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TRPM8 is a thermosensitive ion channel which responds to multiple stimuli such as chemical and thermal stimuli in a multimodal manner. This ion channel has been studied extensively and is of immense importance in understanding the behavioral adaptation of animals across phyla in response to external thermal stimuli. There is a great deal of interest in the pharmacological field also regarding TRPM8, owing to its implication in pain sensation and therapeutics. Initial structural investigation of TRMP8 involved solving the cryo-EM structures of avian TRPM8, but due to species-specific differences in TRPM8-mediated responses to cold stimuli, recent structural investigation pursued mammalian TRPM8 in complex with various agonists, antagonists, and lipids in various conformational states (PMIDs: 29217583; 30733385; 31488702; 36227998; 37857704) All those studies provided important insights into the function of TRMP8. The structural mechanism of cold-induced open state has remained elusive for a long while, and only recently cryo-EM structures of cold-evoked opening of the TRPM8 channel has been reported (PMID: 40661591). The authors in the current study have done extensive cryo-EM analysis of a series of conformational states of TRPM8 and reported the open state structure induced by cold conditions and augmented by type I and II cold agonists. In addition, the authors have attempted extensive functional characterization to delineate the mechanism of polymodal regulation of TRPM8 channels in response to combined action of thermal and chemical stimuli. The experiments carried out in the work is meticulously designed and well executed, with results supporting most of the claims.  The manuscript overall is well written and clear. I have some questions, additional comments and suggestions for improvement/clarity.

Major:

1.    The main question posed in the manuscript was investigating the cold-induced open state, potentiated through cooling agents. An I846V mutant was instrumental in obtaining the open state. I am curious if the authors have considered looking into the difference in channel function behavior of this mutant under given ligand conditions at 20 and 4-8 ⁰C? Since the enhancement of menthol induced activation seems crucial in obtaining the open state, a few lines mentioning the molecular insights into how this mutation potentiates the binding will be helpful, given the structure is already there.

2.    The dynamic mode of menthol binding is interesting. Please consider adding a panel with all the binding poses of menthol superimposed, which can better guide the sequential rotation/movement through different states. Also, as depicted in Figure 1F, only the C₂^M state has a near unambiguous pose. Since the associated maps are not yet released, it is hard to follow the dynamic changes in the orientation of menthol in other maps with only the density features shown as of now. 

3.    AITC, a type II cold agonist for TRPM8 was used in the sample prep throughout, at sub-activating concentration of 0.5 mM and additional 250 µM added during grid vitrification. Can the authors comment on the actual activating concentration and if using higher concentration of AITC could possibly have destabilized the closed state more from the beginning and consequently stabilized the cold induced open state more?

4.    The authors attributed and have shown Phe979 is not involved in gating and have tried to functionally probe its role, but the Phe979Cys lacks channel activity. Have the authors considered examining the surface expression of this mutant to assess if they are being trafficked to membrane normally?

Minor:

1.    Although I am not an expert in electrophysiology, it seems the ephys experiments have been meticulously carried out to validate the ligand binding pocket and gating residues in the pore. I have one query though, in the figure legends, biological replicates are mentioned, but I couldn’t find any details in methods as to what the biological replicates refer to, is it the number of independent experiments (starting from different passage of cells, transfection etc.)?It would be helpful for the authors to clarify this in the text.

2.    It would be helpful if authors can mention the temperature range for activation of TRMP8 in mammals, to help the readers draw a parallel between the physiological temperatures at which TRPM8 functions and the temperature range used in the experiments.    

3.    Please mention at what cryo-EM map level the density feature for the ligand menthol is contoured. This is also suggested for all other panels in which the cryo-EM density features for ligands/residues are shown to improve clarity.

4.    Because the relative spatial positioning of the pore residues defines the various conformational states, the density feature for the corresponding residues (Figure 2A) may be shown, perhaps in supplement.

5.    Figure 1: The panel in Figure E is too dense, and it is hard to judge the quality difference among the different maps from a quick glance. It will be helpful for interpretation if the GSFSC resolutions are provided as inset in the bottom of individual maps. In Panel F, can a marker in terms of distance between two side chain features be included? This will aid in conveying the pore opening through different conformational states to the readers in general. Also, are all the maps contoured at the same level?

6.    Figure S3: The workflow is too dense. To help, the authors could consider splitting into two. For clarity, please mention how the reference map (EMD-27892) was used in heterogenous classification.

In other words, at what resolution reference map was low pass filtered? I am assuming other volumes or bad classes were also used to remove junk particles, what were those volumes? Were any ab-initiomodels generated?

7.    Figure S4: Please mention the number of micrographs for each of the datasets used in image processing, or mention in an additional row in Table S1 to aid reader interpretation.

8.    Figure S1G, S6A, S9A: Please mention what atoms were used for superposition/overlay for clarity.

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.