{"gene":"TRPM8","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":2010,"finding":"TRPM8 is directly gated by cold, menthol, and icilin in planar lipid bilayers, excluding cellular signaling pathways; PI(4,5)P2 is the prime factor impacting TRPM8 gating through direct specific interactions with the channel protein; menthol increases PI(4,5)P2 binding potency and channel activity; icilin activation depends on intracellular calcium; cold-activated TRPM8 exhibits steep temperature dependence (Q10 ~40) with large entropy and enthalpy changes.","method":"Reconstitution in planar lipid bilayers, patch-clamp, phosphoinositide binding assays, in vitro mutagenesis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in lipid bilayers with multiple orthogonal methods, definitively excludes cellular signaling involvement","pmids":["20844147"],"is_preprint":false},{"year":2009,"finding":"TRPM8 channel protein forms a stable complex with inorganic polyphosphate (polyP) and poly-(R)-3-hydroxybutyrate (PHB); enzymatic breakdown of polyP by exopolyphosphatase inhibits TRPM8 channel activity both in cells and in purified channel reconstituted in planar lipid bilayers.","method":"Whole-cell patch-clamp, fluorescent calcium measurements, planar lipid bilayer reconstitution, biochemical co-purification","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in lipid bilayers plus biochemical demonstration of complex, multiple methods","pmids":["19404398"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of TRPM8 in ligand-free, antagonist-bound, and calcium-bound forms reveal a malleable ligand-binding pocket, two nonconducting states (closed and desensitized), direct calcium binding mediating stimulus-evoked desensitization, and large S4-S5 linker rearrangements repositioning S1-S4 and pore domains relative to the TRP helix.","method":"Cryo-electron microscopy structure determination","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structures at multiple states with functional validation, published in high-impact journal","pmids":["31488702"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of mouse TRPM8 in closed, intermediate, and open states reveal two discrete agonist binding sites, state-dependent gate rearrangements, and a disordered-to-ordered transition of the gate-forming S6 helix along the PIP2- and cooling agonist-dependent gating pathway.","method":"Cryo-electron microscopy, electrophysiology","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structures of multiple functional states with electrophysiological validation","pmids":["36227998"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of mouse TRPM8 in ligand-free and Ca2+/icilin-bound forms at 2.5-3.2 Å resolution reveal a short but wide selectivity filter, canonical S4-S5 linker, and show that Ca2+ and icilin bind in the cytosolic-facing cavity of the voltage-sensing-like domain but induce little conformational change; all ligand-bound structures adopt the closed conformation.","method":"Cryo-electron microscopy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structures at 2.5-3.2 Å","pmids":["35662242"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of human TRPM8 in closed state at 2.7 Å resolution reveals the most complete N-terminal pre-melastatin homology region model, lipid binding sites, and icilin binding mode; pore helix S6 register distinguishes closed, desensitized, and open states across TRPM structures.","method":"Cryo-electron microscopy, molecular modeling","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structure of human TRPM8 with structural analysis","pmids":["37857704"],"is_preprint":false},{"year":2024,"finding":"TRPM8 inhibitors bind selectively to the desensitized state of the channel; cold and cooling agonists share a common desensitization pathway; structural determinants for conformational change in TRPM8 desensitization were identified; overlapping mechanisms underlie desensitization and inhibition.","method":"Cryo-electron microscopy, electrophysiology, molecular dynamics simulations","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structures combined with electrophysiology and MD simulations","pmids":["39093967"],"is_preprint":false},{"year":2007,"finding":"Lysophospholipids (LPCs, LPI, LPS) raise the temperature threshold of TRPM8 activation toward normal body temperature, while polyunsaturated fatty acids (e.g., arachidonic acid) inhibit TRPM8 activation by cold, icilin, and menthol; iPLA2 inhibition abolishes cold and icilin responses but not menthol responses of TRPM8.","method":"Patch-clamp, calcium imaging in CHO cells and DRG neurons expressing TRPM8","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple pharmacological and biochemical approaches in heterologous and native cells","pmids":["17376995"],"is_preprint":false},{"year":2006,"finding":"TRPM8 contains two essential cysteine residues flanking the N-linked glycosylation site at Asn-934 in the pore region; mutation of either cysteine abolishes channel function and produces a non-functional homodimer; Asn-934 is the glycosylated residue (complex carbohydrate); TRPM8 forms tetramers consistent with functional TRP channel architecture.","method":"Site-directed mutagenesis, SDS-PAGE, PFO-PAGE, calcium imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with biochemical and functional validation, multiple orthogonal methods","pmids":["17015441"],"is_preprint":false},{"year":2017,"finding":"TRPM8 acts as a Rap1 GTPase inhibitor through a non-channel, pore-independent function; TRPM8 retains Rap1 intracellularly via direct protein-protein interaction, preventing Rap1 cytoplasm-to-plasma membrane trafficking, thereby impairing integrin conformational activation and suppressing endothelial cell migration, tube formation, and spheroid sprouting.","method":"Co-immunoprecipitation, live imaging, dominant-negative/overexpression, migration assays, tube formation assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — direct protein-protein interaction with multiple functional readouts, pore-independence demonstrated","pmids":["28550110"],"is_preprint":false},{"year":2019,"finding":"Activated androgen receptor (AR) interacts with TRPM8 within lipid raft microdomains of the plasma membrane; this AR-TRPM8 interaction inhibits TRPM8 channel activity and promotes prostate cancer cell migration.","method":"Co-immunoprecipitation, lipid raft fractionation, patch-clamp, migration assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP with functional consequence, single lab","pmids":["31501416"],"is_preprint":false},{"year":2020,"finding":"Testosterone (TST) inhibits TRPM8-mediated cold perception through noncanonical engagement of androgen receptor (AR); AR is present on the cell surface and interacts with TRPM8 in response to TST; TST in nanomolar concentrations suppresses TRPM8 currents and single-channel activity in DRG neurons and HEK cells co-expressing TRPM8 and AR, but not TRPM8 alone.","method":"Patch-clamp, calcium imaging, biochemical assays, confocal imaging, behavioral testing in castrated rodents","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including electrophysiology, imaging, and in vivo behavioral assays across two species","pmids":["32277850"],"is_preprint":false},{"year":2019,"finding":"Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P2, sensitizing the channel to inhibition by PI(4,5)P2 depletion upon GPCR activation; constitutively active Gαq inhibits TRPM8 activity independent of PLC; supplementing PI(4,5)P2 via patch pipette reduces Gαq-coupled receptor-mediated TRPM8 inhibition in DRG neurons.","method":"Whole-cell patch-clamp, voltage-sensitive 5'-phosphatase, PI(4,5)P2 supplementation, calcium imaging in DRG neurons","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic and pharmacological tools in both native DRG neurons and heterologous cells, strong mechanistic evidence","pmids":["31127000"],"is_preprint":false},{"year":2013,"finding":"Chloroquine inhibits TRPM8 in 48.8% of TRPM8-positive DRG neurons through direct action of activated Gαq independent of the phospholipase C pathway.","method":"Calcium imaging, pharmacological dissection in DRG neurons","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — functional dissection with selective inhibitors in native neurons, single lab","pmids":["23508958"],"is_preprint":false},{"year":2020,"finding":"Temperature-dependent gating of TRPM8 is driven by a folding-unfolding reaction of the distal C-terminal domain (CTD); progressive deletion of the CTD reduces enthalpy change proportionally; deletion of the last 36 amino acids transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ~4); channel gating involves ~1,900 Å3 change in solute-inaccessible volume matching the coiled-coil void space in cryo-EM structure.","method":"Patch-clamp with progressive CTD deletions, denaturing agent experiments, osmoticant assays, structural analysis","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with biophysical measurements and structural validation, multiple orthogonal methods","pmids":["32747539"],"is_preprint":false},{"year":2019,"finding":"TRPM8 is constitutively tyrosine phosphorylated by Src kinase; Src potentiates TRPM8 activity; Src inhibition with PP2 reduces TRPM8 tyrosine phosphorylation and cold-induced channel activation in HEK293T cells and native DRG neurons.","method":"Co-immunoprecipitation, western blot, calcium imaging, patch-clamp, RNA interference","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — phosphorylation confirmed biochemically with functional validation in heterologous and native cells","pmids":["31729029"],"is_preprint":false},{"year":2021,"finding":"Constitutive phosphorylation of serine residues (S26, S29, S541, S542) in the N-terminus of mouse TRPM8 negatively regulates channel activity; S29A mutation is sufficient to increase TRPM8 cold- and menthol-evoked responses by shifting the voltage activation curve to more negative potentials and increasing the number of active channels at the plasma membrane.","method":"Mass spectrometry, site-directed mutagenesis, calcium imaging, patch-clamp, TIRF microscopy","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 — mass spectrometry identification + mutagenesis + biophysical characterization + TIRF imaging, multiple orthogonal methods","pmids":["34446569"],"is_preprint":false},{"year":2020,"finding":"Chronic morphine activates MOR-PKCβ signaling to phosphorylate TRPM8 at two consensus sites (S1040 and S1041), reducing TRPM8 desensitization and sensitizing TRPM8 responsiveness to cold and menthol; site-directed mutation of S1040/S1041 prevents MOR-induced reduction in TRPM8 desensitization.","method":"Patch-clamp, calcium imaging, site-directed mutagenesis, pharmacological inhibition in DRG neurons and HEK cells","journal":"Molecular brain","confidence":"High","confidence_rationale":"Tier 1 — site-directed mutagenesis of phosphorylation sites combined with electrophysiology in native and heterologous systems","pmids":["32290846"],"is_preprint":false},{"year":2022,"finding":"LCK (lymphocyte-specific protein tyrosine kinase) directly interacts with TRPM8 and phosphorylates it at Y1022, enhancing TRPM8 multimerization and channel current density; 14-3-3ζ also interacts with TRPM8 and promotes multimerization; LCK enhances 14-3-3ζ-TRPM8 binding; Y1022F mutation impairs TRPM8 multimerization and 14-3-3ζ binding; TRPM8 phospho-Y1022 feeds back to inhibit LCK Tyr505 phosphorylation.","method":"Co-immunoprecipitation, patch-clamp, mutagenesis, western blot, functional cancer cell assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — direct interaction confirmed by co-IP with mutagenesis, single lab","pmids":["35665750"],"is_preprint":false},{"year":2017,"finding":"TRPM8 interacts with NF-κB and suppresses NF-κB nuclear localization under cold stress, thereby inhibiting TNFα gene transcription in the mouse