Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a ‘grab and stand’ mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.

To adapt to habitat temperature, vertebrates have developed sophisticated physiological and ecological mechanisms through evolution. Transient receptor potential melastatin 8 (TRPM8) serves as the primary sensor for cold. However, how cold activates TRPM8 and how this sensor is tuned for thermal adaptation remain largely unknown. Here we established a molecular framework of how cold is sensed in TRPM8 with a combination of patch-clamp recording, unnatural amino acid imaging, and structural modeling. We first observed that the maximum cold activation of TRPM8 in eight different vertebrates (i.e., African elephant and emperor penguin) with distinct side-chain hydrophobicity (SCH) in the pore domain (PD) is tuned to match their habitat temperature. We further showed that altering SCH for residues in the PD with solventaccessibility changes leads to specific tuning of the cold response in TRPM8. We also observed that knockin mice expressing the penguin's TRPM8 exhibited remarkable tolerance to cold. Together, our findings suggest a paradigm of thermal adaptation in vertebrates, where the evolutionary tuning of the cold activation in the TRPM8 ion channel through altering SCH and solvent accessibility in its PD largely contributes to the setting of the cold-sensitive/ tolerant phenotype.TRPM8 | cold activation | pore domain | side-chain hydrophobicity | thermal adaptation T o survive and thrive, all living beings have to perceive and adapt to ambient temperature (1), which varies over a wide range from below −50°C in polar areas to above 50°C in deserts (2). Therefore, sophisticated physiological and ecological mechanisms have been developed through evolution to first detect and then adapt to ambient temperature (3-5). The transient receptor potential melastatin 8 (TRPM8) channel is the prototypical sensor for cold in vertebrates (6, 7), which has been validated in both knockout mice (8) and pharmacological studies (9). However, how cold activates TRPM8 remains obscure. From the perspective of channel structure, an earlier study suggested that its C terminus is crucial for cold activation (10), while subsequent work demonstrated that the transmembrane core domain (5) or the pore domain (11) is essential for setting cold response. Although high-resolution structures of TRPM8 have been resolved by cryoelectron microscopy in both the apo and ligand-bound states (12-14), its coldactivated state structure is still unavailable. From the perspective of thermodynamics, large enthalpic (ΔH) and entropic (ΔS) changes are associated with TRPM8 cold activation (15,16). Changes in heat capacity have also been hypothesized to mediate cold activation (17), though experimental evidence for such a hypothesis is limited to voltage-gated potassium channels (18). Interestingly, cold activation of TRPM8 is tuned during evolution in several tested vertebrate species (5,11,19). To understand both the structural and thermodynamic bases of TRPM8 cold activation, we attempted to gain insights from TRPM8 orthologs in vertebrate species inhabiti...