LAUSR

The transient receptor potential melastatin 8 (TRPM8) channel is the primary molecular transducer responsible for the cool sensation elicited by menthol and cold in mammals. TRPM8 activation is controlled by cooling compounds together with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our knowledge of cold sensation and the therapeutic potential of TRPM8 for neuroinflammatory diseases and pain will be enhanced by understanding the structural basis of cooling agonist- and PIP2-dependent TRPM8 activation. We present cryo–electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gating pathway. Our results uncover two discrete agonist sites, state-dependent rearrangements in the gate positions, and a disordered-to-ordered transition of the gate-forming S6—elucidating the molecular basis of chemically induced cool sensation in mammals. Description Chilly reception The sensation of cold stimuli in mammals is mediated by transient receptor potential melastatin 8 (TRPM8) channels that also respond to various chemicals (think of the frosty feel of menthol). The molecular basis for channel activation by cooling agonists has not been clear, in part because prior efforts have used suboptimal agonists or avian channels that cannot be fully opened during structural experiments. Using a combination of cooling agents that does not induce desensitization, Yin et al. determined cryo–electron microscopy structures of the mouse channel in an open state, revealing changes in the pore and gate that are consistent with ion conduction and are supported by electrophysiology and molecular dynamics experiments. The lipid phosphotidylinositol 4,5-bisphosphate plays an important role in sensitizing the channel, revealed in part by closed and intermediate states with one or both agonists absent. —MAF An open-state structure reveals how cold sensing channels can be activated by chemicals. INTRODUCTION Mammals sense cold through drops in temperature or by exposure to particular compounds, such as the menthol found in peppermint. The basis for this cold sensation is through activation of the transient receptor potential melastatin member 8 (TRPM8) ion channels. These channels are expressed in sensory neurons and function as the primary transducer for cool sensation in humans. Channel opening can be achieved by either physical or chemical stimuli, but both modes of stimulation require allosteric binding of the membrane signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). RATIONALE Most previous structural studies were limited to avian TRPM8, which exhibits differential thermal and chemical sensitivities compared with mammalian TRPM8 despite high sequence identity. These studies revealed that PIP2 and cooling compounds bind in the transmembrane channel region but the structures all exhibit nonconducting conformations. How exactly agonist- and PIP2-binding to TRPM8 induces channel opening has therefore remained a mystery. The lack of an open state structure—particularly of mammalian TRPM8—has hampered not only our understanding of cold sensing in humans but also therapeutic developments targeting this important sensory receptor. RESULTS We use single particle cryo–electron microscopy to capture snapshots of mouse TRPM8 structures in closed (C0, C1), intermediate (C2), and open (O) states. We open the channel through application of PIP2 and two types of agonists [type I, cryosim-3 (C3) and type II, allyl isothiocyanate (AITC)]. We show that avian and mammalian TRPM8 employ a common conformational path necessary for PIP2- and ligand-activation but differ in the binding affinity for PIP2 and/or the sensitivity to PIP2-induced structural rearrangements. We reveal that the binding sites for PIP2 and agonists are strategically positioned surrounding the transmembrane helix 4b (S4b), which connects other structural elements critical for channel gating. Our structures, electrophysiology, and molecular dynamics analysis reveal that small local structural changes triggered by PIP2 and cooling agonist binding are propagated and amplified as large rearrangements in the pore domain. During the gating transition, the pore undergoes noncanonical conformational changes that lead to the open state: The pore cavity progressively decreases in size, the surface charge electrostatics gradually becomes more electronegative, and the selectivity filter gradually forms. Concomitantly, the pore-forming helix S6 undergoes substantial helical rotation and translation and a C-terminal coil-to-helix transition, which lead to changes in the intracellular S6 gate positions and dynamics. CONCLUSION In this study, we investigated the mechanism of chemically induced cool sensation in mammals by visualizing the conformational landscape of mouse TRPM8 channel gating as it opens. Our study reveals a molecular mechanism for PIP2- and cooling agonist-mediated TRPM8 activation and clarifies the structural basis for the differential PIP2 sensitivities between TRPM8 orthologs. We unveil noncanonical conformational rearrangements in the pore domain accompanied by substantial state-dependent changes at the intracellular gate positions during TRPM8 gating. We speculate that this design could underlie the sensitivity of TRPM8 toward both physical (cold) and chemical (cooling agonist) stimuli. PIP2- and cooling agonist-dependent activation of TRPM8. (A) The homotetrameric mouse TRPM8 channel embedded in the membrane bilayer. (B) Schematic diagrams illustrating the conformational rearrangements in the pore cavity and the intracellular gate position during TRPM8 gating.