hypothalamus.","method":"Co-immunoprecipitation, nuclear fractionation, qRT-PCR, western blot","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — direct protein interaction demonstrated with mechanistic downstream readout, single lab","pmids":["28332601"],"is_preprint":false},{"year":2018,"finding":"Tacrolimus (FK506) directly gates TRPM8 channels, sensitizing their response to cold by inducing a leftward shift in the voltage-dependent activation curve; this direct gating is demonstrated in purified TRPM8 reconstituted in lipid bilayers; tacrolimus acts independently of calcineurin/PLC signaling and binds at a different site from menthol (TRPM8-Y745H) or icilin (TRPM8-N799A).","method":"Lipid bilayer reconstitution, patch-clamp, calcium imaging, mutagenesis in DRG neurons and heterologous cells","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 — direct gating demonstrated in purified reconstituted channel with site-distinguishing mutagenesis and in vivo confirmation","pmids":["30545944"],"is_preprint":false},{"year":2021,"finding":"TRPM8 gain-of-function mutation p.Arg30Gln (c.89 G>A) enhances channel activation, increases basal current amplitude and intracellular Ca2+, and augments menthol response, contributing to familial trigeminal neuralgia pathogenesis.","method":"Calcium imaging, whole-cell patch-clamp recording of mutant vs. wild-type TRPM8","journal":"Neurology. Genetics","confidence":"Medium","confidence_rationale":"Tier 2 — functional characterization of disease-associated mutation with electrophysiology, single lab","pmids":["33977138"],"is_preprint":false},{"year":2017,"finding":"TRPM8 is required for glioblastoma cell migration, DNA repair, and radioresistance; ionizing radiation activates and upregulates TRPM8-mediated Ca2+ signaling, which interferes with cell cycle control via CaMKII, cdc25C, and cdc2.","method":"RNA interference, patch-clamp, calcium imaging, colony formation, flow cytometry, immunoblot","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional readouts with pathway identification, single lab","pmids":["29221175"],"is_preprint":false},{"year":2017,"finding":"TRPM8 channel activation by menthol in adipocytes induces UCP1 expression and WAT browning through Ca2+ influx and PKA activation; the menthol-induced thermogenic gene expression is blocked by PKA inhibitor KT5720 or calcium chelator BAPTA-AM.","method":"Pharmacological inhibition, mRNA/protein expression analysis, mouse dietary model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological dissection of signaling pathway in cultured cells and in vivo, single lab","pmids":["29088850"],"is_preprint":false},{"year":2017,"finding":"GFRα3 activation sensitizes and upregulates TRPM8 expression and plasma membrane trafficking in DRG neurons; GFRα3 knockdown reduces TRPM8 membrane trafficking and attenuates cold hyperalgesia in CCI rats; TRPM8 inhibition blocks GFRα3 agonist-induced cold hyperalgesia.","method":"siRNA knockdown, western blot, immunofluorescence, behavioral testing, TRPM8 antagonist","journal":"Brain research bulletin","confidence":"Medium","confidence_rationale":"Tier 2 — genetic knockdown with multiple readouts in native tissue, single lab","pmids":["28867384"],"is_preprint":false},{"year":2024,"finding":"TRPM8 secreted RNA in extracellular vesicles (EVs) from prostate cells activates TLR3/NF-κB-mediated sterile inflammatory signaling after EV endocytosis by epithelial cancer cells; translation-defective TRPM8 RNA in xenografts reduces collagen I, increases NK cell infiltration, and expands necrotic areas.","method":"Extracellular vesicle isolation, TLR3/NF-κB reporter assays, xenograft models, immunohistology","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple in vitro and in vivo methods demonstrating non-channel RNA function, single lab","pmids":["38316991"],"is_preprint":false},{"year":2022,"finding":"MHR1-3 domain of TRPM8 confers independent cold sensitivity and is required for pore domain regulation of cold activation; the pore domain underwent positive selection in terrestrial tetrapods; the mature MHR1-3 domain is necessary for pore domain regulatory mechanism in TRPM8 cold activation.","method":"Domain deletion/chimera analysis, electrophysiology, evolutionary analysis","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — domain swapping with functional validation, single study","pmids":["35594403"],"is_preprint":false},{"year":2023,"finding":"TCAF2 (TRP channel-associated factor 2) inhibits TRPM8 expression and activity in tumor pericytes, leading to Wnt5a secretion that activates STAT3 signaling in tumor cells to facilitate EMT and colorectal cancer liver metastasis; TRPM8 agonist menthol suppresses Wnt5a secretion in pericytes.","method":"Gain/loss-of-function, pericyte conditional Tcaf2-KO mice, co-culture assays, proteomic analysis, signaling pathway analysis","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — conditional knockout mice with mechanistic pathway definition, single lab","pmids":["37635201"],"is_preprint":false},{"year":2021,"finding":"TRPM8 channel activation by menthol/icilin in esophageal cancer cells promotes PD-L1 expression via the calcineurin-NFATc3 pathway, leading to immune evasion by reducing CD8+ T cell cytotoxicity.","method":"Overexpression/siRNA knockdown, co-incubation assay with CD8+ T cells, reporter assays, western blot","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 2 — pathway identified with multiple molecular tools, single lab","pmids":["31519770"],"is_preprint":false},{"year":2008,"finding":"TRPM8-expressing DRG neurons constitute a specific functional subpopulation where virtually all EGFPf-positive neurons respond to cold and menthol; these neurons project to superficial layer I of the spinal cord with distinct termination patterns compared to peptidergic fibers, and form unique bush/cluster endings in the superficial epidermis.","method":"Knock-in reporter mouse (TRPM8-EGFPf), calcium imaging, anatomical tracing, immunohistochemistry","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — genetic reporter with functional validation and anatomical tracing, strong evidence for anatomical and functional identity","pmids":["18199758"],"is_preprint":false},{"year":2021,"finding":"Chronic morphine treatment sensitizes TRPM8 to cold and menthol and reduces activation-evoked desensitization via PKCβ phosphorylation of S1040 and S1041; blocking PLC or PKCβ (but not PKA or ROCK) restores desensitization; TRPM8-expressing DRG neurons show hyperexcitability after sustained morphine treatment.","method":"Site-directed mutagenesis (S1040A/S1041A), patch-clamp, pharmacological inhibition, in vivo morphine treatment","journal":"Molecular brain","confidence":"High","confidence_rationale":"Tier 1 — site-directed mutagenesis of phosphorylation sites with electrophysiology confirming mechanism","pmids":["32290846"],"is_preprint":false}],"current_model":"TRPM8 is a homotetrameric, Ca2+-permeable non-selective cation channel that is directly gated by cold temperature, menthol, icilin, and voltage, with PI(4,5)P2 as an obligatory co-activator binding directly to the channel; cryo-EM structures reveal a malleable ligand-binding pocket, two distinct agonist sites, and separate closed and desensitized states driven by Ca2+ binding and large S4-S5 linker rearrangements; temperature sensitivity is mediated by folding-unfolding of the C-terminal coiled-coil domain; channel activity is positively modulated by Src-mediated tyrosine phosphorylation (Y1022 by LCK), tacrolimus (direct gating), polyphosphate association, and lysophospholipids, and negatively regulated by constitutive serine phosphorylation (especially S29), Gαq (which reduces PI(4,5)P2 apparent affinity), androgen receptor interaction, PUFA, and PKCβ-mediated phosphorylation (S1040/S1041) downstream of opioid receptor activation; beyond its channel function, TRPM8 acts as a Rap1 GTPase inhibitor through direct protein-protein interaction to suppress endothelial migration, and its RNA can be secreted in extracellular vesicles to activate TLR3/NF-κB sterile inflammatory signaling."},"narrative":{"teleology":[{"year":2006,"claim":"Establishing that TRPM8 assembles as a tetramer with essential pore-region cysteines flanking the N-glycosylation site at Asn-934 resolved the basic quaternary architecture of the functional channel.","evidence":"Site-directed mutagenesis with SDS-PAGE, PFO-PAGE, and calcium imaging in heterologous cells","pmids":["17015441"],"confidence":"High","gaps":["No high-resolution structure yet available","Stoichiometry of heteromeric assemblies unexplored"]},{"year":2007,"claim":"Demonstrating that lysophospholipids raise the TRPM8 temperature activation threshold toward body temperature while PUFAs inhibit channel activity established a lipid-metabolite regulatory axis for tuning cold sensitivity.","evidence":"Patch-clamp and calcium imaging in CHO cells and DRG neurons expressing TRPM8","pmids":["17376995"],"confidence":"High","gaps":["Binding sites for lysophospholipids and PUFAs on TRPM8 not mapped","In vivo physiological relevance of iPLA2-dependent regulation not tested genetically"]},{"year":2008,"claim":"Genetic reporter tracing revealed that TRPM8-expressing DRG neurons constitute a dedicated cold-sensing population with unique peripheral bush/cluster endings and superficial dorsal horn projections, defining the anatomical substrate of TRPM8-mediated thermosensation.","evidence":"TRPM8-EGFPf knock-in mouse with calcium imaging, anatomical tracing, and immunohistochemistry","pmids":["18199758"],"confidence":"High","gaps":["Whether all cold sensation requires TRPM8 or parallel pathways exist not resolved by this study","Central circuit integration of TRPM8 input not addressed"]},{"year":2009,"claim":"The discovery that TRPM8 forms a stable complex with inorganic polyphosphate and that polyphosphate degradation inhibits channel activity identified an unexpected inorganic co-factor requirement.","evidence":"Planar lipid bilayer reconstitution, biochemical co-purification, whole-cell patch-clamp","pmids":["19404398"],"confidence":"High","gaps":["Polyphosphate binding site on TRPM8 not identified","Physiological polyphosphate levels in sensory neurons not established"]},{"year":2010,"claim":"Reconstitution in planar lipid bilayers definitively established that cold, menthol, and icilin gate TRPM8 directly without requiring cellular signaling intermediates, and that PI(4,5)P₂ is an obligatory co-activator binding the channel protein with agonist-enhanced potency.","evidence":"Purified TRPM8 in planar lipid bilayers, patch-clamp, phosphoinositide binding assays, mutagenesis","pmids":["20844147"],"confidence":"High","gaps":["PI(4,5)P₂ binding site on TRPM8 not structurally resolved","Mechanism coupling cold temperature to channel opening still unknown"]},{"year":2017,"claim":"Multiple studies revealed that TRPM8 functions beyond ion conduction: it acts as a Rap1 GTPase inhibitor through direct pore-independent protein–protein interaction, suppressing endothelial migration, and interacts with NF-κB to suppress its nuclear translocation under cold stress.","evidence":"Co-immunoprecipitation, dominant-negative/overexpression, migration/tube formation assays (Rap1); nuclear fractionation, qRT-PCR (NF-κB)","pmids":["28550110","28332601"],"confidence":"High","gaps":["Structural basis of TRPM8–Rap1 interaction unknown","Relevance of NF-κB interaction beyond hypothalamic cold stress not tested","Whether Rap1 inhibition and NF-κB interaction are coordinated is unexplored"]},{"year":2018,"claim":"Tacrolimus was shown to directly gate TRPM8 independently of calcineurin/PLC at a binding site distinct from menthol and icilin, explaining clinical cold hypersensitivity in transplant patients.","evidence":"Purified TRPM8 in lipid bilayers, patch-clamp, mutagenesis distinguishing Y745H (menthol) and N799A (icilin) sites","pmids":["30545944"],"confidence":"High","gaps":["Structural