trpm8 antagonist  (MedChemExpress)

Journal: Science advances

Article Title: Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

doi: 10.1126/sciadv.adp2211

Figure Lengend Snippet: Fig. 1. Cryo-EM structure determination of TRPM8MM in complex with antagonists. (A) Cartoon diagram of the PIP2 and cooling agonist-dependent gating pathway of TRPM8 channels. Published TRPM8MM structures in the C0, C1, and O states (PDB 8E4P, 8E4N, and 8E4L, respectively) are used for illustration. (B) Chemical structures of TC-I 2014, AMG2850, and AMTB. (C) 3D reconstructions of the conformation A (left; brown) and the C0 state (right; silver-gray). Neighboring protomers are colored in gray. (D) TMD comparison between the conformation A (left; brown) and the C0 state (right; silver-gray). S2-S3 linkers and S3 were omitted for clarity. (E and F) EM density at the VSLD cavity (E) and the S4-S5 linker (F) from the conformation A reconstruction in (C). Densities corresponding to antagonists are colored in lime for TC-I, teal for AMG, and blue for AMTB. Red dashed circles indicate the lack of antagonist densities. Thresholding 0.3 in (E) and 0.6 in (F). (G and I) Binding site and EM densities for TC-I [(G) left, lime sticks], AMG [(G) right, teal sticks], and AMTB [(I) blue sticks]. Densities in magenta mesh are contoured at thresholding 0.3 for TC-I, 0.34 for AMG, and 0.2 for AMTB. (H and J) Summary of current inhibition by 10 μM TC-I or 10 μM AMG (H) or by 25 μM AMTB (J) measured by TEVC recording on the WT and mutant TRPM8MM channels activated by 10 to 30 μM C3 at −60 mV. The antagonist inhibition level is quantified by the percentage of current inhibited by antagonists over full inhibition by 50 μM RR (see Materials and Methods). Values for individual oocytes are shown as open circles with means ± SEM (n = 3 to 8 oocytes). ns > 0.05, **P < 0.01, ***P < 0.001, using one-way ANOVA followed by Dunnett’s post hoc test.

Article Snippet: C3 or menthol (Sigma- Aldrich) and TRPM8 antagonists TC- I 2014 (Tocris Bioscience), AMG2850 (Alomone Labs), AMTB hydrochloride (Sigma- Aldrich), and ruthenium red (RR; Sigma- Aldrich) were applied using a gravity- fed perfusion system for the corresponding measurements.

Techniques: Cryo-EM Sample Prep, Comparison, Binding Assay, Inhibition, Mutagenesis

Journal: Science advances

Article Title: Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

doi: 10.1126/sciadv.adp2211

Figure Lengend Snippet: Fig. 2. Molecular details of state-dependent inhibition. (A) Mean normalized concentration-response relations for TC-I (left), AMG (middle), and AMTB (right) against TRPM8MM activation by 1 mM menthol in the presence of increasing concentrations of extracellular Ca2+ in the TEVC recording buffer. Data are shown as means ± SEM. n = 4, 5, and 4 oocytes for measurement with 0, 0.5, and 2 mM Ca2+ in the left panel, respectively; n = 4, 5, and 5 oocytes for measurement with 0, 1, and 3 mM Ca2+ in the middle panel, respectively; n = 5, 5, and 6 oocytes for measurement with 0, 0.5, and 2 mM Ca2+ in the right panel, respectively. The continuous curves were fit to the Hill equation with IC50 values indicated in the figure. (B) Thermodynamic model of TRPM8 desensitization and inhibitor binding for the equilibria (Kn) among C, O, D, OI, and DI states and the associated thermodynamic coupling (c). Red arrows indicate transition among the thermodynamically favored states based on fit values. (C) AMG dose- response data fit with the thermodynamic model for 0, 1, and 3 mM extracellular Ca2+. Data are from (A), taking into account the initial level of desensitization. Data points for individual replicate (n ≥ 4) shown. (D) Model fit quality as SSQ plotted as a function of fixed coupling parameter (c) value. (E) Calcium dependence of desensitization (K3) for AMG and TC-I. Error bars represent 95% confidence intervals. (F) Shown in surface (upper) and cylinder (lower) representations, comparison of the antagonist bind- ing site above the S4-S5 linker in TRPM8 channels adopting the C0 (PDB 8E4P), C1 (PDB 8E4N), O (PDB 8E4L), and D (current study) states. Red dashed lines highlight changes in the size of the binding pocket. TC-I and AMG shown as spheres, and residues involved in antagonist binding shown as sticks.

Article Snippet: C3 or menthol (Sigma- Aldrich) and TRPM8 antagonists TC- I 2014 (Tocris Bioscience), AMG2850 (Alomone Labs), AMTB hydrochloride (Sigma- Aldrich), and ruthenium red (RR; Sigma- Aldrich) were applied using a gravity- fed perfusion system for the corresponding measurements.

Techniques: Inhibition, Concentration Assay, Activation Assay, Binding Assay, Comparison

Journal: Science advances

Article Title: Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

doi: 10.1126/sciadv.adp2211

Figure Lengend Snippet: Fig. 4. Mechanism of TRPM8 desensitization. (A) Aligned at the VSLD, comparison of the O (blue) the D (brown) states at the pore, viewed from the extracellular side. Red circle indicates the S6 gate residue Val976. (B and C) Ion permeation pathway (B) and pore radii (C) in the O state and the D state. (D to F) Comparison of the O and the D states at the interfacial cavity for PIP2 binding [(D) and (E)] and at S4b and the S4-S5 linker (F). Val976 shown as yellow spheres. Asterisks denote the π helix position. Red dashed lines and arrows in (E) indicate the structural rearrangements from the O to the D state. ⊗ symbol denotes the ion conduction pathway. (G) Comparison of the O and the D states at the interaction network among S4, the S4-S5 linker, and the neighboring pore domain (S5′ and S6′) with Phe869′ highlighted in red and Ile857, Leu860, and Leu970′ in teal. (H) Mean normalized concentration-response relations for AMG against the WT (n = 3) and F869A (n = 3) in the presence of 0 or 100 μM extracellular Ca2+. Representative current traces shown in fig. S14E. (I) Mean normalized concentration-response relations for AMTB against the WT (black trace; n = 3) and F869A (red trace; n = 3). Representative current traces shown in fig. S14F. (J) Representative current traces of the WT and mutant TRPM8MM channels activated by 200 μM menthol (upper) and cold (lower) at −60 mV in HEK293T cells. Dashed lines indicate the zero-current level. (K and L) Summary of currents remaining after desensitization for the WT and mutant TRPM8MM channels activated by menthol (K) and cold (L) (n = 3 to 5). *P < 0.05, **P < 0.01, ***P < 0.001, using one-way ANOVA followed by Dunnett’s post hoc test. Data are means ± SEM in (H), (I), (K), and (L).

Article Snippet: C3 or menthol (Sigma- Aldrich) and TRPM8 antagonists TC- I 2014 (Tocris Bioscience), AMG2850 (Alomone Labs), AMTB hydrochloride (Sigma- Aldrich), and ruthenium red (RR; Sigma- Aldrich) were applied using a gravity- fed perfusion system for the corresponding measurements.

Techniques: Comparison, Residue, Binding Assay, Concentration Assay, Mutagenesis

Journal: Science advances

Article Title: Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

doi: 10.1126/sciadv.adp2211

Figure Lengend Snippet: Fig. 5. Mechanism of TRPM8 inhibition. (A to E) Comparison of the pore domain (S5′ and S6′) between the O (A) and the D states [(B) and (D)]. Trp877 on S5 is rotated and exposed to the membrane-facing side, and the π helix position on S6 is shifted in the D state [(C) and (E)]. TC-I or AMG binding stabilizes the alternate π helix and the ro- tated Trp877 positions in the D state [(B) and (D)]. Dashed lines indicate interactions that stabilize the specific helical configurations. (F) AMTB interaction stabilizes the VSLD in the D state. Residue side chains and antagonists shown as sticks.

Article Snippet: C3 or menthol (Sigma- Aldrich) and TRPM8 antagonists TC- I 2014 (Tocris Bioscience), AMG2850 (Alomone Labs), AMTB hydrochloride (Sigma- Aldrich), and ruthenium red (RR; Sigma- Aldrich) were applied using a gravity- fed perfusion system for the corresponding measurements.

Techniques: Inhibition, Comparison, Membrane, Binding Assay, Residue

Journal: Science advances

Article Title: Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

doi: 10.1126/sciadv.adp2211

Figure Lengend Snippet: Fig. 7. Summary of ligand binding and ligand-dependent gating of TRPM8 channels. (A) The binding sites for PIP2 (1), type I agonists (2), type II agonists (3), Ca2+ ions (4), competitive antagonists (5), and noncompetitive agonists (6) are located surrounding the S4b in TRPM8. Representative ligands, PIP2 (red), C3 (yellow), AITC (teal), TC-I (lime), and Ca2+ (green), are shown in surface or sphere and highlighted by dashed lines. (B) Conformational changes of S4b, S5, and the S4-S5 linker in the closed C0 (silver-gray), closed C1 (yellow), O (blue), and D (brown) states. Secondary structures and S4-S5 linker motions indicated by lines and arrows. PIP2, C3, AITC, and TC-I shown as sticks and Ca2+ ions as spheres. Neighboring protomers colored in white. (C) Structural diagram illustrating the conformational change from the O state to the D state and the D state–dependent inhibition by antagonists. Ligands and S6 gate residue Val976 shown as sticks and Ca2+ as green spheres. Insets show antagonist binding highlighted in yellow shades and red arrows.

