Multiscale Modeling of Icilin and Calcium Binding to Human TRPM8 Ion Channel: Insights From MD, MMPBSA, and QM/MM Analyses.

The transient receptor potential melastatin 8 (TRPM8) channel has been established as the principal molecular sensor of cold in mammals and has emerged as a promising pharmacological target for pain therapy. Whereas menthol activates TRPM8 independently of intracellular Ca2+, the synthetic agonist icilin strictly requires this ion as a binding cofactor. The mechanistic basis of this ion-dependent agonism remains incompletely understood. In this work, a multiscale computational framework integrating comparative modeling, molecular dynamics simulations (MD), binding free energy decomposition (MMPBSA), and hybrid QM/MM calculations was applied to elucidate the structural and electronic determinants of icilin recognition in the human TRPM8 channel. The results indicate that the Ca2+ ion does not substantially alter overall binding affinity but reorganizes the hydrogen-bonding and coordination network, incorporating icilin into its coordination sphere while redistributing electron density among critical residues. This reorganization stabilizes coupling between the voltage sensor-like domain and the TRP domain, supporting an allosteric model in which Ca2+ functions as an enhancer of conformational communication rather than as a simple affinity booster. Non-covalent interaction (NCI) and natural bond orbital (NBO) analyses identified residues such as E782, D802, and E1004 as key sites coordinating Ca2+ within the TRPM8 cavity. Overall, these findings advance the mechanistic understanding of TRPM8 activation and emphasize the role of ion-mediated polarization. Importantly, although Ca2+ modulation is a conserved feature across several thermoTRPs, the underlying structural mechanisms are specific to each channel. Beyond fundamental insights, this framework provides a mechanistic basis for the rational design of selective TRPM8 modulators.