Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep 27;365(6460):1434-1440.
doi: 10.1126/science.aax6672. Epub 2019 Sep 5.

Structural insights into TRPM8 inhibition and desensitization

Melinda M Diver  1 , Yifan Cheng  2   3 , David Julius  4
Affiliations

Structural insights into TRPM8 inhibition and desensitization

Melinda M Diver et al. Science. .

Abstract

The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary detector of environmental cold and an important target for treating pathological cold hypersensitivity. Here, we present cryo-electron microscopy structures of TRPM8 in ligand-free, antagonist-bound, or calcium-bound forms, revealing how robust conformational changes give rise to two nonconducting states, closed and desensitized. We describe a malleable ligand-binding pocket that accommodates drugs of diverse chemical structures, and we delineate the ion permeation pathway, including the contribution of lipids to pore architecture. Furthermore, we show that direct calcium binding mediates stimulus-evoked desensitization, clarifying this important mechanism of sensory adaptation. We observe large rearrangements within the S4-S5 linker that reposition the S1-S4 and pore domains relative to the TRP helix, leading us to propose a distinct model for modulation of TRPM8 and possibly other TRP channels.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.. Structure of TRPM8 in two distinct states.
(A-B) Side view (A) and top view (B) of the cryo-EM density map of the desensitized state of TRPM8 with subunits differentiated by color. (C) Monomer of the desensitized state with secondary structure elements colored blue-to-red from N-terminus to C-terminus and domains labeled. (D) Ribbon representation of the closed state of TRPM8 (TC-I 2014-bound) with a single subunit colored red. Horizontal lines indicate the approximate boundaries of the cell membrane. (E) Ribbon representation of the desensitized state of TRPM8 with a single subunit colored blue. The outer pore loop is colored yellow. (F) Close-up view of the outer pore loop. Conserved residues important for selectivity (Asp907, Asp908, and Asp910), disulfide bond formation (Cys919 and Cys930), and N-linked glycosylation (Asp924) are drawn as sticks. Nitrogen, blue; oxygen, red; sulfur, green. Density (blue mesh, 4σ contour) for the N-linked glycan is shown.
Fig. 2.
Fig. 2.. Binding site for hydrophobic modulators of TRPM8.
(A) Density within the AMTB-binding site. The map is contoured at 3σ (gray mesh). Stick representation of AMTB-bound TRPM8 with the antagonist AMTB colored teal. (B) Gray surface indicates the shape of the binding pocket, as dictated by residues lining the cavity. AMTB is shown as spheres. (C) Interactions with AMTB (transparent surface and sticks with teal carbon atoms). Nitrogen, blue; oxygen, red. (D) Density within the TC-I 2014-binding site. The map is contoured at 3σ (gray mesh). Stick representation of TC-I 2014-bound TRPM8 with the antagonist TC-I 2014 colored yellow. (E) Gray surface indicates the shape of the binding pocket, as dictated by residues lining the cavity. TC-I 2014 is shown as spheres. (F) Interactions with TC-I 2014 (transparent surface and sticks with yellow carbon atoms). (G) The antagonists AMTB and TC-I 2014 adopt distinct poses in the ligand-binding pocket. (H) Unassigned density (gray mesh, 8σ contour) observed in the ligand-binding pocket of ligand-free TRPM8. (I) Binding site for hydrophobic modulators in TRPM8. The dashed region demarcates the binding pocket. (J) Structure of TRPV1 highlighting the resiniferatoxin (RTX) binding site. The dashed region demarcates the binding site for hydrophobic modulators in TRPV1.
Fig. 3.
Fig. 3.. TRPM8 calcium-binding site.
(A) Sequence alignment of the calcium-binding site within the TRPM subfamily. The conserved binding site is shared by TRPM2, TRPM4, TRPM5, and TRPM8. Residues that coordinate the calcium ion are indicated with a star. Alignment was made with ClustalW. (B) Ribbon representation of calcium-bound TRPM8 with a single subunit colored blue and the calcium ion colored pink (sphere). Horizontal lines indicate the approximate boundaries of the cell membrane. (C) Interactions with calcium (pink sphere). Nitrogen, blue; oxygen, red. (D and E) Structure-function analysis of the role of the calcium-binding site in TRPM8 desensitization. In oocytes expressing pmTRPM8, application of menthol (100 μM) evoked inward currents for which desensitization was observed in the presence of calcium. However, upon mutating several of the residues important for calcium coordination, TRPM8 desensitization was reduced. The A796G mutation was introduced to all constructs to sensitize avian TRPM8 to the agonist icilin. Voltage was held at −60 mV. Desensitization is reported as Ifinal/Iinitial (maximum currents ranged from 0.5–4 mA) for the application of menthol in the presence of calcium. Data represent n=6 or 7 oocytes. Asterisk indicates p < 0.01 comparing wild type and each mutant construct with unpaired two-tailed Student’s t-tests. Representative traces are shown in (D). (F) Structure-function analysis of the role of the calcium-binding site in co-agonism with icilin. In oocytes expressing pmTRPM8, application of menthol (100 μM) or icilin (10 μM), in the presence of calcium, evoked inward currents. However, upon mutating residues proposed to be important for calcium coordination, there was no longer a response to icilin. The A796G mutation was introduced to all constructs to sensitize avian TRPM8 to the agonist icilin. Voltage was held at −60 mV. Data represent n=7 to 12 oocytes. Two asterisks indicate p < 1×10−8 comparing wild type and each mutant construct with unpaired two-tailed Student’s t-tests.
Fig. 4.
Fig. 4.. Conformational changes associated with ligand binding to a shared pocket.
(A) Superimposition of the closed (TC-I 2014-bound; red) and desensitized (blue) TRPM8 structures. (B and C) Structural changes associated with TRPM8 channel gating. Transition from the closed (B) to desensitized (C) state is accompanied by formation of a S4-S5 linker, a local α-to-π-helical transition in S6, and rearrangement of the TRP domain, which alter the lower gate. (D) In the closed state, an ordered acyl chain (orange) is present along the ion permeation pathway, thereby increasing the hydrophobicity of the pore. Green spheres indicate ions observed in the vicinity of the acyl chains. (E) Close-up view, orthogonal to (D) with ion densities (gray mesh; 3σ contour). Inset shows density for acyl chain (blue mesh, 3σ contour). (F) Cation binding sites (green spheres) are in close proximity to negatively charged residues contributed by S6. Nitrogen, blue; oxygen, red. (G) In the desensitized state, a stabilizing lipid packs between the pore helix and S6 of the neighboring subunit (modeled as CHS). Inset shows lipid density (blue mesh, 4σ contour).
Fig. 5.
Fig. 5.. Ion pore.
(A - D) Ion conduction pathway with front and rear subunits removed for clarity and a representation (gray surface) of the minimal radial distance from the center of the pore to the nearest van der Waals protein or lipid contact. Residues lining the selectivity filter and lower gate are shown as sticks for the EGTA-bound (A), AMTB-bound (B), TC-I 2014-bound (C), and calcium-bound (D) states. (E - F) Close-up view of the lower gate of closed (TC-I 2014-bound) (E) and desensitized (F) TRPM8. The residues and acyl chain (closed confirmation only) that form the hydrophobic seal are shown as sticks.
Fig. 6.
Fig. 6.. Gating movements in TRPM8.
In the presence of antagonists (left panel), TRPM8 is closed and density corresponding to an acyl chain is present within the ion conduction pathway. In the presence of calcium (right panel), the channel is desensitized. Structural rearrangements associated with transitioning from closed to desensitized states include a rigid-body tilt of the S1-S4 domain; formation of a canonical S4-S5 linker; shifts of S5, the pore helix, and S6; stabilization of the outer pore loop; and tilting of the TRP domain, such that it is parallel to the membrane bilayer. This transition is accompanied by introduction of a 310-helix in S4 and a π-helix in both the pore helix and S6.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McKemy DD, Neuhausser WM, Julius D, Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002). - PubMed
    1. Peier AM et al., A TRP Channel that Senses Cold Stimuli and Menthol. Cell 108, 705–715 (2002). - PubMed
    1. Bautista DM et al., The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204–208 (2007). - PubMed
    1. Dhaka A et al., TRPM8 Is Required for Cold Sensation in Mice. Neuron 54, 371–378 (2007). - PubMed
    1. Colburn RW et al., Attenuated Cold Sensitivity in TRPM8 Null Mice. Neuron 54, 379–386 (2007). - PubMed

Publication types