identity of the tacrolimus binding pocket not resolved","Whether tacrolimus gating shares the PIP₂-dependent pathway is unclear"]},{"year":2019,"claim":"Cryo-EM structures of TRPM8 in ligand-free, antagonist-bound, and Ca²⁺-bound states resolved the architecture of the malleable ligand-binding pocket, revealed two non-conducting states (closed and desensitized), and showed that Ca²⁺ binding drives desensitization through large S4–S5 linker rearrangements.","evidence":"Cryo-electron microscopy at multiple functional states","pmids":["31488702"],"confidence":"High","gaps":["Open-state structure not captured","PI(4,5)P₂ binding mode not resolved in these structures"]},{"year":2019,"claim":"The mechanism of Gαq-mediated TRPM8 inhibition was established: Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P₂ independently of PLC activity, sensitizing the channel to inhibition upon receptor-mediated PI(4,5)P₂ depletion.","evidence":"Whole-cell patch-clamp with voltage-sensitive phosphatase, PI(4,5)P₂ supplementation, calcium imaging in DRG neurons","pmids":["31127000","23508958"],"confidence":"High","gaps":["Gαq binding site on TRPM8 not mapped","Whether other Gα subunits modulate PI(4,5)P₂ affinity is untested"]},{"year":2019,"claim":"Src-family kinase-mediated constitutive tyrosine phosphorylation was shown to positively regulate TRPM8, as Src inhibition reduced both phosphorylation and cold-induced channel activation.","evidence":"Co-immunoprecipitation, western blot, calcium imaging, patch-clamp, RNAi in HEK293T cells and DRG neurons","pmids":["31729029"],"confidence":"Medium","gaps":["Specific tyrosine residue(s) phosphorylated by Src not identified in this study","Relationship to LCK-mediated Y1022 phosphorylation not clarified"]},{"year":2020,"claim":"The molecular basis of TRPM8 temperature sensitivity was resolved: a folding–unfolding equilibrium of the C-terminal coiled-coil domain generates the large enthalpy change underlying steep temperature dependence (Q10 ~40), with progressive CTD deletion proportionally reducing enthalpy and the last 36 residues being essential.","evidence":"Patch-clamp with progressive CTD deletions, denaturing agents, osmoticant assays, structural analysis","pmids":["32747539"],"confidence":"High","gaps":["Atomic-level description of the folding transition at different temperatures not available","How CTD folding couples to pore opening is structurally unresolved"]},{"year":2020,"claim":"Chronic morphine was found to sensitize TRPM8 by PKCβ-mediated phosphorylation at S1040 and S1041, reducing activation-evoked desensitization; this mechanism links opioid receptor signaling to cold hypersensitivity.","evidence":"Site-directed mutagenesis (S1040A/S1041A), patch-clamp, pharmacological inhibition in DRG neurons and HEK cells, in vivo morphine treatment","pmids":["32290846"],"confidence":"High","gaps":["Whether S1040/S1041 phosphorylation alters TRPM8 structure is unknown","Clinical relevance in opioid-induced cold allodynia not validated in patients"]},{"year":2021,"claim":"Mass spectrometry-guided mutagenesis identified constitutive N-terminal serine phosphorylation (especially S29) as a tonic brake on TRPM8 activity: S29A shifts voltage-dependent activation to more negative potentials and increases active channel density at the plasma membrane.","evidence":"Mass spectrometry, site-directed mutagenesis, calcium imaging, patch-clamp, TIRF microscopy","pmids":["34446569"],"confidence":"High","gaps":["Kinase(s) responsible for constitutive S29 phosphorylation not identified","Whether S29 phosphorylation is dynamically regulated in vivo is unknown"]},{"year":2021,"claim":"A gain-of-function TRPM8 variant p.Arg30Gln was identified in a family with trigeminal neuralgia; the mutation enhances basal current, intracellular Ca²⁺, and menthol responsiveness, establishing TRPM8 as a disease gene for inherited facial pain.","evidence":"Calcium imaging and whole-cell patch-clamp of mutant vs. wild-type TRPM8","pmids":["33977138"],"confidence":"Medium","gaps":["Single family studied; prevalence among trigeminal neuralgia patients unknown","Structural consequence of R30Q substitution not modeled"]},{"year":2022,"claim":"Multiple cryo-EM studies captured closed, intermediate, and open states of TRPM8, revealing two discrete agonist binding sites, a disordered-to-ordered S6 gate transition along the PIP₂/agonist gating pathway, and showing that Ca²⁺/icilin occupy the cytosol-facing VSLD cavity with minimal conformational change.","evidence":"Cryo-EM at 2.5–3.2 Å resolution with electrophysiological validation","pmids":["36227998","35662242"],"confidence":"High","gaps":["Full open-state structure with PIP₂ simultaneously bound not obtained","Structural basis for temperature-induced gating transition not captured"]},{"year":2022,"claim":"LCK was identified as a direct TRPM8 kinase phosphorylating Y1022, which enhances channel multimerization and recruits 14-3-3ζ; phospho-Y1022 feeds back to inhibit LCK Tyr505 phosphorylation, establishing a reciprocal regulatory loop.","evidence":"Co-immunoprecipitation, patch-clamp, site-directed mutagenesis (Y1022F), western blot","pmids":["35665750"],"confidence":"Medium","gaps":["In vivo significance of LCK–TRPM8 loop not demonstrated","Whether 14-3-3ζ binding alters TRPM8 trafficking or gating kinetics is unclear"]},{"year":2024,"claim":"Cryo-EM and functional studies demonstrated that TRPM8 inhibitors bind selectively to the desensitized state and that cold- and agonist-induced desensitization share overlapping structural mechanisms, unifying inhibition and desensitization pathways.","evidence":"Cryo-EM, electrophysiology, molecular dynamics simulations","pmids":["39093967"],"confidence":"High","gaps":["Whether state-selective inhibitor design can achieve therapeutic selectivity in vivo is untested","Kinetics of closed-to-desensitized transitions under physiological conditions not measured"]},{"year":2024,"claim":"TRPM8 RNA packaged in extracellular vesicles was found to activate TLR3/NF-κB sterile inflammatory signaling in recipient epithelial cancer cells, revealing a non-channel, RNA-level function of the TRPM8 gene product.","evidence":"Extracellular vesicle isolation, TLR3/NF-κB reporter assays, xenograft models with translation-defective TRPM8 RNA","pmids":["38316991"],"confidence":"Medium","gaps":["Whether TRPM8 RNA is selectively sorted into EVs is not established","Physiological relevance outside tumor xenograft context unknown","Whether other TRP channel mRNAs share this TLR3-activating property is untested"]},{"year":null,"claim":"Key open questions include the structural basis of temperature-induced conformational change at atomic resolution, the identity of kinases maintaining constitutive S29 phosphorylation, the structural determinants of the Rap1-binding interface, and whether the non-channel functions (Rap1 inhibition, NF-κB interaction, EV-RNA signaling) operate in physiological cold sensing or are context-restricted.","evidence":"","pmids":[],"confidence":"High","gaps":["No cryo-EM structure capturing the temperature-induced gating transition","Kinase responsible for constitutive S29 phosphorylation unidentified","TRPM8–Rap1 binding interface not structurally mapped","Physiological integration of channel and non-channel functions not addressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,2,3,4,5,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,11,16,29]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,12,23]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[29]},{"term_id":"R-HSA-9709957","term_label":"Sensory Perception","supporting_discovery_ids":[0,14,29]}],"complexes":[],"partners":["RAP1A","LCK","YWHAZ","AR","GNAQ","SRC","TCAF2"],"other_free_text":[]},"mechanistic_narrative":"TRPM8 is a cold- and menthol-activated, Ca²⁺-permeable non-selective cation channel that serves as the principal molecular transducer of innocuous cold sensation in somatosensory neurons. The channel assembles as a homotetramer requiring PI(4,5)P₂ as an obligatory co-activator; menthol increases PI(4,5)P₂ binding potency, while Gαq reduces its apparent affinity, sensitizing the channel to inhibition upon GPCR activation [PMID:20844147, PMID:31127000]. Cryo-EM structures reveal a malleable ligand-binding pocket with two discrete agonist sites, Ca²⁺-dependent desensitization involving large S4–S5 linker rearrangements, and a disordered-to-ordered transition of the S6 gate helix during activation, while temperature sensitivity is conferred by a folding–unfolding equilibrium of the C-terminal coiled-coil domain [PMID:31488702, PMID:36227998, PMID:32747539]. Beyond its channel function, TRPM8 acts as a Rap1 GTPase inhibitor through direct protein–protein interaction to suppress endothelial cell migration independently of ion conduction, and a gain-of-function p.Arg30Gln mutation causes familial trigeminal neuralgia [PMID:28550110, PMID:33977138]."},"prefetch_data":{"uniprot":{"accession":"Q7Z2W7","full_name":"Transient receptor potential cation channel subfamily M member 8","aliases":["Long transient receptor potential channel 6","LTrpC-6","LTrpC6","Transient receptor potential p8","Trp-p8"],"length_aa":1104,"mass_kda":127.7,"function":"Non-selective ion channel permeable to monovalent and divalent cations, including Na(+), K(+), and Ca(2+), with higher permeability for Ca(2+). Activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. Activated by cool temperatures (<23-28 degrees Celsius) and by chemical ligands evoking a sensation of coolness, such as menthol and icilin therefore plays a central role in the detection of environmental cold temperatures (PubMed:15306801, PubMed:15852009, PubMed:16174775, PubMed:25559186, PubMed:37857704). TRPM8 is a voltage-dependent channel; its activation by cold or chemical ligands shifts its voltage thresholds towards physiological membrane potentials, leading to the opening of the channel (PubMed:15306801). In addition to its critical role in temperature sensing, regulates basal tear secretion by sensing evaporation-induced cooling and changes in osmolality (By similarity). May plays a role in prostate cancer cell migration (PubMed:16174775, PubMed:25559186) Negatively regulates menthol- and cold-induced channel activity by stabilizing the closed state of the channel Negatively regulates menthol- and cold-induced channel activity by stabilizing the closed state of the channel","subcellular_location":"Cell membrane; Membrane raft; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q7Z2W7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRPM8","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRPM8","total_profiled":1310},"omim":[{"mim_id":"616252","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 2; TCAF2","url":"https://www.omim.org/entry/616252"},{"mim_id":"616251","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 1; TCAF1","url":"https://www.omim.org/entry/616251"},{"mim_id":"607066","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY V, MEMBER 3; TRPV3","url":"https://www.omim.org/entry/607066"},{"mim_id":"606678","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY M, MEMBER 8; TRPM8","url":"https://www.omim.org/entry/606678"},{"mim_id":"604775","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY A, MEMBER 1; TRPA1","url":"https://www.omim.org/entry/604775"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"liver","ntpm":29.4},{"tissue":"prostate","ntpm":75.4}],"url":"https://www.proteinatlas.org/search/TRPM8"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q7Z2W7","domains":[{"cath_id":"3.40.50.450","chopping":"54-347_388-415","consensus_level":"medium","plddt":82.4516,"start":54,"end":415},{"cath_id":"-","chopping":"471-535_554-678","consensus_level":"medium","plddt":84.8221,"start":471,"end":678},{"cath_id":"-","chopping":"862-1003","consensus_level":"high","plddt":83.2884,"start":862,"end":1003},{"cath_id":"1.20.120","chopping":"735-848","consensus_level":"medium","plddt":87.5684,"start":735,"end":848}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z2W7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z2W7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z2W7-F1-predicted_aligned_error_v6.png","plddt_mean":80.