Structures of a mammalian TRPM8 in closed state

Abstract

Transient receptor potential melastatin 8 (TRPM8) channel is a Ca²⁺-permeable non-selective cation channel that acts as the primary cold sensor in humans. TRPM8 is also activated by ligands such as menthol, icilin, and phosphatidylinositol 4,5-bisphosphate (PIP₂), and desensitized by Ca²⁺. Here we have determined electron cryo-microscopy structures of mouse TRPM8 in the absence of ligand, and in the presence of Ca²⁺ and icilin at 2.5-3.2 Å resolution. The ligand-free state TRPM8 structure represents the full-length structure of mammalian TRPM8 channels with a canonical S4-S5 linker and the clearly resolved selectivity filter and outer pore loop. TRPM8 has a short but wide selectivity filter which may account for its permeability to hydrated Ca²⁺. Ca²⁺ and icilin bind in the cytosolic-facing cavity of the voltage-sensing-like domain of TRPM8 but induce little conformational change. All the ligand-bound TRPM8 structures adopt the same closed conformation as the ligand-free structure. This study reveals the overall architecture of mouse TRPM8 and the structural basis for its ligand recognition.

Fig. 1. Structure determinations of MmTRPM8.

a The 3D reconstructions of MmTRPM8 colored by local resolutions in Å. b The Gold standard Fourier Shell Correlation (FSC) curves of the final 3D reconstructions of MmTRPM8 in different ligand-bound states. Source data are provided as a Source Data file. c Local maps of MmTRPM8 structures in the regions of pore helix, selectivity filter, and outer pore loop at the contour level of 3.5 σ.

Fig. 2. Overall structure of MmTRPM8_{LMNG-ligand-free}.

a The 3D reconstruction of MmTRPM8_{LMNG-ligand-free} with each subunit individually colored. b The cartoon representation of MmTRPM8_{LMNG-ligand-free} with each subunit individually colored. c Cartoon representation of one subunit of MmTRPM8_{LMNG-ligand-free} with domains colored by the rainbow. d The topology of one MmTRPM8 subunit.

Fig. 3. Ion conduction pore of MmTRPM8_{LMNG-ligand-free}.

a Ion conduction pore of MmTRPM8_{LMNG-ligand-free} with front and rear subunits removed for clarity. b Top view of the surface electrostatic potential at the external entrance of MmTRPM8_{LMNG-ligand-free}. c The selectivity filter of MmTRPM8_{LMNG-ligand-free}. Dash lines show atom-to-atom cross distances in Å. The 4.7 Å distance shows that the Gln914 side chain does not form a hydrogen bond with Gly913 from its neighboring subunit. d Sphere-shaped densities in the selectivity filters of MmTRPM8_{LMNG-ligand-free} and MmTRPM8_{nanodisc-Ca-icilin-PIP2}. e The selectivity filter of MmTRPM4 (PDB: 6BCJ). The Gln973 side chain forms a strong hydrogen bond with the carbonyl of Gly972 from its neighboring subunit.

Fig. 4. Interactions between VSLD and the pore domain in MmTRPM8_{LMNG-ligand-free}.

a Hydrophobic interactions between S4 (cyan) and S5 from the adjacent subunit (yellow). b The structure of S4 and S4-S5 linker. The 3₁₀ helix in S4 is highlighted by orange color. c Interactions between TRP helix and S4-S5 linker. The polar residues Glu1004 and Tyr1005 in the C-terminal end of TRP helix point to the cytosolic-facing cavity of VSLD.

Fig. 5. The Ca²⁺ binding site in MmTRPM8.

