Activation mechanism of the mouse cold-sensing TRPM8 channel by cooling agonist and PIP2

Abstract

The transient receptor potential melastatin 8 (TRPM8) channel is the primary molecular transducer responsible for the cool sensation elicited by menthol and cold in mammals. TRPM8 activation is controlled by cooling compounds together with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂). Our knowledge of cold sensation and the therapeutic potential of TRPM8 for neuroinflammatory diseases and pain will be enhanced by understanding the structural basis of cooling agonist- and PIP₂-dependent TRPM8 activation. We present cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP₂-dependent gating pathway. Our results uncover two discrete agonist sites, state-dependent rearrangements in the gate positions, and a disordered-to-ordered transition of the gate-forming S6-elucidating the molecular basis of chemically induced cool sensation in mammals.

Figure 1.. Functional characterization and structure determination of the mouse TRPM8_MM channel.

(A) Chemical structures of 1-diisopropylphosphorylnonane (C3) and allyl isothiocyanate (AITC). (B) (Left) TRPM8_MM current-voltage (I-V) plots obtained from 600-ms voltage ramps, following the sequential application of 300 μM C3 then 50 μM TRPM8 antagonist AMG2850. (Right) Mean normalized concentration-response relations for C3. Data are shown as mean ± SEM (n = 6). The curves are fit to the Hill equation with EC₅₀ = 10.3 ± 0.4 μM, and s (slope) = 1.59 ± 0.1. (C) Representative time course recording of TRPM8_MM currents elicited by 300 μM C3, 0.5 mM AITC, 300 μM C3/2 mM AITC, and 50 μM AMG2850, as indicated by the colored horizontal lines. The dotted line indicates the zero-current level. The voltage was initially held at −60 mV and ramped to +60 mV over 300 ms every 2 seconds; plotted here are the currents at −60 mV. (D) Conductance-voltage (G-V) relationships of TRPM8_MM in the absence of agonist (control, black; n = 5) and in the presence of 300 μM C3 only (blue; n = 7) and 300 μM C3/2 mM AITC (red; n = 7), respectively. Error bars indicate SEM. (E) Cryo-EM reconstructions of TRPM8_MM and TRPM8_FA channels in the C₀, C₁, C₂, and O states, as indicated. Thresholding, 0.25 (purple), 0.36 (silver-gray), 0.22 (orange), 0.2 (light orange), 0.24 (green), and 0.22 (blue). (F) Close-up view at the S6 gate of 3D reconstructions from (E), viewed from the intracellular side, at thresholding 0.24, 0.25, 0.18, and 0.22, respectively. (G) Close-up view of the EM densities for the S4-S5 junction, S6, and TRP domain in the O state from (E), at thresholding 0.24. (H) Close-up view of the EM densities for PIP₂ (red), C3 (yellow), AITC (teal) from the dashed box in the O state from (E), at thresholding 0.25. All thresholding values were obtained at the isosurface level.

Figure 2.. PIP₂ binding primes the TRPM8_MM channel.

(A to C) (Top) EM densities (gray mesh) for PIP₂ (red sticks) in PIP₂-TRPM8_MM, TRPM8_MM (A), and PIP₂-TRPM8_FA (B). CHS (yellow sticks) binds to apo-TRPM8_MM (C) at the interfacial cavity. Densities are contoured at 0.16 and 0.15 (A), 0.31 (B), and 0.22 to 0.24 (C) thresholding, respectively. Residue sidechains are shown in sticks. (Bottom) Surface representations showing the two PIP₂ binding modes [(A) and (B)] and CHS (C) in the interfacial cavity. PIP₂ is shown as red spheres. (D to E) Conformations of S4b and S5 in apo-TRPM8_MM (D) and PIP₂-TRPM8_MM (E). Arrows indicate key structural differences. PIP₂ (red) and residues are shown in sticks.

Figure 3.. Ligand binding to TRPM8_MM.

(A and C) EM densities for C3 [(A) yellow] and AITC [(C) teal] in the O state PIP₂-C₃-AITC-TRPM8_MM structure. Ligands and residue sidechains are shown as sticks. Densities are contoured at thresholding 0.11 for C3 [(A) yellow mesh], 0.200 for AITC [(C) teal mesh], and 0.27 for TRPM8_MM channel (gray mesh), respectively. (B and D) Representative TEVC recordings on wildtype and mutant TRPM8_MM channels at −60 mV (lower traces) and +60 mV (upper traces). Horizontal colored lines denote the application of 2 mM AITC (red), 300 μM C3 (blue), and 50 μM AMG2850 (AMG) (black). The dotted lines denote the zero current. Summary of the current magnitudes in response to 300 μM C3 relative to 2 mM AITC at +60 mV from experiments in the left panels [(B), rightmost panel] and the current magnitudes in response to 2 mM AITC relative to 1 mM menthol at +60 mV [(D) rightmost panel], respectively. Values for individual oocytes are shown as open circles along with mean ± S.E.M. (n = 3 to 10 oocytes). P-values are calculated by two-tailed Student’s t-test. (E) Comparison of conformational changes at S4b and S5 upon PIP₂, C3, and AITC binding reveals that AITC cannot bind to TRPM8 in the C₀ state (right). Ligands and residue sidechains are shown in sticks. (F) PIP₂, C3, and AITC bind surrounding the S4b helix (blue cylinder). TMD helices are shown in either cylinder (left) or surface (right). The neighboring pore domain (indicated by an apostrophe) is colored gray.