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRPM8","jax_strain_url":"https://www.jax.org/strain/search?query=TRPM8"},"sequence":{"accession":"Q7Z2W7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7Z2W7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7Z2W7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z2W7"}},"corpus_meta":[{"pmid":"18199758","id":"PMC_18199758","title":"Visualizing cold spots: TRPM8-expressing sensory neurons and their projections.","date":"2008","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/18199758","citation_count":262,"is_preprint":false},{"pmid":"15311065","id":"PMC_15311065","title":"Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract.","date":"2004","source":"The Journal of urology","url":"https://pubmed.ncbi.nlm.nih.gov/15311065","citation_count":193,"is_preprint":false},{"pmid":"17376995","id":"PMC_17376995","title":"Modulation of the cold-activated channel TRPM8 by lysophospholipids and polyunsaturated fatty acids.","date":"2007","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/17376995","citation_count":147,"is_preprint":false},{"pmid":"19812688","id":"PMC_19812688","title":"The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic pain.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19812688","citation_count":140,"is_preprint":false},{"pmid":"20844147","id":"PMC_20844147","title":"Gating of transient receptor potential melastatin 8 (TRPM8) channels activated by cold and chemical agonists in planar lipid bilayers.","date":"2010","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/20844147","citation_count":134,"is_preprint":false},{"pmid":"15893591","id":"PMC_15893591","title":"TRPM8 protein localization in trigeminal ganglion and taste papillae.","date":"2005","source":"Brain research. Molecular brain research","url":"https://pubmed.ncbi.nlm.nih.gov/15893591","citation_count":132,"is_preprint":false},{"pmid":"31488702","id":"PMC_31488702","title":"Structural insights into TRPM8 inhibition and desensitization.","date":"2019","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/31488702","citation_count":124,"is_preprint":false},{"pmid":"17517374","id":"PMC_17517374","title":"Increased TRPA1, TRPM8, and TRPV2 expression in dorsal root ganglia by nerve injury.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17517374","citation_count":122,"is_preprint":false},{"pmid":"19404398","id":"PMC_19404398","title":"Inorganic polyphosphate modulates TRPM8 channels.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19404398","citation_count":121,"is_preprint":false},{"pmid":"17517434","id":"PMC_17517434","title":"Characterisation of TRPM8 as a pharmacophore receptor.","date":"2007","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/17517434","citation_count":108,"is_preprint":false},{"pmid":"31141957","id":"PMC_31141957","title":"Recent Progress in TRPM8 Modulation: An Update.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31141957","citation_count":93,"is_preprint":false},{"pmid":"20482834","id":"PMC_20482834","title":"Estrogen regulation of TRPM8 expression in breast cancer cells.","date":"2010","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/20482834","citation_count":84,"is_preprint":false},{"pmid":"23508958","id":"PMC_23508958","title":"Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23508958","citation_count":78,"is_preprint":false},{"pmid":"32610057","id":"PMC_32610057","title":"TRPM8 channels: A review of distribution and clinical role.","date":"2020","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/32610057","citation_count":77,"is_preprint":false},{"pmid":"28358322","id":"PMC_28358322","title":"Development of TRPM8 Antagonists to Treat Chronic Pain and Migraine.","date":"2017","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/28358322","citation_count":74,"is_preprint":false},{"pmid":"27634619","id":"PMC_27634619","title":"TRPM8 and Migraine.","date":"2016","source":"Headache","url":"https://pubmed.ncbi.nlm.nih.gov/27634619","citation_count":73,"is_preprint":false},{"pmid":"18441098","id":"PMC_18441098","title":"Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures.","date":"2008","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18441098","citation_count":73,"is_preprint":false},{"pmid":"28550110","id":"PMC_28550110","title":"TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1.","date":"2017","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28550110","citation_count":73,"is_preprint":false},{"pmid":"26512697","id":"PMC_26512697","title":"Roles of TRPM8 Ion Channels in Cancer: Proliferation, Survival, and Invasion.","date":"2015","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/26512697","citation_count":72,"is_preprint":false},{"pmid":"19295141","id":"PMC_19295141","title":"Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel.","date":"2009","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/19295141","citation_count":70,"is_preprint":false},{"pmid":"24756721","id":"PMC_24756721","title":"TRPM8.","date":"2014","source":"Handbook of experimental pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/24756721","citation_count":69,"is_preprint":false},{"pmid":"17217067","id":"PMC_17217067","title":"TRPM8.","date":"2007","source":"Handbook of experimental pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/17217067","citation_count":69,"is_preprint":false},{"pmid":"30046815","id":"PMC_30046815","title":"TRPV1 and TRPM8 Channels and Nocifensive Behavior in a Rat Model for Dry Eye.","date":"2018","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/30046815","citation_count":68,"is_preprint":false},{"pmid":"36227998","id":"PMC_36227998","title":"Activation mechanism of the mouse cold-sensing TRPM8 channel by cooling agonist and PIP2.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/36227998","citation_count":68,"is_preprint":false},{"pmid":"22860192","id":"PMC_22860192","title":"Modulation of thermoreceptor TRPM8 by cooling compounds.","date":"2012","source":"ACS chemical neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/22860192","citation_count":67,"is_preprint":false},{"pmid":"21411765","id":"PMC_21411765","title":"Scraping through the ice: uncovering the role of TRPM8 in cold transduction.","date":"2011","source":"American journal of physiology. Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21411765","citation_count":67,"is_preprint":false},{"pmid":"29288650","id":"PMC_29288650","title":"Cooling Relief of Acute and Chronic Itch Requires TRPM8 Channels and Neurons.","date":"2017","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/29288650","citation_count":66,"is_preprint":false},{"pmid":"34445208","id":"PMC_34445208","title":"TRPM8 Channels: Advances in Structural Studies and Pharmacological Modulation.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34445208","citation_count":64,"is_preprint":false},{"pmid":"22061619","id":"PMC_22061619","title":"Regulation of TRPM8 channel activity.","date":"2011","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/22061619","citation_count":62,"is_preprint":false},{"pmid":"26645885","id":"PMC_26645885","title":"Reciprocal effects of capsaicin and menthol on thermosensation through regulated activities of TRPV1 and TRPM8.","date":"2015","source":"The journal of physiological sciences : JPS","url":"https://pubmed.ncbi.nlm.nih.gov/26645885","citation_count":57,"is_preprint":false},{"pmid":"29088850","id":"PMC_29088850","title":"Dietary menthol-induced TRPM8 activation enhances WAT \"browning\" and ameliorates diet-induced obesity.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29088850","citation_count":54,"is_preprint":false},{"pmid":"17015441","id":"PMC_17015441","title":"The cold and menthol receptor TRPM8 contains a functionally important double cysteine motif.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17015441","citation_count":53,"is_preprint":false},{"pmid":"30942489","id":"PMC_30942489","title":"Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits.","date":"2019","source":"The Journal of comparative neurology","url":"https://pubmed.ncbi.nlm.nih.gov/30942489","citation_count":52,"is_preprint":false},{"pmid":"21128593","id":"PMC_21128593","title":"Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists.","date":"2010","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21128593","citation_count":52,"is_preprint":false},{"pmid":"21290296","id":"PMC_21290296","title":"TRPM8 in health and disease: cold sensing and beyond.","date":"2011","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/21290296","citation_count":50,"is_preprint":false},{"pmid":"26207981","id":"PMC_26207981","title":"Differential Contribution of TRPA1, TRPV4 and TRPM8 to Colonic Nociception in Mice.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26207981","citation_count":50,"is_preprint":false},{"pmid":"24358160","id":"PMC_24358160","title":"Functional expression of TRPM8 and TRPA1 channels in rat odontoblasts.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24358160","citation_count":49,"is_preprint":false},{"pmid":"24037916","id":"PMC_24037916","title":"The combination of TRPM8 and TRPA1 expression causes an invasive phenotype in lung cancer.","date":"2013","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/24037916","citation_count":49,"is_preprint":false},{"pmid":"27437828","id":"PMC_27437828","title":"Transient Receptor Potential Melastatin 8 Channel (TRPM8) Modulation: Cool Entryway for Treating Pain and Cancer.","date":"2016","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27437828","citation_count":48,"is_preprint":false},{"pmid":"20079339","id":"PMC_20079339","title":"Distinct expression of cold receptors (TRPM8 and TRPA1) in the rat nodose-petrosal ganglion complex.","date":"2010","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/20079339","citation_count":48,"is_preprint":false},{"pmid":"28651550","id":"PMC_28651550","title":"A novel TRPM8 agonist relieves dry eye discomfort.","date":"2017","source":"BMC ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/28651550","citation_count":45,"is_preprint":false},{"pmid":"30445735","id":"PMC_30445735","title":"TRPM8 Channels and Dry Eye.","date":"2018","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/30445735","citation_count":43,"is_preprint":false},{"pmid":"18955132","id":"PMC_18955132","title":"Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells.","date":"2008","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/18955132","citation_count":43,"is_preprint":false},{"pmid":"24278689","id":"PMC_24278689","title":"TRPM7 and TRPM8 Ion Channels in Pancreatic Adenocarcinoma: Potential Roles as Cancer Biomarkers and Targets.","date":"2012","source":"Scientifica","url":"https://pubmed.ncbi.nlm.nih.gov/24278689","citation_count":42,"is_preprint":false},{"pmid":"35662242","id":"PMC_35662242","title":"Structures of a mammalian TRPM8 in closed state.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35662242","citation_count":40,"is_preprint":false},{"pmid":"28332601","id":"PMC_28332601","title":"TRPM8 in the negative regulation of TNFα expression during cold stress.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28332601","citation_count":40,"is_preprint":false},{"pmid":"30545944","id":"PMC_30545944","title":"The Immunosuppressant Macrolide Tacrolimus Activates Cold-Sensing TRPM8 Channels.","date":"2018","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/30545944","citation_count":40,"is_preprint":false},{"pmid":"31501416","id":"PMC_31501416","title":"TRPM8-androgen receptor association within lipid rafts promotes prostate cancer cell migration.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/31501416","citation_count":39,"is_preprint":false},{"pmid":"25460045","id":"PMC_25460045","title":"Thyronamine induces TRPM8 channel activation in human conjunctival epithelial cells.","date":"2014","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/25460045","citation_count":38,"is_preprint":false},{"pmid":"31873179","id":"PMC_31873179","title":"Reduced TRPM8 expression underpins reduced migraine risk and attenuated cold pain sensation in humans.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31873179","citation_count":37,"is_preprint":false},{"pmid":"20934218","id":"PMC_20934218","title":"TRPM8 mediates cold and menthol allergies associated with mast cell activation.","date":"2010","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/20934218","citation_count":37,"is_preprint":false},{"pmid":"30567389","id":"PMC_30567389","title":"Activation of TRPV1 and TRPM8 Channels in the Larynx and Associated Laryngopharyngeal Regions Facilitates the Swallowing Reflex.","date":"2018","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/30567389","citation_count":37,"is_preprint":false},{"pmid":"29221175","id":"PMC_29221175","title":"TRPM8 is required for survival and radioresistance of glioblastoma cells.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29221175","citation_count":36,"is_preprint":false},{"pmid":"30613832","id":"PMC_30613832","title":"Role of Transient Receptor Potential Channels Trpv1 and Trpm8 in Diabetic Peripheral Neuropathy.","date":"2017","source":"Journal of diabetes and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/30613832","citation_count":34,"is_preprint":false},{"pmid":"32532587","id":"PMC_32532587","title":"Current View of Ligand and Lipid Recognition by the Menthol Receptor TRPM8.","date":"2020","source":"Trends in biochemical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32532587","citation_count":32,"is_preprint":false},{"pmid":"27038374","id":"PMC_27038374","title":"TRPM8 Ion Channels as Potential Cancer Biomarker and Target in Pancreatic Cancer.","date":"2016","source":"Advances in protein chemistry and structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/27038374","citation_count":32,"is_preprint":false},{"pmid":"20378345","id":"PMC_20378345","title":"(-)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels.","date":"2010","source":"Bioorganic & medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/20378345","citation_count":31,"is_preprint":false},{"pmid":"34853378","id":"PMC_34853378","title":"Therapeutic potential of TRPM8 antagonists in prostate cancer.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/34853378","citation_count":31,"is_preprint":false},{"pmid":"19507192","id":"PMC_19507192","title":"Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.","date":"2009","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19507192","citation_count":31,"is_preprint":false},{"pmid":"21723782","id":"PMC_21723782","title":"TRPM8 and dyspnea: from the frigid and fascinating past to the cool future?","date":"2011","source":"Current opinion in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/21723782","citation_count":29,"is_preprint":false},{"pmid":"30316690","id":"PMC_30316690","title":"Silencing of TRPM8 inhibits aggressive tumor phenotypes and enhances gemcitabine sensitivity in pancreatic cancer.","date":"2018","source":"Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.]","url":"https://pubmed.ncbi.nlm.nih.gov/30316690","citation_count":29,"is_preprint":false},{"pmid":"37033630","id":"PMC_37033630","title":"Therapeutic potential of TRPM8 channels in cancer treatment.","date":"2023","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37033630","citation_count":28,"is_preprint":false},{"pmid":"26163239","id":"PMC_26163239","title":"Identification of the cold receptor TRPM8 in the nasal mucosa.","date":"2015","source":"American journal of rhinology & allergy","url":"https://pubmed.ncbi.nlm.nih.gov/26163239","citation_count":28,"is_preprint":false},{"pmid":"28943398","id":"PMC_28943398","title":"Cold-sensing TRPM8 channel participates in circadian control of the brown adipose tissue.","date":"2017","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/28943398","citation_count":28,"is_preprint":false},{"pmid":"27845197","id":"PMC_27845197","title":"Sustained Morphine Administration Induces TRPM8-Dependent Cold Hyperalgesia.","date":"2016","source":"The journal of pain","url":"https://pubmed.ncbi.nlm.nih.gov/27845197","citation_count":28,"is_preprint":false},{"pmid":"33071240","id":"PMC_33071240","title":"Menthol to Induce Non-shivering Thermogenesis via TRPM8/PKA Signaling for Treatment of Obesity.","date":"2021","source":"Journal of obesity & metabolic syndrome","url":"https://pubmed.ncbi.nlm.nih.gov/33071240","citation_count":27,"is_preprint":false},{"pmid":"35897101","id":"PMC_35897101","title":"Activation of peripheral TRPM8 mitigates ischemic stroke by topically applied menthol.","date":"2022","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/35897101","citation_count":27,"is_preprint":false},{"pmid":"31519770","id":"PMC_31519770","title":"TRPM8 facilitates proliferation and immune evasion of esophageal cancer cells.","date":"2019","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/31519770","citation_count":25,"is_preprint":false},{"pmid":"27483288","id":"PMC_27483288","title":"TRPV1 and TRPM8 in Treatment of Chronic Cough.","date":"2016","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/27483288","citation_count":25,"is_preprint":false},{"pmid":"20932258","id":"PMC_20932258","title":"The emerging pharmacology of TRPM8 channels: hidden therapeutic potential underneath a cold surface.","date":"2011","source":"Current pharmaceutical biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/20932258","citation_count":25,"is_preprint":false},{"pmid":"32747539","id":"PMC_32747539","title":"A folding reaction at the C-terminal domain drives temperature sensing in TRPM8 channels.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32747539","citation_count":24,"is_preprint":false},{"pmid":"29926226","id":"PMC_29926226","title":"TRPM8 and prostate: a cold case?","date":"2018","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/29926226","citation_count":24,"is_preprint":false},{"pmid":"37857704","id":"PMC_37857704","title":"Structure of human TRPM8 channel.","date":"2023","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/37857704","citation_count":23,"is_preprint":false},{"pmid":"39093967","id":"PMC_39093967","title":"Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.","date":"2024","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/39093967","citation_count":23,"is_preprint":false},{"pmid":"35594403","id":"PMC_35594403","title":"The acquisition of cold sensitivity during TRPM8 ion channel evolution.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35594403","citation_count":23,"is_preprint":false},{"pmid":"32277850","id":"PMC_32277850","title":"Testosterone-androgen receptor: The steroid link inhibiting TRPM8-mediated cold sensitivity.","date":"2020","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32277850","citation_count":23,"is_preprint":false},{"pmid":"37635201","id":"PMC_37635201","title":"TCAF2 in Pericytes Promotes Colorectal Cancer Liver Metastasis via Inhibiting Cold-Sensing TRPM8 Channel.","date":"2023","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/37635201","citation_count":22,"is_preprint":false},{"pmid":"33147416","id":"PMC_33147416","title":"The cool things to know about TRPM8!","date":"2020","source":"Channels (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/33147416","citation_count":22,"is_preprint":false},{"pmid":"35361751","id":"PMC_35361751","title":"AMTB, a TRPM8 antagonist, suppresses growth and metastasis of osteosarcoma through repressing the TGFβ signaling pathway.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35361751","citation_count":22,"is_preprint":false},{"pmid":"37373205","id":"PMC_37373205","title":"Human Osteoarthritic Chondrocytes Express Nineteen Different TRP-Genes-TRPA1 and TRPM8 as Potential Drug Targets.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37373205","citation_count":22,"is_preprint":false},{"pmid":"33977138","id":"PMC_33977138","title":"Trigeminal Neuralgia TRPM8 Mutation: Enhanced Activation, Basal [Ca2+]i and Menthol Response.","date":"2021","source":"Neurology. Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33977138","citation_count":21,"is_preprint":false},{"pmid":"27236325","id":"PMC_27236325","title":"Activation of Cold-Sensitive Channels TRPM8 and TRPA1 Inhibits the Proliferative Airway Smooth Muscle Cell Phenotype.","date":"2016","source":"Lung","url":"https://pubmed.ncbi.nlm.nih.gov/27236325","citation_count":21,"is_preprint":false},{"pmid":"31127000","id":"PMC_31127000","title":"Gαq Sensitizes TRPM8 to Inhibition by PI(4,5)P2 Depletion upon Receptor Activation.","date":"2019","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31127000","citation_count":21,"is_preprint":false},{"pmid":"33090595","id":"PMC_33090595","title":"TRPM8 channel augments T-cell activation and proliferation.","date":"2020","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/33090595","citation_count":20,"is_preprint":false},{"pmid":"19997638","id":"PMC_19997638","title":"Comparative effects of heterologous TRPV1 and TRPM8 expression in rat hippocampal neurons.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19997638","citation_count":20,"is_preprint":false},{"pmid":"35743115","id":"PMC_35743115","title":"TRPM8 as an Anti-Tumoral Target in Prostate Cancer Growth and Metastasis Dissemination.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35743115","citation_count":19,"is_preprint":false},{"pmid":"26111800","id":"PMC_26111800","title":"Function and postnatal changes of dural afferent fibers expressing TRPM8 channels.","date":"2015","source":"Molecular pain","url":"https://pubmed.ncbi.nlm.nih.gov/26111800","citation_count":19,"is_preprint":false},{"pmid":"25375115","id":"PMC_25375115","title":"Menthol inhibits detrusor contractility independently of TRPM8 activation.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25375115","citation_count":19,"is_preprint":false},{"pmid":"38316991","id":"PMC_38316991","title":"Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.","date":"2024","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/38316991","citation_count":18,"is_preprint":false},{"pmid":"31729029","id":"PMC_31729029","title":"Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation.","date":"2019","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31729029","citation_count":18,"is_preprint":false},{"pmid":"38892000","id":"PMC_38892000","title":"Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38892000","citation_count":17,"is_preprint":false},{"pmid":"32290846","id":"PMC_32290846","title":"Chronic morphine regulates TRPM8 channels via MOR-PKCβ signaling.","date":"2020","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/32290846","citation_count":17,"is_preprint":false},{"pmid":"35489198","id":"PMC_35489198","title":"Both heat-sensitive TRPV4 and cold-sensitive TRPM8 ion channels regulate microglial activity.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35489198","citation_count":17,"is_preprint":false},{"pmid":"34066977","id":"PMC_34066977","title":"Oxaliplatin Causes Transient Changes in TRPM8 Channel Activity.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34066977","citation_count":17,"is_preprint":false},{"pmid":"34446569","id":"PMC_34446569","title":"Constitutive Phosphorylation as a Key Regulator of TRPM8 Channel Function.","date":"2021","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/34446569","citation_count":17,"is_preprint":false},{"pmid":"32943886","id":"PMC_32943886","title":"TRPM8 Inhibition Regulates the Proliferation, Migration and ROS Metabolism of Bladder Cancer Cells.","date":"2020","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32943886","citation_count":17,"is_preprint":false},{"pmid":"28039451","id":"PMC_28039451","title":"High-throughput proteome analysis reveals targeted TRPM8 degradation in prostate cancer.