Temperature-Dependent Menthol Binding Across TRPM8 Conformational States

Cold sensation is mediated by TRPM8, a polymodal ion channel activated by temperature, 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 binding and channel activation. Polymodal ion channels further challenge site-centric views of ligand binding, as function emerges from coupled conformational equilibria and environment-dependent membrane partitioning. Here, we investigate temperature-dependent menthol distribution in TRPM8 using flooding molecular dynamics simulations of open and closed channel conformations across a physiological temperature range. This approach reveals distributed, low-affinity interactions that are not captured by discrete binding-site models. Below the TRPM8 activation threshold (<300 K), menthol preferentially accumulates in intracellular and interfacial regions, whereas above this threshold it redistributes toward transmembrane regions, indicating that temperature-dependent activation regimes reshape ligand partitioning at the protein–membrane interface. Across all conditions, menthol behaves as a low-affinity multisite ligand sampling a continuum of metastable interaction regions, consistent with experimentally identified interaction regions spanning the N-terminal domain, pore domain, voltage sensor-like domain (VSLD), and C-terminal region. A two-state allosteric model indicates that these temperature-dependent occupancy patterns shift the open–closed equilibrium of TRPM8. Together, these results suggest that temperature regulates function not only through channel energetics but also by modulating ligand interactions at the protein–membrane interface. This work identifies temperature-dependent ligand partitioning as a key physicochemical determinant of ligand efficacy in polymodal ion channels and provides molecular-level insight into menthol modulation of TRPM8.