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

Francisco José Taberner,Ainara López-Córdoba,Gregorio Fernández-Ballester,Yuri Korchev,Antonio Ferrer-Montiel

doi:10.1074/jbc.m114.577478

Abstract

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.

Highlights

The gating mechanism of transient receptor potential melastatin 8 (TRPM8) channels remains elusive
Mutations in the S6-TRP Box Linker Region of TRPM8 Modulate Channel Gating—To investigate the contribution of the linker region between the cytosolic end of the S6 and the TRP box in TRPM8 channels, we used a chimeric approach whereby short fragments of TRPM8 were cumulatively replaced by the cognate region of TRPV1 (Fig. 1B)
Cumulative data indicate that the TRP domain, a cytosolic protein region adjacent to the S6 transmembrane segment plays a pivotal role in channel gating

Summary

Background

The gating mechanism of transient receptor potential melastatin 8 (TRPM8) channels remains elusive. The TRP domain is a moderately conserved region of about 30 amino acids that contains the highly conserved 6-mer segment referred to as the TRP box (Fig. 1) This protein domain is a pivotal molecular determinant of TRP channel structure and activity, as it contributes to their tetrameric assembly and to regulate channel responses to all activating stimuli [12, 21,22,23,24,25,26]. To further understand the functional role of the TRP domain in channel gating and unveil molecular determinants of this process, we have investigated the contribution of the S6-TRP box linker region in TRPM8 channel activation (Fig. 1). Our findings provide novel insights on the mechanism of channel gating in the TRP channel family, and substantiate the importance of the TRP domain in transmitting the energy of stimuli recognition to gate opening

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION