Research Article

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

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Science  01 Mar 2019:
Vol. 363, Issue 6430, eaav9334
DOI: 10.1126/science.aav9334

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Cool mechanism for sensing cool

In humans, cold is primarily sensed by transient receptor potential melastatin member 8 (TRPM8), a calcium channel. Yin et al. present cryo–electron microscopy structures of TRPM8 with cooling agents, membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), and calcium. Structural and functional analyses showed that the PIP2 binding site in TRPM8 is completely different from PIP2 sites in other TRP channels. The binding of PIP2 and cooling agents allosterically enhance each other and activate the channel opening. Thus, the activation mechanism of TRPM8 is distinct from that used by other TRP channels.

Science, this issue p. eaav9334

Structured Abstract


Transient receptor potential melastatin member 8 (TRPM8) is a calcium-permeable ion channel that serves as the principal detector of cold in humans. Natural and synthetic cooling agents, such as menthol and icilin, also activate TRPM8, and this process forms the basis of chemically induced cool sensation in humans (see panel A in the figure). TRPM8 recognizes menthol and icilin by distinct mechanisms, as intracellular Ca2+ is necessary for recognition of icilin but not menthol. Moreover, activation of TRPM8 requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which allosterically increases the potency of the agonists.


Despite extensive functional studies, many questions regarding TRPM8 remain unanswered: (i) How are menthol and icilin recognized by TRPM8, and why is Ca2+ required for icilin binding? (ii) Where is the PIP2 binding site located, and why is it required for TRPM8 activation? (iii) What is the structural basis for the allosteric coupling between PIP2 and cooling agents? The recent ligand-free TRPM8 structure provided a structural context for previously conducted functional studies. However, it could not facilitate answers to questions about the principles of distinct cooling agent recognition, PIP2 binding, and cooling agent-PIP2 coupling in TRPM8.


We present two cryo–electron microscopy structures of an avian TRPM8 channel in complex with the supercooling agonist icilin, Ca2+, and PIP2, and with the menthol analog WS-12 and PIP2, respectively. These structures reveal that the binding pocket for cooling agents is located at the cavity formed by the voltage-sensor like domain (VSLD) and the TRP domain (see panel B in the figure). They illustrate the structural bases for the recognition of menthol and icilin by TRPM8 and explain why Ca2+ is required for icilin binding in TRPM8. These structures and subsequent functional studies unveil the unanticipated location for PIP2 binding at the membrane interfacial cavity established by multiple key subdomains in TRPM8 (see panel B in the figure). Notably, PIP2 can bind to the interfacial cavity in two different modes: partially or fully engaged. Furthermore, structural analyses reveal the molecular basis for the allosteric coupling between PIP2 and cooling agonists in TRPM8. The binding sites for PIP2 and cooling agents are positioned strategically on opposing sides of the transmembrane segment 4 (S4) in the VSLD cavity, from where each triggers a structural rearrangement that favors binding of the other. Binding of cooling agents and PIP2 leads to substantial conformation rearrangements of the VLSD and pore, which culminate in the opening of the Ca2+-permeable pathway of TRPM8 (see panel C in the figure).


In this study, we show that TRPM8 possesses an intricate structural design for sensing cooling agents and lipids. The mechanisms used by TRPM8 are in stark contrast to those observed in the heat and vanilloid receptor TRPV1. First, the TRPM8 agonist-binding site is located at the VSLD cavity, which enables the cooling agents to directly control the TRP domain to open the intracellular gate. Second, PIP2 binding in TRPM8 engages subdomains from both the transmembrane domain and the cytoplasmic domain at an interlayer nexus. Thus, PIP2 facilitates cooling agent sensing allosterically and mediates structural rearrangements during channel gating, which account for the stringent PIP2 dependence in TRPM8 channels.

Structural basis of cooling agent and lipid sensing in the TRPM8 channel.

(A) TRPM8 is a polymodal calcium-permeable channel regulated by various physical and chemical stimuli. (B) Location of the binding site for cooling agents and membrane lipid PIP2. MHR4, melastatin homology region 4. (C) Ligand-induced structural rearrangements suggest allosteric coupling between cooling agents and PIP2. R, Arg; H, His.


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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