RESEARCH ARTICLE SUMMARY◥

STRUCTURAL BIOLOGY

Structural basis of cooling agentand lipid sensing by thecold-activated TRPM8 channelYing Yin, Son C. Le, Allen L. Hsu, Mario J. Borgnia, Huanghe Yang, Seok-Yong Lee*

INTRODUCTION: Transient receptor po-tential melastatin member 8 (TRPM8) is acalcium-permeable ion channel that servesas the principal detector of cold in humans.Natural and synthetic cooling agents, suchas menthol and icilin, also activate TRPM8,and this process forms the basis of chemicallyinduced cool sensation in humans (see panelA in the figure). TRPM8 recognizes mentholand icilin by distinct mechanisms, as in-tracellular Ca2+ is necessary for recognitionof icilin but not menthol. Moreover, activa-tion of TRPM8 requires the membrane lipid

phosphatidylinositol 4,5-bisphosphate (PIP2),which allosterically increases the potency ofthe agonists.

RATIONALE: Despite extensive functionalstudies, many questions regarding TRPM8remain unanswered: (i) How are mentholand icilin recognized by TRPM8, and why isCa2+ required for icilin binding? (ii) Where isthe PIP2 binding site located, and why is itrequired for TRPM8 activation? (iii) What isthe structural basis for the allosteric couplingbetween PIP2 and cooling agents? The recent

ligand-free TRPM8 structure provided a struc-tural context for previously conducted func-tional studies. However, it could not facilitateanswers to questions about the principles ofdistinct cooling agent recognition, PIP2 bind-ing, and cooling agent-PIP2 coupling in TRPM8.

RESULTS: We present two cryo–electron mi-croscopy structures of an avian TRPM8 channelin complex with the supercooling agonist icilin,Ca2+, and PIP2, and with the menthol analogWS-12 and PIP2, respectively. These structuresreveal that the bindingpocket for cooling agentsis 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 rec-ognition of menthol andicilin by TRPM8 and ex-plainwhyCa2+ is requiredfor icilinbinding inTRPM8.These structures and sub-sequent functional studies

unveil the unanticipated location for PIP2binding at the membrane interfacial cavityestablished by multiple key subdomains inTRPM8 (see panel B in the figure). Notably,PIP2 can bind to the interfacial cavity in twodifferent modes: partially or fully engaged.Furthermore, structural analyses reveal themolecular basis for the allosteric coupling be-tween PIP2 and cooling agonists in TRPM8.The binding sites for PIP2 and cooling agentsare positioned strategically on opposing sidesof the transmembrane segment 4 (S4) in theVSLD cavity, fromwhere each triggers a struc-tural rearrangement that favors binding of theother. Binding of cooling agents and PIP2 leadsto substantial conformation rearrangementsof the VLSD and pore, which culminate inthe opening of the Ca2+- permeable pathwayof TRPM8 (see panel C in the figure).

CONCLUSION: In this study, we show thatTRPM8 possesses an intricate structural de-sign for sensing cooling agents and lipids. Themechanisms used by TRPM8 are in stark con-trast to those observed in the heat and vanilloidreceptor TRPV1. First, the TRPM8 agonist-binding site is located at the VSLD cavity,which enables the cooling agents to directlycontrol the TRP domain to open the intra-cellular gate. Second, PIP2 binding in TRPM8engages subdomains from both the trans-membranedomain and the cytoplasmic domainat an interlayer nexus. Thus, PIP2 facilitatescooling agent sensing allosterically andmediatesstructural rearrangements during channel gating,which account for the stringent PIP2 dependencein TRPM8 channels.▪

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Yin et al., Science 363, 945 (2019) 1 March 2019 1 of 1

The list of author affiliations is available in the full article online.*Corresponding author. Email: seok-yong.lee@duke.eduCite this article as Y. Yin et al., Science 363, eaav9334 (2019).DOI: 10.1126/science.aav9334

R850 TRP

S4aS5

H844

R841

R997

TRP

S4aS5

R841

H844

R997

R850

S4b

Icilin

Ca 2+

PI(4,5)P2

S4b

TRPMHR4’

MHR4

pre-S1

PI(4,5)P 2

VSLD

S1 S2 S3

S4

Icilin

A B

C

ColdCooling

agent

Lipid

Ca 2+

Ca 2+

Structural basis of cooling agent and lipid sensing in the TRPM8 channel. (A) TRPM8is a polymodal calcium-permeable channel regulated by various physical and chemicalstimuli. (B) Location of the binding site for cooling agents and membrane lipid PIP2. MHR4,melastatin homology region 4. (C) Ligand-induced structural rearrangements suggestallosteric coupling between cooling agents and PIP2. R, Arg; H, His.

