a recent preprint by the Julius group (doi: 10.1101/2025.06.06.658377) that suggested Phe979 as the gate. In Fig. 2C, Val976 does not appear to project toward the pore pathway in the closed or pre-open state, whereas Phe979 does. Could the author explain why they think Val976 is the gate residue instead of Phe979?

3. All cryo-EM structures in this study were reconstructed under C4 symmetry. The authors may consider processing the maps without symmetry imposition to see if there is asymmetric ligand binding status or different subunit conformation.

4. Except for the C1A state, all other structures, including the open state, were obtained under co-application of cold and menthol. Meanwhile, the model in Fig. 6 shows separate cold-driven and menthol-driven activation mechanisms. Could the authors revise the figure to more accurately reflect the present structural study?

5. The authors have utilized multiple ligands and temperature conditions to determine the structures across various functional states. It would be helpful if they could provide a summary table that lists all sample preparation conditions along with their functional states.

Minor Points

1. The naming and annotation scheme of receptor structures is difficult to follow, as the same ligand label “M” is used to nearly all states without relevant discussion of its necessity. A shorter naming scheme placed earlier in the text may reduce confusion.

2. Contour levels are missing for the cryo-EM density maps shown in the figures. Providing the values would be helpful for comparing the ligand densities and key residues across different functional states.

3. Line 1 of page 6, rapamycin was introduced in some reconstructions, but its use and expected impact on stabilizing channel opening are not clearly justified. The authors may consider explaining the intended rationale of this compound.

4. The abbreviation “PH” appears on page 8 for the first time without definition. It would improve reader understanding if the authors could define this term earlier with related information.

5. It would be helpful for reader to understand the rationale if the authors could provide Kd or Ki, and fold-excess of the applied RR (Fig. 3F–G).

6. There are some typos throughout the text that the authors could correct to improve readability:

Figure 1: the legends for panels C and D are reversed.

Page 9: “4.6A” should be “4.6 Å.”

Page 14: “Fig. 3 and 6” should be “Figs. 3 and 6.”

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.