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. 2019 Mar 1;363(6430):eaav9334.
doi: 10.1126/science.aav9334. Epub 2019 Feb 7.

Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel

Ying Yin  1 , Son C Le  1 , Allen L Hsu  2 , Mario J Borgnia  1   2 , Huanghe Yang  1 , Seok-Yong Lee  3
Affiliations

Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel

Ying Yin et al. Science. .

Abstract

Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca2+)-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with the menthol analog WS-12 and PIP2 Our structures reveal the binding sites for cooling agonists and PIP2 in TRPM8. Notably, PIP2 binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP2 and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Structures and functions of TRPM8 in complex with PIP2 and cooling compounds.
(A and B) Cryo-EM reconstructions and (C and D) structures of the class 1 icilin-PIP2-Ca2+ complex [(A) and (C)] and the WS-12-PIP2 complex [(B) and (D)], viewed from the membrane plane and from the extracellular side. Ligands are highlighted in surface representations. Gray bars indicate division of channel layers. (E and F) Functional characterization of the TRPM8FA_AG (E) and TRPM8FA (F) channels by icilin, WS-12 and Ca2+. Channel activation was elicited via application of 20 µM icilin or 40 µM WS-12 in the presence or absence of 12.5 µM Ca2+ to the cytoplasmic side of inside-out patches. Time course of the peak current (I) amplitudes at +100 mV from the voltage (V) ramp (−100 mV to +100 mV) is shown on the left and representative current traces are shown on the right. Quantifications of current amplitudes are shown in fig. S2B.
Figure 2.
Figure 2.. Structural basis of icilin recognition and its Ca2+ dependence in TRPM8.
(A and B) Close-up views of the VSLD cavity showing key residues interacting with icilin [(A), blue sticks] and Ca2+ [(B), green sphere]. S2 and the S2-S3 linker are omitted in (A) for clarity. (C and D) Viewed orthogonally to the membrane (C) or from the intracellular side (D), comparison of the conformational changes in the VSLD cavity upon icilin and Ca2+ binding (blue) with the apo TRPM8 structure (yellow, PDB 6BPQ). The icilin molecule (blue) and Ca2+ ion (green) are shown as spheres.
Figure 3.
Figure 3.. The binding site for menthol analog WS-12 in TRPM8.
(A) WS-12 binding in the VSLD cavity (left) and close-up view of the binding site (right). PIP2 (red) and WS-12 (green) molecules are shown as sticks and highlighted with surfaces in the left panel. The S2-S3 linker and S3 are omitted in right panel. (B) Comparison of the VSLD cavity in the apo TRPM8 (yellow), WS-12-PIP2-complex (green), and class 1 icilin-PIP2-Ca2+ complex (blue) structures. Agonists are shown as spheres. The green sphere in the right panel represents Ca2+. Grey surfaces represent the binding pocket shaped by residues lining the VSLD cavity. The S2-S3 linker and S3 are omitted. (C) Representative recordings showing differential channel activation by icilin and WS-12 in TRPM8FA_AG and TRPM8FA_AG with the His844→Ala (H844A) mutation in response to 20 µM icilin or 40 µM WS-12 in the presence of 12.5 µM Ca2+. The membrane was held at +60 mV. (D) Icilin- and WS-12-evoked current ratio from recordings in (C). A two-tailed unpaired Student’s t-test was used for the comparison; ****P < 0.0001. (E) Conductance-voltage (G-V) relationship of TRPM8FA_AG and TRPM8FA_AG H844A in response to 20 µM icilin (left) or 40 µM WS-12 (right) as measured by their normalized peak tail currents (see Methods and fig. S10 for details). Numbers of individual recordings are noted in parentheses. Error bars indicate SEM.
Figure 4.
Figure 4.. PIP2 binding in TRPM8.
A) Functional characterization of TRPM8FA_AG by icilin and PIP2. 20 µM icilin in the presence of 12.5 µM Ca2+ was used to activate TRPM8FA_AG channels using inside-out patches. Membrane PIP2 was depleted by poly-L-lysine (PLL, 50 µg/ml) in the absence of icilin and Ca2+. 40 µM diC8-PIP2 was applied together with icilin and Ca2+ to recover channel activity. Time course of the peak current amplitudes at +100 mV from the voltage ramp (−100 mV to +100 mV) is shown on the left, and representative current traces are shown on the right. Quantifications of current amplitudes are in fig. S2D. (B) Global and close-up views (inset) of the PIP2 binding site in the class 1 icilin-PIP2-Ca2+ complex structure. PIP2 and icilin molecules are shown as sticks and surfaces. Gray bars indicate division of channel layers. (C) Key residues in the interfacial cavity that interact with PIP2. PIP2 and amino acid side chains are shown as sticks. (D) Conductance-voltage (G-V) relationship of K605Q, R688Q, R850Q, and R997Q mutant TRPM8FA_AG channels in response to 20 µM icilin and 12.5 µM Ca2+, as measured by their normalized peak tail currents (see Methods and fig. S11A for details). Numbers of individual recordings are noted in parentheses. Error bars indicate SEM. (E) Comparison of the PIP2 binding site in TRPM8 (left) and the phosphatidylinositol binding site in TRPV1 (right, PDB 5IRZ). Lipid molecules are shown as red sticks.
Figure 5.
Figure 5.. Comparison of PIP2 binding in TRPM8 complex structures.
(A to D) Side-by-side comparison of the PIP2 binding site in the ligand-free TRPM8 [(A), yellow], WS-12-PIP2 complex [(B), green], class 2 icilin-PIP2-Ca2+ complex [(C), purple], and class 1 icilin-PIP2-Ca2+ complex structures [(D), blue]. PIP2 was not modeled in (C) owing to the weak EM density. (E) Overlay of the PIP2 binding site shown in (A) to (D). PIP2 molecules are omitted for clarity. (F and G) Surface representations comparing PIP2 binding in the WS-12-PIP2 complex (F) and in the class 1 icilin-PIP2-Ca2+ complex (G) structures. PIP2 molecules are shown as spheres.
Figure 6.
Figure 6.. Allosteric coupling between agonists and PIP2.
(A) Aligned at TMD, comparison between the ligand-free TRPM8 (yellow) and the class 1 icilin-PIP2-Ca2+ complex (blue) structures shows that the binding of PIP2 and icilin (in sticks and transparent surfaces colored red and blue, respectively) induces secondary structure rearrangements in S4b and the S4-S5 junction. (B) Side-by-side comparison illustrating the allosteric coupling between PIP2 and icilin.
Figure 7.
Figure 7.. Structural rearrangements in response to the binding of PIP2 and agonists.
(A) As viewed from the intracellular side, comparison of the ligand-free TRPM8 (yellow) and the class 1 icilin-PIP2-Ca2+ complex (blue) structures reveals a large rotation of VSLD. (B and C) As viewed from the membrane (B) and from the extracellular side (C), overlay of the TMD in the ligand-free TRPM8 (yellow) and the class 1 icilin-PIP2-Ca2+ complex structure (blue). Arrows indicate the movement of VSLD, PH, and the neighboring S6 (S6’). (D) Close-up views of the region highlighted by the dashed square in (C), comparing differences in the interaction network between VSLD (Trp798 in S3) and the pore (S6’) in the two structures. (E) Comparison of S6 helices and PH in TRPM8 apo (yellow) and complex structures (WS-12, green; class 2 icilin-PIP2-Ca2+ complex, purple; class 1 icilin-PIP2-Ca2+ complex structure, blue). Residues at the narrowest point at the S6 gate are shown in orange spheres with diagonal distances indicated in angstroms. S6 and PH in the class 1 icilin-PIP2-Ca2+ complex structure were modeled as poly-alanine.
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References

    1. Farooqi AA et al., TRPM channels: Same ballpark, different players, and different rules in immunogenetics. Immunogenetics 63, 773–787 (2011). - PubMed
    1. Fleig A, Penner R, The TRPM ion channel subfamily: molecular, biophysical and functional features. Trends Pharmacol Sci 25, 633–639 (2004). - PubMed
    1. McKemy DD, TRPM8: The cold and menthol receptor in TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades, Liedtke WB, Heller S, Eds. (CRC Press/Taylor & Francis, 2007), pp. 177–188. - PubMed
    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

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