Multiscale Modeling of Icilin and Calcium Binding to Human TRPM8 Ion Channel: Insights From MD , MMPBSA

ABSTRACT

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 Ca ²⁺ , 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 Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ within the TRPM8 cavity. Overall, these findings advance the mechanistic understanding of TRPM8 activation and emphasize the role of ion‐mediated polarization. Importantly, although Ca ²⁺ 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.