Molecular determinants of TRPM8 function: key clues for a cool modulation

Abstract

Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21 years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

Keywords: WS-12; cold; cryo-EM structures; icilin; ion channel; menthol.

FIGURE 1

Menthol and WS-12 binding site in the VSLD of FaTRPM8. (A). Representation of the TRPM8 system embedded in the lipid membrane and water. The four subunits are shown in surface representation. One monomer with secondary structure elements is shown for reference (S1 is highlighted in cyan, S2 in white, S3 in light green, S4 in dark green, S5 and S6 in orange, and the TRP domain in red). The dotted box indicates the ligand binding site of TRPM8 formed by the S1-S4 transmembrane segments (VSLD) and the TRP domain. (B). Close-up view of the WS-12 binding in the VSLD cavity (PDB ID: 6NR2). Key residues involved in menthol-/WS-12-dependent TRPM8 activation mentioned in the text and Table 2 are shown. Residue numbers correspond to FaTRPM8 (dark green) and MmTRPM8 (brown). For clarity, the S2 and S3 transmembrane segments were omitted. The side chain of residue L1008 was added using Pymol v2.5.4. (C). General description of the computational modeling of menthol binding by Xu et al., 2020, using a model based on the FaTRPM8 in complex with WS-12 (PDB ID: 6NR2). For clarity, S2 and S3 were omitted, and the side chain of residue L1008 was added using Pymol v2.5.4. (D). Alignment of S1-S6 and TRP domain of Parus major, Ficedula albicollis, Mus musculus, and Homo sapiens orthologs of TRPM8 using Jalview (Waterhouse et al., 2009). Numbers correspond to residues in MmTRPM8 channel.

FIGURE 2

Icilin and Ca²⁺ binding sites in the VSLD of MmTRPM8. (A). Icilin binding site of MmTRPM8 (PDB ID: 7WRE). Relevant residues to icilin-evoked TRPM8 responses are indicated (Table 3). The S1 transmembrane segment is highlighted in cyan, S3 in light green, S4 in dark green, and the TRP domain in red. For clarity the S2 was omitted. (B). Ca²⁺ ion binding site. Ca²⁺ (depicted as a pink sphere) is coordinated by side chains E782, Q785, N799, and D802 from S2 and S3. In addition, side chain Y793 within the S2-S3 linker is represented (PDB ID: 7WRE) (Visualization in Pymol v2.5.4).

FIGURE 3

TRPM8 PI(_4,5)P₂ interacting site. PI_(4,5)P₂ binding site of MmTRPM8 (PDB ID: 8E4N). Residues interacting with PI_(4,5)P₂ are shown. R688 at the pre-S1 (yellow), R850, located at the junction between S4 (dark green) and S5 (orange), R998 within the TRP domain (red), and R605 from the MHR4 of the adjacent subunit (green) (Visualization in Pymol v2.5.4).

FIGURE 4

Pore domain of MmTRPM8. (A). Representation of MmTRPM8’s ion conduction pathway with the front and rear subunits removed for clarity. Residues of mutants exhibiting major alterations in the responses of TRPM8 by cold or chemical agonists are shown as sticks (PDB ID: 7WRE). (B). Close-up of the side chains of residues Q914, G913, and F912 in MmTRPM8 in the open state, which form the selectivity filter (PDB ID: 8E4L). (C). Close-up view of the lower gate. Gate residues V976, M978, F979, and V983 are shown as sticks (PDB ID: 8E4L) (Visualization in Pymol v2.5.4).