Current View of Ligand and Lipid Recognition by the Menthol Receptor TRPM8

Abstract

Transient receptor potential (TRP) melastatin member 8 (TRPM8), which is a calcium-permeable ion channel, functions as the primary molecular sensor of cold and menthol in humans. Recent cryoelectron microscopy (cryo-EM) studies of TRPM8 have shown distinct structural features in its architecture and domain assembly compared with the capsaicin receptor TRP vanilloid member 1 (TRPV1). Moreover, ligand-bound TRPM8 structures have uncovered unforeseen binding sites for both cooling agonists and membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. These complex structures unveil the molecular basis of cooling agonist sensing by TRPM8 and the allosteric role of PI(4,5)P₂ in agonist binding for TRPM8 activation. Here, we review the recent advances in TRPM8 structural biology and investigate the molecular principles governing the distinguishing role of TRPM8 as the evolutionarily conserved menthol receptor.

Keywords: PI(4,5)P(2) regulation; allosteric coupling; cold receptor; cooling agent; menthol receptor; transient receptor potential ion channels.

Figure 1.. Unique structural features of TRPM8.

(A) Structure models showing the architecture of TRPM8 (left; PDB ID: 6BPQ) and TRPV1 (right; PDB ID: 3J5P) channels. The transmembrane domains (TMDs) are embedded in the plasma membrane (gray box). The cytoplasmic domain (CD) of TRPM8 can be further divided into the top (CD top) and the bottom (CD bottom) layers. (B and C) Atomic models of a single protomer from TRPM8 (B) and TRPV1 (C), respectively. Abbreviations: ARD, ankyrin repeat domain; CC, coiled coil; CTDH, C-terminal domain helix; MHR, melastatin homology region; PH, pore helix; VSLD, voltage-sensor-like domain. (D) Cartoon diagrams delineating the topology of TRPM8 (left) and TRPV1 (right). Subdomains labeled as in B and C. (E-G) Comparison of the transmembrane domain (TMD) in the apo TRPM8_FA (E,; PDB ID: 6BPQ), TRPM8_FA in complex with PI(4,5)P₂/icilin/Ca²⁺ (F; PDB ID: 6NR3), and the apo TRPV1 structures (G; PDB ID: 3J5P). The configurations of secondary structure in the C-terminus of S4 (S4b), S5, and S6 are indicated by arrows for comparison.

Figure 2.. The novel binding site for agonists and antagonists in TRPM8.

(A) The locations of ligand binding in TRPM8 (left) and TRPV1 (right) embedded in the plasma membrane (gray box), which are highlighted by dashed lines. The ligand binding site in TRPM8 is located in the voltage-sensor-like domain cavity (VSLD cavity) formed by the VSLD and the TRP domain. The double-knot toxin (DkTx, pink surface representation) binds atop TRPV1 channel. For each channel, one protomer is shown as in Figure 1B and C, while the rest are shown in white surface representation. (B) Close-up view of the binding site for resiniferatoxin (RTx) in TRPV1. Magenta spheres represent the RTx molecule. (C-E) Comparison of the binding of vanilloid agonist RTx (C; PDB ID: 5IRX), phosphatidylinositol lipid (D; PDB ID: 5IRZ), and antagonist capsazepine (E; PDB ID: 5IS0) in TRPV1 channel. Dashed lines indicate interactions mediated by R557 in S4 and E570 in S4-S5 linker. Ligands are shown as sticks. (F) Comparison of the VSLD cavity in the ligand-free TRPM8_FA (yellow; PDB ID: 6BPQ), TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ complex (blue; PDB ID: 6NR3), TRPM8_FA-PI(4,5)P₂/WS-12 complex (green; PBD ID: 6NR2), TRPM8_PM-AMTB complex (wheat; PDB ID: 6O6R), and TRPM8_PM-TCI-2014 complex (pink; PDB ID: 6O72). Key residues for ligand binding are shown in sticks. Spheres represent agonists, antagonists, and Ca²⁺ ion. In panels for ligand-bound structures, S2 is omitted for clarity. (G-H) Ca²⁺ binding in the VSLD cavity of TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ (G) and TRPM8_PM-Ca²⁺ (H; PDB ID: 6O77) complex structures. Green spheres represent Ca²⁺ ions. Dashed lines indicate the ion coordination.

Figure 3.. Molecular basis of ligand recognition by TRPM8.