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28039451","citation_count":17,"is_preprint":false},{"pmid":"31654626","id":"PMC_31654626","title":"Pharmacological inhibition of TRPM8-induced gene transcription.","date":"2019","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/31654626","citation_count":16,"is_preprint":false},{"pmid":"35665750","id":"PMC_35665750","title":"The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35665750","citation_count":16,"is_preprint":false},{"pmid":"28867384","id":"PMC_28867384","title":"Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.","date":"2017","source":"Brain research bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/28867384","citation_count":16,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46507,"output_tokens":6951,"usd":0.121893},"stage2":{"model":"claude-opus-4-6","input_tokens":10716,"output_tokens":4275,"usd":0.240682},"total_usd":0.362575,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"TRPM8 is directly gated by cold, menthol, and icilin in planar lipid bilayers, excluding cellular signaling pathways; PI(4,5)P2 is the prime factor impacting TRPM8 gating through direct specific interactions with the channel protein; menthol increases PI(4,5)P2 binding potency and channel activity; icilin activation depends on intracellular calcium; cold-activated TRPM8 exhibits steep temperature dependence (Q10 ~40) with large entropy and enthalpy changes.\",\n      \"method\": \"Reconstitution in planar lipid bilayers, patch-clamp, phosphoinositide binding assays, in vitro mutagenesis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in lipid bilayers with multiple orthogonal methods, definitively excludes cellular signaling involvement\",\n      \"pmids\": [\"20844147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TRPM8 channel protein forms a stable complex with inorganic polyphosphate (polyP) and poly-(R)-3-hydroxybutyrate (PHB); enzymatic breakdown of polyP by exopolyphosphatase inhibits TRPM8 channel activity both in cells and in purified channel reconstituted in planar lipid bilayers.\",\n      \"method\": \"Whole-cell patch-clamp, fluorescent calcium measurements, planar lipid bilayer reconstitution, biochemical co-purification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in lipid bilayers plus biochemical demonstration of complex, multiple methods\",\n      \"pmids\": [\"19404398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of TRPM8 in ligand-free, antagonist-bound, and calcium-bound forms reveal a malleable ligand-binding pocket, two nonconducting states (closed and desensitized), direct calcium binding mediating stimulus-evoked desensitization, and large S4-S5 linker rearrangements repositioning S1-S4 and pore domains relative to the TRP helix.\",\n      \"method\": \"Cryo-electron microscopy structure determination\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures at multiple states with functional validation, published in high-impact journal\",\n      \"pmids\": [\"31488702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of mouse TRPM8 in closed, intermediate, and open states reveal two discrete agonist binding sites, state-dependent gate rearrangements, and a disordered-to-ordered transition of the gate-forming S6 helix along the PIP2- and cooling agonist-dependent gating pathway.\",\n      \"method\": \"Cryo-electron microscopy, electrophysiology\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures of multiple functional states with electrophysiological validation\",\n      \"pmids\": [\"36227998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of mouse TRPM8 in ligand-free and Ca2+/icilin-bound forms at 2.5-3.2 Å resolution reveal a short but wide selectivity filter, canonical S4-S5 linker, and show that Ca2+ and icilin bind in the cytosolic-facing cavity of the voltage-sensing-like domain but induce little conformational change; all ligand-bound structures adopt the closed conformation.\",\n      \"method\": \"Cryo-electron microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structures at 2.5-3.2 Å\",\n      \"pmids\": [\"35662242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of human TRPM8 in closed state at 2.7 Å resolution reveals the most complete N-terminal pre-melastatin homology region model, lipid binding sites, and icilin binding mode; pore helix S6 register distinguishes closed, desensitized, and open states across TRPM structures.\",\n      \"method\": \"Cryo-electron microscopy, molecular modeling\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structure of human TRPM8 with structural analysis\",\n      \"pmids\": [\"37857704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRPM8 inhibitors bind selectively to the desensitized state of the channel; cold and cooling agonists share a common desensitization pathway; structural determinants for conformational change in TRPM8 desensitization were identified; overlapping mechanisms underlie desensitization and inhibition.\",\n      \"method\": \"Cryo-electron microscopy, electrophysiology, molecular dynamics simulations\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures combined with electrophysiology and MD simulations\",\n      \"pmids\": [\"39093967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Lysophospholipids (LPCs, LPI, LPS) raise the temperature threshold of TRPM8 activation toward normal body temperature, while polyunsaturated fatty acids (e.g., arachidonic acid) inhibit TRPM8 activation by cold, icilin, and menthol; iPLA2 inhibition abolishes cold and icilin responses but not menthol responses of TRPM8.\",\n      \"method\": \"Patch-clamp, calcium imaging in CHO cells and DRG neurons expressing TRPM8\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacological and biochemical approaches in heterologous and native cells\",\n      \"pmids\": [\"17376995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPM8 contains two essential cysteine residues flanking the N-linked glycosylation site at Asn-934 in the pore region; mutation of either cysteine abolishes channel function and produces a non-functional homodimer; Asn-934 is the glycosylated residue (complex carbohydrate); TRPM8 forms tetramers consistent with functional TRP channel architecture.\",\n      \"method\": \"Site-directed mutagenesis, SDS-PAGE, PFO-PAGE, calcium imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with biochemical and functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"17015441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPM8 acts as a Rap1 GTPase inhibitor through a non-channel, pore-independent function; TRPM8 retains Rap1 intracellularly via direct protein-protein interaction, preventing Rap1 cytoplasm-to-plasma membrane trafficking, thereby impairing integrin conformational activation and suppressing endothelial cell migration, tube formation, and spheroid sprouting.\",\n      \"method\": \"Co-immunoprecipitation, live imaging, dominant-negative/overexpression, migration assays, tube formation assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct protein-protein interaction with multiple functional readouts, pore-independence demonstrated\",\n      \"pmids\": [\"28550110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Activated androgen receptor (AR) interacts with TRPM8 within lipid raft microdomains of the plasma membrane; this AR-TRPM8 interaction inhibits TRPM8 channel activity and promotes prostate cancer cell migration.\",\n      \"method\": \"Co-immunoprecipitation, lipid raft fractionation, patch-clamp, migration assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with functional consequence, single lab\",\n      \"pmids\": [\"31501416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Testosterone (TST) inhibits TRPM8-mediated cold perception through noncanonical engagement of androgen receptor (AR); AR is present on the cell surface and interacts with TRPM8 in response to TST; TST in nanomolar concentrations suppresses TRPM8 currents and single-channel activity in DRG neurons and HEK cells co-expressing TRPM8 and AR, but not TRPM8 alone.\",\n      \"method\": \"Patch-clamp, calcium imaging, biochemical assays, confocal imaging, behavioral testing in castrated rodents\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including electrophysiology, imaging, and in vivo behavioral assays across two species\",\n      \"pmids\": [\"32277850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P2, sensitizing the channel to inhibition by PI(4,5)P2 depletion upon GPCR activation; constitutively active Gαq inhibits TRPM8 activity independent of PLC; supplementing PI(4,5)P2 via patch pipette reduces Gαq-coupled receptor-mediated TRPM8 inhibition in DRG neurons.\",\n      \"method\": \"Whole-cell patch-clamp, voltage-sensitive 5'-phosphatase, PI(4,5)P2 supplementation, calcium imaging in DRG neurons\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and pharmacological tools in both native DRG neurons and heterologous cells, strong mechanistic evidence\",\n      \"pmids\": [\"31127000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Chloroquine inhibits TRPM8 in 48.8% of TRPM8-positive DRG neurons through direct action of activated Gαq independent of the phospholipase C pathway.\",\n      \"method\": \"Calcium imaging, pharmacological dissection in DRG neurons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional dissection with selective inhibitors in native neurons, single lab\",\n      \"pmids\": [\"23508958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Temperature-dependent gating of TRPM8 is driven by a folding-unfolding reaction of the distal C-terminal domain (CTD); progressive deletion of the CTD reduces enthalpy change proportionally; deletion of the last 36 amino acids transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ~4); channel gating involves ~1,900 Å3 change in solute-inaccessible volume matching the coiled-coil void space in cryo-EM structure.\",\n      \"method\": \"Patch-clamp with progressive CTD deletions, denaturing agent experiments, osmoticant assays, structural analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with biophysical measurements and structural validation, multiple orthogonal methods\",\n      \"pmids\": [\"32747539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPM8 is constitutively tyrosine phosphorylated by Src kinase; Src potentiates TRPM8 activity; Src inhibition with PP2 reduces TRPM8 tyrosine phosphorylation and cold-induced channel activation in HEK293T cells and native DRG neurons.\",\n      \"method\": \"Co-immunoprecipitation, western blot, calcium imaging, patch-clamp, RNA interference\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation confirmed biochemically with functional validation in heterologous and native cells\",\n      \"pmids\": [\"31729029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Constitutive phosphorylation of serine residues (S26, S29, S541, S542) in the N-terminus of mouse TRPM8 negatively regulates channel activity; S29A mutation is sufficient to increase TRPM8 cold- and menthol-evoked responses by shifting the voltage activation curve to more negative potentials and increasing the number of active channels at the plasma membrane.\",\n      \"method\": \"Mass spectrometry, site-directed mutagenesis, calcium imaging, patch-clamp, TIRF microscopy\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mass spectrometry identification + mutagenesis + biophysical characterization + TIRF imaging, multiple orthogonal methods\",\n      \"pmids\": [\"34446569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Chronic morphine activates MOR-PKCβ signaling to phosphorylate TRPM8 at two consensus sites (S1040 and S1041), reducing TRPM8 desensitization and sensitizing TRPM8 responsiveness to cold and menthol; site-directed mutation of S1040/S1041 prevents MOR-induced reduction in TRPM8 desensitization.