a The VSLD of MmTRPM8_LMNG-Ca harbors a Ca²⁺ binding site. The Ca²⁺ density is shown at the level of 0.013 in UCSF chimera. b The absence of density at the potential Ca²⁺ binding site in VSLD in the map of MmTRPM8_{LMNG-ligand-free} at the level of 0.013 in UCSF chimera. c The RMSD of the Ca²⁺ plotted against simulation time. The distances between the Ca²⁺ and CG atoms in Glu782 (d), Asn799 (e), and Asp802 (f) plotted against simulation time. g Summary of MmTRPM8 activated by menthol and icilin. In HEK293T cells expressing WT or mutant MmTRPM8, the application of menthol (2 mM) or icilin (10 μM) elicited outward currents. For the mutants proposed to be important for Ca²⁺ coordination, most of them were no longer activated by icilin. WT and mutants were colored in blue and grey, respectively. For WT MmTRPM8, n = 3; for Q785K, n = 7, P = 2.25E−06; for Q785Y, n = 6, P = 2.41E−07; for N799R, n = 8, P = 1.78E−05; for N799L, n = 5, P = 3.46E−04; for D802K, n = 4, P = 1.99E−04; for E782H, n = 3, P = 1.23E−05; for E782M, n = 3, P = 1.34E−05; for E782R, n = 3, P = 1.41E−05; for Q785H, n = 3, P = 1.06E−05; for Q785L, n = 3, P = 1.04E−05; for Q785M, n = 3, P = 2.81E−05; for Q785N, n = 3, P = 1.14E−05; for Q785R, n = 3, P = 1.61E−05; for N799H, n = 3, P = 2.79E−05; for N799I, n = 3, P = 5.56E−05; for N799K, n = 3, P = 1.31E−05; for D802E, n = 3, P = 1.21E−05; for D802M, n = 3, P = 1.02E−05; for D802R, n = 3, P = 1.33E−05. Two-sided t-test; *** denotes P < 0.001. All data points are mean ± s.e.m. h Summary of MmTRPM8 and mutants activated by icilin alone (10 μM) or in the presence of Ca²⁺ (1 mM). WT and mutants were colored in blue and grey, respectively. For WT MmTRPM8, n = 3; for Q785K, n = 7, P = 1.62E−10; for Q785Y, n = 6, P = 3.87E−07; for N799R, n = 8, P = 1.02E−10; for N799L, n = 5, P = 1.03E−07; for D802K, n = 4, P = 4.98E−06. Two-sided t-test; *** denotes P < 0.001). All data points are mean ± s.e.m. For c–h, source data are provided as a Source Data file.

Fig. 6. Structural basis and MD simulation for the icilin recognition.

a The Ca²⁺ and icilin binding sites in the VSLD of MmTRPM8_{LMNG-Ca-icilin}. b The icilin density in MmTRPM8_{LMNG-Ca-icilin} at the level of 0.015 in UCSF chimera. c Interactions between MmTRPM8 and Ca²⁺ and icilin. d The binding of Ca²⁺ and icilin induces local conformational change revealed by structural comparison of MmTRPM8_{LMNG-Ca-icilin} (salmon) and MmTRPM8_{LMNG-ligand-free} (cyan). e The RMSD of icilin plotted against simulation time. f The distance between the N21 atom in icilin and CG atom in Phe839 plotted against simulation time. g The RMSD of Ca²⁺ plotted against simulation time. h The distance between the Ca²⁺ and CG atom in Asp802 plotted against simulation time. i–k Concentration-dependent icilin activation of Asp802, Phe839, and Arg842 mutants in comparison with WT MmTRPM8 in whole-cell patch-clamp recordings (For WT MmTRPM8, n = 5; for D802A, D802I, D802L, D802S, D802N, D802Q, F839R, F839Y, R842K, R842N, and R842Q, n = 3). All data points are mean ± s.e.m. For e–k, source data are provided as a Source Data file.

Fig. 7. MmTRPM8 structures are in closed states.

a Ion conduction pore of MmTRPM8_{LMNG-ligand-free} with front and rear subunits removed for clarity. Central pathway is marked with a dotted mesh. b Pore radii along the central axis in six MmTRPM8 structures. Source data are provided as a Source Data file. c Structural comparison of the pore domain in MmTRPM8_{LMNG-ligand-free} and the closed MmTRPM4 (PDB: 6BCJ). For clarity, the front and rear subunits are omitted. The rectangular box indicates the activation gate. d Structural comparison of the pore domain in MmTRPM8_{LMNG-ligand-free} and the closed MmTRPM7 (PDB: 5ZX5). Structures of the activation gates in MmTRPM8_{LMNG-ligand-free} (e), closed MmTRPM4 (f), and closed MmTRPM7 (g). Dash lines show diagonal atom-to-atom distances of Cα atoms from constriction-lining residues (in Å).