Temperature-Dependent Menthol Binding across TRPM8 Conformational States

Cold sensation is mediated by TRPM8, a polymodal ion channel activated by low temperatures, pH, voltage, and cooling compounds such as menthol. Despite its central role in thermosensation and pharmacology, the molecular basis of menthol-mediated activation remains unclear, particularly how temperature influences ligand interactions and channel modulation. This polymodal behavior challenges conventional views of ligand–protein recognition, as channel activity emerges from the interplay between conformational equilibria, ligand partitioning, and physicochemical properties. Here, we investigated the influence of temperature on menthol distribution around TRPM8 using coarse-grained flooding molecular dynamics simulations of experimentally resolved open and closed conformations over a physiological temperature range (279-310 K). To enable these simulations, we developed and parameterized a new Martini 3-compatible coarse-grained model for menthol. Menthol exhibited marked temperature-dependent redistribution across the membrane and protein surface, preferentially accumulating in intracellular and aqueous regions at temperatures that favor TRPM8 activation, whereas at warmer temperatures it partitioned predominantly into the lipid phase. Redistribution was also observed at the protein surface, with interaction regions consistent with experimentally identified sites spanning the N-terminal domain, pore domain, voltage sensor-like domain, and C-terminal region. A two-state allosteric model further indicated that these temperature-dependent occupancy changes shift the open-closed equilibrium of TRPM8 independently of the channel's intrinsic thermosensitivity. By isolating the contribution of ligand partitioning from the complex network of coupled stimuli that regulate polymodal channels, this approach provides a unique framework for dissecting individual physicochemical determinants of channel modulation. Together, these findings identify temperature-dependent ligand partitioning as a key determinant of menthol efficacy and provide new molecular insight into TRPM8 modulation.

This work was recently published in Cirqueira, Leonardo et al. Journal of the American Chemical Society (2026) 148 (27): 29195–29205. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants 2023/07855-3 and 2024/09222-0.