\",\n      \"method\": \"Patch-clamp, calcium imaging, site-directed mutagenesis, pharmacological inhibition in DRG neurons and HEK cells\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-directed mutagenesis of phosphorylation sites combined with electrophysiology in native and heterologous systems\",\n      \"pmids\": [\"32290846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LCK (lymphocyte-specific protein tyrosine kinase) directly interacts with TRPM8 and phosphorylates it at Y1022, enhancing TRPM8 multimerization and channel current density; 14-3-3ζ also interacts with TRPM8 and promotes multimerization; LCK enhances 14-3-3ζ-TRPM8 binding; Y1022F mutation impairs TRPM8 multimerization and 14-3-3ζ binding; TRPM8 phospho-Y1022 feeds back to inhibit LCK Tyr505 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, patch-clamp, mutagenesis, western blot, functional cancer cell assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction confirmed by co-IP with mutagenesis, single lab\",\n      \"pmids\": [\"35665750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPM8 interacts with NF-κB and suppresses NF-κB nuclear localization under cold stress, thereby inhibiting TNFα gene transcription in the mouse hypothalamus.\",\n      \"method\": \"Co-immunoprecipitation, nuclear fractionation, qRT-PCR, western blot\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct protein interaction demonstrated with mechanistic downstream readout, single lab\",\n      \"pmids\": [\"28332601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Tacrolimus (FK506) directly gates TRPM8 channels, sensitizing their response to cold by inducing a leftward shift in the voltage-dependent activation curve; this direct gating is demonstrated in purified TRPM8 reconstituted in lipid bilayers; tacrolimus acts independently of calcineurin/PLC signaling and binds at a different site from menthol (TRPM8-Y745H) or icilin (TRPM8-N799A).\",\n      \"method\": \"Lipid bilayer reconstitution, patch-clamp, calcium imaging, mutagenesis in DRG neurons and heterologous cells\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct gating demonstrated in purified reconstituted channel with site-distinguishing mutagenesis and in vivo confirmation\",\n      \"pmids\": [\"30545944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPM8 gain-of-function mutation p.Arg30Gln (c.89 G>A) enhances channel activation, increases basal current amplitude and intracellular Ca2+, and augments menthol response, contributing to familial trigeminal neuralgia pathogenesis.\",\n      \"method\": \"Calcium imaging, whole-cell patch-clamp recording of mutant vs. wild-type TRPM8\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional characterization of disease-associated mutation with electrophysiology, single lab\",\n      \"pmids\": [\"33977138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPM8 is required for glioblastoma cell migration, DNA repair, and radioresistance; ionizing radiation activates and upregulates TRPM8-mediated Ca2+ signaling, which interferes with cell cycle control via CaMKII, cdc25C, and cdc2.\",\n      \"method\": \"RNA interference, patch-clamp, calcium imaging, colony formation, flow cytometry, immunoblot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts with pathway identification, single lab\",\n      \"pmids\": [\"29221175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPM8 channel activation by menthol in adipocytes induces UCP1 expression and WAT browning through Ca2+ influx and PKA activation; the menthol-induced thermogenic gene expression is blocked by PKA inhibitor KT5720 or calcium chelator BAPTA-AM.\",\n      \"method\": \"Pharmacological inhibition, mRNA/protein expression analysis, mouse dietary model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological dissection of signaling pathway in cultured cells and in vivo, single lab\",\n      \"pmids\": [\"29088850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GFRα3 activation sensitizes and upregulates TRPM8 expression and plasma membrane trafficking in DRG neurons; GFRα3 knockdown reduces TRPM8 membrane trafficking and attenuates cold hyperalgesia in CCI rats; TRPM8 inhibition blocks GFRα3 agonist-induced cold hyperalgesia.\",\n      \"method\": \"siRNA knockdown, western blot, immunofluorescence, behavioral testing, TRPM8 antagonist\",\n      \"journal\": \"Brain research bulletin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockdown with multiple readouts in native tissue, single lab\",\n      \"pmids\": [\"28867384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRPM8 secreted RNA in extracellular vesicles (EVs) from prostate cells activates TLR3/NF-κB-mediated sterile inflammatory signaling after EV endocytosis by epithelial cancer cells; translation-defective TRPM8 RNA in xenografts reduces collagen I, increases NK cell infiltration, and expands necrotic areas.\",\n      \"method\": \"Extracellular vesicle isolation, TLR3/NF-κB reporter assays, xenograft models, immunohistology\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple in vitro and in vivo methods demonstrating non-channel RNA function, single lab\",\n      \"pmids\": [\"38316991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MHR1-3 domain of TRPM8 confers independent cold sensitivity and is required for pore domain regulation of cold activation; the pore domain underwent positive selection in terrestrial tetrapods; the mature MHR1-3 domain is necessary for pore domain regulatory mechanism in TRPM8 cold activation.\",\n      \"method\": \"Domain deletion/chimera analysis, electrophysiology, evolutionary analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain swapping with functional validation, single study\",\n      \"pmids\": [\"35594403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TCAF2 (TRP channel-associated factor 2) inhibits TRPM8 expression and activity in tumor pericytes, leading to Wnt5a secretion that activates STAT3 signaling in tumor cells to facilitate EMT and colorectal cancer liver metastasis; TRPM8 agonist menthol suppresses Wnt5a secretion in pericytes.\",\n      \"method\": \"Gain/loss-of-function, pericyte conditional Tcaf2-KO mice, co-culture assays, proteomic analysis, signaling pathway analysis\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional knockout mice with mechanistic pathway definition, single lab\",\n      \"pmids\": [\"37635201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPM8 channel activation by menthol/icilin in esophageal cancer cells promotes PD-L1 expression via the calcineurin-NFATc3 pathway, leading to immune evasion by reducing CD8+ T cell cytotoxicity.\",\n      \"method\": \"Overexpression/siRNA knockdown, co-incubation assay with CD8+ T cells, reporter assays, western blot\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway identified with multiple molecular tools, single lab\",\n      \"pmids\": [\"31519770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRPM8-expressing DRG neurons constitute a specific functional subpopulation where virtually all EGFPf-positive neurons respond to cold and menthol; these neurons project to superficial layer I of the spinal cord with distinct termination patterns compared to peptidergic fibers, and form unique bush/cluster endings in the superficial epidermis.\",\n      \"method\": \"Knock-in reporter mouse (TRPM8-EGFPf), calcium imaging, anatomical tracing, immunohistochemistry\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic reporter with functional validation and anatomical tracing, strong evidence for anatomical and functional identity\",\n      \"pmids\": [\"18199758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Chronic morphine treatment sensitizes TRPM8 to cold and menthol and reduces activation-evoked desensitization via PKCβ phosphorylation of S1040 and S1041; blocking PLC or PKCβ (but not PKA or ROCK) restores desensitization; TRPM8-expressing DRG neurons show hyperexcitability after sustained morphine treatment.\",\n      \"method\": \"Site-directed mutagenesis (S1040A/S1041A), patch-clamp, pharmacological inhibition, in vivo morphine treatment\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-directed mutagenesis of phosphorylation sites with electrophysiology confirming mechanism\",\n      \"pmids\": [\"32290846\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPM8 is a homotetrameric, Ca2+-permeable non-selective cation channel that is directly gated by cold temperature, menthol, icilin, and voltage, with PI(4,5)P2 as an obligatory co-activator binding directly to the channel; cryo-EM structures reveal a malleable ligand-binding pocket, two distinct agonist sites, and separate closed and desensitized states driven by Ca2+ binding and large S4-S5 linker rearrangements; temperature sensitivity is mediated by folding-unfolding of the C-terminal coiled-coil domain; channel activity is positively modulated by Src-mediated tyrosine phosphorylation (Y1022 by LCK), tacrolimus (direct gating), polyphosphate association, and lysophospholipids, and negatively regulated by constitutive serine phosphorylation (especially S29), Gαq (which reduces PI(4,5)P2 apparent affinity), androgen receptor interaction, PUFA, and PKCβ-mediated phosphorylation (S1040/S1041) downstream of opioid receptor activation; beyond its channel function, TRPM8 acts as a Rap1 GTPase inhibitor through direct protein-protein interaction to suppress endothelial migration, and its RNA can be secreted in extracellular vesicles to activate TLR3/NF-κB sterile inflammatory signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TRPM8 is a cold- and menthol-activated, Ca²⁺-permeable non-selective cation channel that serves as the principal molecular transducer of innocuous cold sensation in somatosensory neurons. The channel assembles as a homotetramer requiring PI(4,5)P₂ as an obligatory co-activator; menthol increases PI(4,5)P₂ binding potency, while Gαq reduces its apparent affinity, sensitizing the channel to inhibition upon GPCR activation [PMID:20844147, PMID:31127000]. Cryo-EM structures reveal a malleable ligand-binding pocket with two discrete agonist sites, Ca²⁺-dependent desensitization involving large S4–S5 linker rearrangements, and a disordered-to-ordered transition of the S6 gate helix during activation, while temperature sensitivity is conferred by a folding–unfolding equilibrium of the C-terminal coiled-coil domain [PMID:31488702, PMID:36227998, PMID:32747539]. Beyond its channel function, TRPM8 acts as a Rap1 GTPase inhibitor through direct protein–protein interaction to suppress endothelial cell migration independently of ion conduction, and a gain-of-function p.Arg30Gln mutation causes familial trigeminal neuralgia [PMID:28550110, PMID:33977138].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that TRPM8 assembles as a tetramer with essential pore-region cysteines flanking the N-glycosylation site at Asn-934 resolved the basic quaternary architecture of the functional channel.\",\n      \"evidence\": \"Site-directed mutagenesis with SDS-PAGE, PFO-PAGE, and calcium imaging in heterologous cells\",\n      \"pmids\": [\"17015441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure yet available\", \"Stoichiometry of heteromeric assemblies unexplored\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that lysophospholipids raise the TRPM8 temperature activation threshold toward body temperature while PUFAs inhibit channel activity established a lipid-metabolite regulatory axis for tuning cold sensitivity.