(A) Comparison of the VSLD cavity in the TRPM8_FA-Apo (yellow), TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ complex (blue), TRPM8_FA-PI(4,5)P₂/WS-12 complex (green), TRPM8_PM-AMTB complex (wheat), and TRPM8_PM-TC-I 2014 complex (pink). Gray transparent surfaces represent the shape of the cavity mediated by residues lining the binding pocket. Arrowheads point the flexible arginine and histidine residues in S4 (R841 and H844 in TRPM8_FA; R832 and H835 in TRPM8_PM). Ligands are shown in spheres. PDB IDs indicated in Figure 2F legend. (B) Overlay of the VSLD cavity in the ligand bound TRPM8 structures, showing the flexibility of the key ligand-binding residues. Color coding is the same as in (A). Residue numbering refers to TRPM8_FA. (C-E) Comparison of the VSLD cavity in the TRPM8_FA-Apo (C, yellow) and the TRPM8_FAPI(4,5)P₂/icilin/Ca²⁺ complex (D, blue) structures, showing the conformational changes induced by icilin (purple spheres) and Ca²⁺ (green sphere) binding; especially S4b undergoes a α-to-3₁₀ helical transition. Superimposition of the two structures (E) suggests the Ca²⁺ coordination facilitates the icilin binding; the A805G mutation confers flexibility in S3 for the assembly of Ca²⁺ coordination and widens the VSLD cavity to accommodate the icilin molecule. (F) Comparison of the ligand-induced conformational changes in S4 and S5 between TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ (blue) and TRPM8_PM-Ca²⁺ structures (purple) with TRPM8_FA-Apo (yellow). Ligand binding induces bending of S5 in TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ (blue) and TRPM8_PM-Ca²⁺. The S4b in TRPM8_PM-Ca²⁺ remains α-helical, whereas that in TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ transitions to a 3₁₀-helix.

Figure 4.. Structural basis of the PI(4,5)₂ dependence of TRPM8.

(A) Comparison of the binding site for phosphatidylinositol lipid (PtdIns) (left; PDB ID: 5IRZ) in TRPV1 and that for PI(4,5)P₂ in TRPM8 (right; PDB ID: 6NR3). Lipid molecules are shown as spheres. (B) Residues interacting with PI(4,5)P₂ (yellow sticks) in the interfacial cavity in TRPM8. (C) Comparison of the PI(4,5)P₂ binding site in TRPM8_FA-Apo (yellow), TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ complex (blue), and TRPM8_FA-PI(4,5)P₂/WS-12 complex (green). Structural rearrangements of S4 and S5 in the TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ complex reposition R850 closer to the PI(4,5)P₂ binding site. PDB IDs indicated in Figure 2F legend. (D) Surface representations showing two different conformations of the interfacial cavity which enable partially (left, TRPM8_FA-PI(4,5)P₂/WS-12 complex) and fully (right, TRPM8_FA-PI(4,5)P₂/icilin/Ca²⁺ complex) engagement of PI(4,5)P₂ with the TRPM8 channel. PI(4,5)P₂ is shown in spheres. (E) Global (left columns) and close-up (right columns) views comparing the intra- and inter-subunit domain interfaces in TRPM8 (PDB ID: 6NR3), TRPM4 (PDB ID: 5WP6), and TRPM2 (PDB ID: 6PUS). Tight association between the pre-S1 domain and the neighboring MHR4 domains contributes to the PI(4,5)P₂ binding site in TRPM8. (F) Distinct but adjacent binding sites for PI(4,5)P₂ (yellow sticks) and icilin (purple sticks) (PDB ID: 6NR3) illustrate the allosteric coupling between the lipid and cooling agonists for TRPM8 activation.

Figure 5.. Regulatory functions of cytoplasmic domains in TRPM and TRPV channels.

(A) Residues in TRPM8 (red spheres) that have been implicated for direct interactions with Gα_q mapped to the MHR1/2 and MHR3 domains. (B) The ligand binding sites for ATP in TRPM4 (left; PDB ID: 6BCO) and ADP-ribose (ADPR) in TRPM2 (right; PDB ID: 6PUS) are located in the cytoplasmic domains of the channel. (C) In TRPV3, state-dependent secondary structure changes from loop (left; PDB ID: 6MHO) to helix (right; PDB ID: 6OT5) in the distal C-terminal domain (CTD) triggers rearrangements in the inter-subunit interfaces, which are propagated to the TRP domain and lead to channel activation. (D) Inter-subunit interface between the distal CTD and the neighboring ankyrin repeat domain (ARD) in the TRPV1-Apo structure (PDB ID: 3J5P). (E) Structural comparison of the less-well defined CTD and ARD in the TRPV1-Apo with those in the closed and the open states of TRPV3. TRPV3_WT-Apo: light orange, PDB 6MHO; TRPV3_K169A-2-APB: teal, PDB 6OT5; TRPV1-Apo: magenta, PDB 3J5P.

Figure 6.. The design principle of synergetic actions of PI(4,5)P₂ and cooling agonists on TRPM8 activation.

(A and B) Comparison of the distinct binding sites for lipid and agonists in TRPM8 (A) and TRPV1 (B) channels. The gray box represents the plasma membrane. Cytoplasmic domains of the channels are simplified. (C) Schematic diagram depicting a discrete binding site for menthol in the VSLD cavity and the strategic position of PI(4,5)P₂ binding for allosteric coupling with cooling agonist in TRPM8. In contrast, capsaicin and membrane lipid share the same binding pocket above the S4-S5 linker in TRPV1, where PtdIns is proposed to act as a competitive antagonist for vanilloid activation based on the structure. The locations representative of ligand binding in TRPM8 and TRPV1 are shown as green and pink ovals, respectively. Cytoplasmic domains are omitted for simplicity.