Figure 4.. Structural changes in the pore domain and S6 gate position during gating.

(A) Viewed from the membrane plane (top) and from the extracellular side (bottom), electrostatic potential of the pore-lining surface in the C₀, C₁, C₂, and O states, calculated by APBS (74) implemented in PyMOL. S6 helices are shown in cartoon and gating residues as sticks. The scale bar indicates electrostatics from −15 to +15 kT/e. (B) Pore radii calculated using the HOLE program (75) as colored in (A) (see methods). Regions spanning the selectivity filter, pore cavity, and S6 gate in distinct conformation states are denoted. The dotted line denotes a 2.0 Å-radius. (C) Ion permeation pathway in the C₀, C₁, C₂, and O states shown as gray surfaces. (D to G) Close-up views of the S6 gate from the membrane plane (top panels) and from the extracellular side (bottom panels) for the C₀ state [(D) purple], C₁ state [(E) orange], C₂ state [(F) green], and O state [(G) blue]. Diagonal distances in Å between opposing gating residues are labeled in the top panels. Gray meshes represent EM densities at the S6 gate region, contoured at 0.19 (D), 0.16 (E), 0.16 (F), and 0.16 (G) thresholding, respectively. Gating residues are shown in sticks and colored red for Met⁹⁷⁸, orange for Phe⁹⁷⁹ and Val⁹⁸³, and blue for Val⁹⁷⁶. The apo-TRPM8_MM (purple), PIP₂-TRPM8_MM (orange), PIP₂-C3-TRPM8_MM (green), and PIP₂-C3-AITC-TRPM8_MM (blue) structures are used for illustration and analysis.

Figure 5.. State-dependent interface in the O state of TRPM8_MM.

(A) Side-by-side comparison of the S6 rearrangement in the C₀, C₁, C₂, and O state structures viewed from the membrane plane. Gating residues are shown as sticks and colored as in Fig. 4. The ⨂ symbol next to the gate residue indicates the location of the pore. Teal colored spheres represent residues from every helical turn along S6. S1 to S5 and PH are colored transparent gray for clarity. Gray bars indicate the membrane bilayer position. The number of helical turns in S6 is denoted. (B) G-V relationships of gating residue mutants. The data were fit with Boltzmann functions, with extrapolation to +400 mV. Bar chart (rightmost panel) quantifying the effect of mutations on the V_1/2 of activation with respect to wildtype (ΔV_1/2) as mean ± SEM (n = 4 to 11 oocytes). V_1/2 of V983T is far right-shifted so its ΔV_1/2 is omitted in the bar chart. (C) Close-up extracellular view at the MDQK interface sliced from (A). Gray cartoon and (´) represent structural domains from the neighboring protomer. ⨂ indicates the location of the ion conduction pore. (D) G-V relationship for the double mutant cycle analysis. G-V curves for D866N, M978D, Q987E, Q987E/M978T were obtained with G_max values measured in the presence of menthol (fig. S14). Coupling energy ΔΔzFV_1/2 (mean ± S.E.M.) was calculated using Equations #2 to #7 in methods. (E) Bar chart quantifying the difference of V_1/2 between mutant and wildtype (ΔV_1/2) as mean ± S.E.M. (n = 4 to 11 oocytes).

Figure 6.. Structural basis of ligand-dependent gating of TRPM8_MM.

(A) Structural overlay of the C₁ (orange) and O (blue) states at S4b. C3 (yellow) and residue sidechains are shown in sticks. Arrows indicate downward movement and sidechain rotations. (B) Comparison of the C₁ (orange) and O (blue) state structures at the TMD, aligned at the VSLD. Arrows indicate movement in S5, PH, and S6 by the marked distances. Key residues for comparison are shown as spheres. Dashed lines (right panel) compare the change in the bending points (Ile⁸⁶⁵ and Phe⁸⁶⁸ as spheres) on S5. C3 and AITC are shown as sticks for orientation reference. S1 to S3 are removed for clarity. (C) Comparison of two neighboring protomers in the C₁ (orange and light orange) and O state (blue and light blue) at the TMDs. The channel is shown as cylinders and ligands as sticks. S1 to S3 are removed in chain A and only S5 and S6 are shown in chain B for clarity. Dashed regions are zoomed in for comparison in (D to F). Alignment was done using the entire tetrameric channel. (D to F) Close-up views showing side-by-side comparison of the C₁ (orange) and O state (blue) structures at inter- and intra-subunit interfaces marked in (C). Sidechains are shown in sticks [(D) and (F)] or spheres (E). Dashed lines indicate the minimum distances between the corresponding residues. (G) (Left) G-V relationship of mutants at interfaces as indicated. (Right) Bar chart quantifying the difference of V_1/2 between mutant and wildtype (ΔV_1/2) as mean ± S.E.M. (n = 4 to 6 oocytes).