\",\n      \"evidence\": \"Patch-clamp and calcium imaging in CHO cells and DRG neurons expressing TRPM8\",\n      \"pmids\": [\"17376995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding sites for lysophospholipids and PUFAs on TRPM8 not mapped\", \"In vivo physiological relevance of iPLA2-dependent regulation not tested genetically\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genetic reporter tracing revealed that TRPM8-expressing DRG neurons constitute a dedicated cold-sensing population with unique peripheral bush/cluster endings and superficial dorsal horn projections, defining the anatomical substrate of TRPM8-mediated thermosensation.\",\n      \"evidence\": \"TRPM8-EGFPf knock-in mouse with calcium imaging, anatomical tracing, and immunohistochemistry\",\n      \"pmids\": [\"18199758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether all cold sensation requires TRPM8 or parallel pathways exist not resolved by this study\", \"Central circuit integration of TRPM8 input not addressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The discovery that TRPM8 forms a stable complex with inorganic polyphosphate and that polyphosphate degradation inhibits channel activity identified an unexpected inorganic co-factor requirement.\",\n      \"evidence\": \"Planar lipid bilayer reconstitution, biochemical co-purification, whole-cell patch-clamp\",\n      \"pmids\": [\"19404398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Polyphosphate binding site on TRPM8 not identified\", \"Physiological polyphosphate levels in sensory neurons not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Reconstitution in planar lipid bilayers definitively established that cold, menthol, and icilin gate TRPM8 directly without requiring cellular signaling intermediates, and that PI(4,5)P₂ is an obligatory co-activator binding the channel protein with agonist-enhanced potency.\",\n      \"evidence\": \"Purified TRPM8 in planar lipid bilayers, patch-clamp, phosphoinositide binding assays, mutagenesis\",\n      \"pmids\": [\"20844147\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PI(4,5)P₂ binding site on TRPM8 not structurally resolved\", \"Mechanism coupling cold temperature to channel opening still unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Multiple studies revealed that TRPM8 functions beyond ion conduction: it acts as a Rap1 GTPase inhibitor through direct pore-independent protein–protein interaction, suppressing endothelial migration, and interacts with NF-κB to suppress its nuclear translocation under cold stress.\",\n      \"evidence\": \"Co-immunoprecipitation, dominant-negative/overexpression, migration/tube formation assays (Rap1); nuclear fractionation, qRT-PCR (NF-κB)\",\n      \"pmids\": [\"28550110\", \"28332601\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TRPM8–Rap1 interaction unknown\", \"Relevance of NF-κB interaction beyond hypothalamic cold stress not tested\", \"Whether Rap1 inhibition and NF-κB interaction are coordinated is unexplored\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Tacrolimus was shown to directly gate TRPM8 independently of calcineurin/PLC at a binding site distinct from menthol and icilin, explaining clinical cold hypersensitivity in transplant patients.\",\n      \"evidence\": \"Purified TRPM8 in lipid bilayers, patch-clamp, mutagenesis distinguishing Y745H (menthol) and N799A (icilin) sites\",\n      \"pmids\": [\"30545944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural identity of the tacrolimus binding pocket not resolved\", \"Whether tacrolimus gating shares the PIP₂-dependent pathway is unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Cryo-EM structures of TRPM8 in ligand-free, antagonist-bound, and Ca²⁺-bound states resolved the architecture of the malleable ligand-binding pocket, revealed two non-conducting states (closed and desensitized), and showed that Ca²⁺ binding drives desensitization through large S4–S5 linker rearrangements.\",\n      \"evidence\": \"Cryo-electron microscopy at multiple functional states\",\n      \"pmids\": [\"31488702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Open-state structure not captured\", \"PI(4,5)P₂ binding mode not resolved in these structures\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The mechanism of Gαq-mediated TRPM8 inhibition was established: Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P₂ independently of PLC activity, sensitizing the channel to inhibition upon receptor-mediated PI(4,5)P₂ depletion.\",\n      \"evidence\": \"Whole-cell patch-clamp with voltage-sensitive phosphatase, PI(4,5)P₂ supplementation, calcium imaging in DRG neurons\",\n      \"pmids\": [\"31127000\", \"23508958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Gαq binding site on TRPM8 not mapped\", \"Whether other Gα subunits modulate PI(4,5)P₂ affinity is untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Src-family kinase-mediated constitutive tyrosine phosphorylation was shown to positively regulate TRPM8, as Src inhibition reduced both phosphorylation and cold-induced channel activation.\",\n      \"evidence\": \"Co-immunoprecipitation, western blot, calcium imaging, patch-clamp, RNAi in HEK293T cells and DRG neurons\",\n      \"pmids\": [\"31729029\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific tyrosine residue(s) phosphorylated by Src not identified in this study\", \"Relationship to LCK-mediated Y1022 phosphorylation not clarified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The molecular basis of TRPM8 temperature sensitivity was resolved: a folding–unfolding equilibrium of the C-terminal coiled-coil domain generates the large enthalpy change underlying steep temperature dependence (Q10 ~40), with progressive CTD deletion proportionally reducing enthalpy and the last 36 residues being essential.\",\n      \"evidence\": \"Patch-clamp with progressive CTD deletions, denaturing agents, osmoticant assays, structural analysis\",\n      \"pmids\": [\"32747539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level description of the folding transition at different temperatures not available\", \"How CTD folding couples to pore opening is structurally unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Chronic morphine was found to sensitize TRPM8 by PKCβ-mediated phosphorylation at S1040 and S1041, reducing activation-evoked desensitization; this mechanism links opioid receptor signaling to cold hypersensitivity.\",\n      \"evidence\": \"Site-directed mutagenesis (S1040A/S1041A), patch-clamp, pharmacological inhibition in DRG neurons and HEK cells, in vivo morphine treatment\",\n      \"pmids\": [\"32290846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S1040/S1041 phosphorylation alters TRPM8 structure is unknown\", \"Clinical relevance in opioid-induced cold allodynia not validated in patients\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mass spectrometry-guided mutagenesis identified constitutive N-terminal serine phosphorylation (especially S29) as a tonic brake on TRPM8 activity: S29A shifts voltage-dependent activation to more negative potentials and increases active channel density at the plasma membrane.\",\n      \"evidence\": \"Mass spectrometry, site-directed mutagenesis, calcium imaging, patch-clamp, TIRF microscopy\",\n      \"pmids\": [\"34446569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase(s) responsible for constitutive S29 phosphorylation not identified\", \"Whether S29 phosphorylation is dynamically regulated in vivo is unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A gain-of-function TRPM8 variant p.Arg30Gln was identified in a family with trigeminal neuralgia; the mutation enhances basal current, intracellular Ca²⁺, and menthol responsiveness, establishing TRPM8 as a disease gene for inherited facial pain.\",\n      \"evidence\": \"Calcium imaging and whole-cell patch-clamp of mutant vs. wild-type TRPM8\",\n      \"pmids\": [\"33977138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family studied; prevalence among trigeminal neuralgia patients unknown\", \"Structural consequence of R30Q substitution not modeled\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Multiple cryo-EM studies captured closed, intermediate, and open states of TRPM8, revealing two discrete agonist binding sites, a disordered-to-ordered S6 gate transition along the PIP₂/agonist gating pathway, and showing that Ca²⁺/icilin occupy the cytosol-facing VSLD cavity with minimal conformational change.\",\n      \"evidence\": \"Cryo-EM at 2.5–3.2 Å resolution with electrophysiological validation\",\n      \"pmids\": [\"36227998\", \"35662242\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full open-state structure with PIP₂ simultaneously bound not obtained\", \"Structural basis for temperature-induced gating transition not captured\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"LCK was identified as a direct TRPM8 kinase phosphorylating Y1022, which enhances channel multimerization and recruits 14-3-3ζ; phospho-Y1022 feeds back to inhibit LCK Tyr505 phosphorylation, establishing a reciprocal regulatory loop.\",\n      \"evidence\": \"Co-immunoprecipitation, patch-clamp, site-directed mutagenesis (Y1022F), western blot\",\n      \"pmids\": [\"35665750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo significance of LCK–TRPM8 loop not demonstrated\", \"Whether 14-3-3ζ binding alters TRPM8 trafficking or gating kinetics is unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cryo-EM and functional studies demonstrated that TRPM8 inhibitors bind selectively to the desensitized state and that cold- and agonist-induced desensitization share overlapping structural mechanisms, unifying inhibition and desensitization pathways.\",\n      \"evidence\": \"Cryo-EM, electrophysiology, molecular dynamics simulations\",\n      \"pmids\": [\"39093967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether state-selective inhibitor design can achieve therapeutic selectivity in vivo is untested\", \"Kinetics of closed-to-desensitized transitions under physiological conditions not measured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"TRPM8 RNA packaged in extracellular vesicles was found to activate TLR3/NF-κB sterile inflammatory signaling in recipient epithelial cancer cells, revealing a non-channel, RNA-level function of the TRPM8 gene product.\",\n      \"evidence\": \"Extracellular vesicle isolation, TLR3/NF-κB reporter assays, xenograft models with translation-defective TRPM8 RNA\",\n      \"pmids\": [\"38316991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TRPM8 RNA is selectively sorted into EVs is not established\", \"Physiological relevance outside tumor xenograft context unknown\", \"Whether other TRP channel mRNAs share this TLR3-activating property is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis of temperature-induced conformational change at atomic resolution, the identity of kinases maintaining constitutive S29 phosphorylation, the structural determinants of the Rap1-binding interface, and whether the non-channel functions (Rap1 inhibition, NF-κB interaction, EV-RNA signaling) operate in physiological cold sensing or are context-restricted.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM structure capturing the temperature-induced gating transition\", \"Kinase responsible for constitutive S29 phosphorylation unidentified\", \"TRPM8–Rap1 binding interface not structurally mapped\", \"Physiological integration of channel and non-channel functions not addressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 11, 16, 29]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 12, 23]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [29]},\n      {\"term_id\": \"R-HSA-9709957\", \"supporting_discovery_ids\": [0, 14, 29]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"RAP1A\",\n      \"LCK\",\n      \"YWHAZ\",\n      \"AR\",\n      \"GNAQ\",\n      \"SRC\",\n      \"TCAF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}