The region adjacent to the C-end of the inner gate in transient receptor potential melastatin 8 (TRPM8) channels plays a central role in allosteric channel activation.

The ability of transient receptor potential (TRP) channels to sense and respond to environmental and endogenous cues is crucial in animal sensory physiology. The molecular mechanism of channel gating is yet elusive. The TRP box, a conserved region in the N-end of the C terminus domain, has been signaled as pivotal for allosteric activation in TRP channels. Here, we have examined the role of the linker region between the TRPM8 inner gate and the TRP box (referred to as the S6-TRP box linker) to identify structural determinants of channel gating. Stepwise substitutions of segments in the S6-TRP box linker of TRPM8 channel with the cognate TRPV1 channel sequences produced functional chimeric channels, and identified Tyr(981) as a central molecular determinant of channel function. Additionally, mutations in the 986-990 region had a profound impact on channel gating by voltage and menthol, as evidenced by the modulation of the conductance-to-voltage (G-V) relationships. Simulation of G-V curves using an allosteric model for channel activation revealed that these mutations altered the allosteric constants that couple stimuli sensing to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1 structural model, showed that Tyr(981) may lie in a hydrophobic pocket at the end of the S6 transmembrane segment and is involved in inter-subunit interactions with residues from neighbor subunits. The 986-990 region holds intrasubunit interactions between the TRP domain and the S4-S5 linker. These findings substantiate a gating mechanism whereby the TRP domain acts as a coupling domain for efficient channel opening. Furthermore, they imply that protein-protein interactions of the TRP domain may be targets for channel modulation and drug intervention.