Article Snippet: C3 or menthol (Sigma- Aldrich) and TRPM8 antagonists TC- I 2014 (Tocris Bioscience), AMG2850 (Alomone Labs), AMTB hydrochloride (Sigma- Aldrich), and ruthenium red (RR; Sigma- Aldrich) were applied using a gravity- fed perfusion system for the corresponding measurements.

Techniques: Ligand Binding Assay, Binding Assay, Inhibition, Residue

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Characterization of transient receptor potential melastatin 8 (TRPM8) immunoreactivity in the mouse cerebellum. ( A ) Green fluorescent protein (GFP) expression in the cerebellum of TRPM8-GFP mice. ( B ) TRPM8 expression in the whole cerebellum using light sheet fluorescent microscopy. ( C ) Double labeling of TRPM8 (green) with calbindin, parvalbumin, vesicular GABA transporter (VGAT), vesicular glutamate transporter 2 (VGLUT2), γ-aminobutyric acid (GABA) A receptor (GABA A -R), hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) (red) in the cerebellum of TRPM8-EGFP mice. Dotted squares indicate the magnified areas. Scale bars: 100 μm ( A ), 20 μm ( C ).

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Expressing, Microscopy, Labeling, Binding Assay

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Effects of transient receptor potential melastatin 8 (TRPM8) deficiency on the rotarod, open field, and footprint tests. ( A ) Latency to fall off the rotarod in wild-type (WT) and TRPM8-deficient (TRPM8KO) mice. Data are presented as the mean ± standard error (SE) for 9–10 mice per group. * P < 0.05 compared with WT. ( B ) Distance traveled in the chamber, average movement speed (velocity), and percentage of time spent in the central and peripheral areas in WT and TRPM8KO mice. Data are presented as the mean ± SE for 10 mice per group. * P < 0.05 compared with WT. NS, not significant. ( C ) Quantification of footprints in terms of stride, sway, and stance in WT and TRPM8KO mice. Data are presented as the mean ± SE for 7 mice per group. NS, not significant.

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques:

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Effects of transient receptor potential melastatin 8 (TRPM8) deficiency on cerebellar weight, structure, and γ-aminobutyric acid (GABA)-related factors. ( A ) Cerebellar weights in wild-type (WT) and TRPM8-deficient (TRPM8KO) mice. ( B ) Representative images of hematoxylin and eosin staining (left) and Nissl staining (right) in WT and TRPM8KO mice. ( C ) Quantification of the number and soma size of Purkinje cells (calbindin-positive cells), number of parvalbumin-positive cells, and number of vesicular GABA transporter (VGAT)-positive puncta in WT and TRPM8KO mice. ( D ) Representative western blotting images of glutamate decarboxylase (GAD)65 and GAD67 proteins from WT and TRPM8KO mice, and quantitative western blotting results. ( E ) Comparison of GABA levels in WT and TRPM8KO mice. Data are presented as the mean ± standard error for 5–7 mice. NS, not significant. Scale bars: 500 μm ( B ), 20 μm ( C ).

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Staining, Western Blot, Comparison

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Effects of transient receptor potential melastatin 8 (TRPM8) antagonist (RQ-00203078) administration on motor coordination. ( A ) Latency to fall off the rotarod in mice that were intraperitoneally treated with vehicle (control) or RQ-00203078. Data are presented as the mean ± standard error (SE) for 8–10 mice per group. * P < 0.05 compared with vehicle. ( B ) Latency to fall off the rotarod in mice that were treated with vehicle or RQ-00203078 via intracerebellar administration. Data are presented as the mean ± SE for 8 mice per group. * P < 0.05 compared with vehicle. ( C ) Double labeling of TRPM8 with c-Fos and γ-aminobutyric acid (GABA) in the cerebellum of a TRPM8-EGFP mouse. Scale bars: 20 μm.

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Control, Labeling

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Schematic diagram of how transient receptor potential melastatin 8 (TRPM8) channels on inhibitory γ-aminobutyric acid (GABA)ergic neurons may contribute to motor coordination by modulating synaptic transmission at Purkinje cell–interneuron synapses. This image was generated using the Motifolio illustration toolkit (Motifolio Inc., Ellicott City, MD, USA).

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Transmission Assay, Generated