Structure of human TRPM8 channel

Abstract

TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It is a therapeutic target for anticancer drug development, and its modulators can be utilized for several pathological conditions. Here, we present a cryo-electron microscopy structure of a human TRPM8 channel in the closed state that was solved at 2.7 Å resolution. Our structure comprises the most complete model of the N-terminal pre-melastatin homology region. We also visualized several lipids that are bound by the protein and modeled how the human channel interacts with icilin. Analyses of pore helices in available TRPM structures showed that all these structures can be grouped into different closed, desensitized and open state conformations based on the register of the pore helix S6 which positions particular amino acid residues at the channel constriction.

Fig. 1. Overall structure and domain composition of human TRPM8 channel.

a Cryo-electron microscopy electrostatic potential composite HsTRPM8 map colored according to the channel subunits (olive, red, orange, and yellow; three views). b Atomic model of HsTRPM8 in cartoon representation, colored as in a. c Domain composition of HsTRPM8. Residue numbers at the boundaries between particular elements are given. d Structure of a single subunit with domains colored as in c. A contour of the cryo-EM composite map for the entire tetramer is shown in gray (three views). Dotted lines in a, b indicate the approximate boundaries of the cell membrane. MHR melastatin homology region, VSLD voltage-sensor-like domain, TMD transmembrane domain, TRP TRP helix, CTD C-terminal domain. See also Supplementary Figs. 1 and 2.

Fig. 2. Configuration of the HsTRPM8 TMD in a closed C₀ state.

a The pore of HsTRPM8, composed of helices S5, PH, and S6 (two views). Hydrophobic residues M978 and F979 closing the gate and the F912 and G913 from the selectivity filter are shown as sticks. In the upper panel, front and rear subunits were removed for clarity. Gray surface represents pore radius calculated using the HOLE program. b Comparison of the pore of HsTRPM8 (red) and MmTRPM8 (light blue) (PDB ID: 7WRA). c–e Comparison of TMDs of HsTRPM8 (red) and MmTRPM8 (light blue) after superimposition of the corresponding pore regions. Insets show details of differences in the conformation of TRP helix (d) and S4-S5 linker (e). PH pore helix, TRP TRP helix. See also Supplementary Figs. 3 and 4.

Fig. 3. Ligand-binding pockets in HsTRPM8, modeling, and validation of icilin binding.

a HsTRPM8 composite map (top view) with non-protein densities colored blue. b VLSD cavity with unidentified non-protein densities. c Densities between helices S5 and S6 are assigned to undecane and Na⁺ ions. d Densities between helix S5 and VSLD assigned to two molecules of cholesteryl hemisuccinate (CHS) and one phosphatidylcholine (POPC). e–h Modeling of icilin bound to VSLD cavity of HsTRPM8 in standard (e, f) and rotated (g, h) orientation. Predicted interacting residues are labeled and displayed as sticks. i HsTRPM8 activation assay: concentration-response curves of HsTRPM8 variants activated by selected agonists. HEK293 cells were transiently transfected with constructs coding indicated HsTRPM8 variants or empty vectors (MOCK). Transfected cells were then stimulated with increasing concentrations of the indicated agonists. Dose response curve with a variable slope was fitted to a measured fluorescence signal of the Fluo-8 NW dye normalized to the cellular responses prior to agonist stimulation (ΔF/F₀). Error bars correspond to SD from n = 3 (WS-3) or n = 4 (Icilin, Cooling Agent-10, Menthol) replicates. For each curve, EC₅₀ value is given with 95% confidence intervals. See also Supplementary Figs. 2e and 5, and Supplementary Table 1.

Fig. 4. Structure of the pre-MHR and MHR1/2 domains of HsTRPM8.

a Atomic structure of the pre-MHR and MHR1/2 from one subunit of the HsTRPM8 is shown in cartoon representation with helices in red, α-strands in green, and loops in yellow. A contour of the composite cryo-EM map for the entire tetramer is shown in gray. The inset shows a focused MHR1/2 map in the same color code. b, c Comparison of pre-MHR from HsTRPM8 reported herein (red) with other human TRPM channels (b) and orthologous TRPM8 structures (c) (colored as in the figure keys).