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RESEARCH ARTICLE◥

STRUCTURAL BIOLOGY

Structural basis of cooling agentand lipid sensing by thecold-activated TRPM8 channelYing Yin1, Son C. Le1, Allen L. Hsu2, Mario J. Borgnia1,2,Huanghe Yang1, Seok-Yong Lee1*

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 andmembrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lackof structural informationhas thus far precluded amechanistic understanding of ligand and lipid sensing by TRPM8.Usingcryo–electron microscopy, we determined the structures of TRPM8 in complex with thesynthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with thementhol analogWS-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 ofallosteric coupling between PIP2 and agonists.This study provides a platform for understandingthe molecular mechanism of TRPM8 activation by cooling agents.

The transient receptor potential melastatin(TRPM) family, part of the TRP channelsuperfamily, is composed of eightmembers(TRPM1 to TRPM8) and is involved invarious processes such as temperature

and redox sensing (1–3). Both in vivo and in vitrostudies have shown that TRPM8 acts as a coldandmenthol receptor (4–8). In addition, TRPM8plays crucial roles in cold-related pain and mi-graine (9, 10) and mediates menthol-inducedanalgesia of acute and inflammatory pain (11).Therefore, TRPM8 is a therapeutic target fortreatments of cold allodynia, chronic pain, andmigraine (12–15).As a polymodal sensor, TRPM8 gating is

shaped bymultiple physicochemical stimuli. Thechannel is activated by exposure to cold temper-atures or menthol and is allosterically modu-lated by phosphatidylinositol 4,5-bisphosphate(PIP2) and Ca

2+. TRPM8 activation requires PIP2;at high concentrations, PIP2 alone appears to besufficient for channel activation, whereas itsdepletion desensitizes the channel (16–19). Fur-thermore, binding of PIP2 and cooling com-pounds is allosterically coupled, as binding ofone increases the potency of the other (17, 20).Although intracellular Ca2+ is required for ac-tivation of several TRPM channels (21, 22), it isnot necessary for cold- or menthol-dependentTRPM8 activation (4, 23).

Because of the physiological and therapeuticimportance of TRPM8, many studies have fo-cused on dissecting the mechanisms by whichcooling agents bind and gate the channel.(12, 13, 18, 24–28). Despite their apparent over-lapping binding sites in TRPM8 (24), the naturalcooling compound menthol and the syntheticcooling compound icilin activate the channelvia distinct mechanisms (26). First, Ca2+ is re-quired for TRPM8 activation by icilin but notmenthol (23). Second, intracellular pH does notaffect menthol-dependent TRPM8 gating butdoesmodulate the effect of icilin on the channel(29). Third, although menthol is a commonagonist for all known orthologs of TRPM8, icilindoes not activate avian TRPM8 channels, whichrequire a mutation of Ala to Gly (Ala805→Gly)on the transmembrane segment 3 (S3) to inducesensitivity to icilin (23).The recent cryo–electron microscopy (cryo-

EM) structure of the ligand-free TRPM8 offeredthe first opportunity to place residues previouslyidentified as important for agonist binding in astructural context (30). However, this structurecould not elucidate the specific mechanisms bywhich natural and synthetic cooling agents bindto and activate the channel or how PIP2 allo-sterically affects their potency. To answer thesequestions, we determined two cryo-EM struc-tures of TRPM8: one in complex with icilin, PIP2,and Ca2+; the other in complex with the mentholanalog WS-12 and PIP2 (Fig. 1, A to D). Ourstructural and functional studies identify a dis-tinct binding site for PIP2 and unveil the struc-tural basis formenthol, icilin, and Ca2+ recognitionby TRPM8. Moreover, structural analyses provideinsights into the allosteric coupling between lipid

and agonists and illuminate the principles ofligand-dependent gating in TRPM8.

Structure determination of TRPM8 incomplex with PIP2 and cooling agents

The previously developed TRPM8 constructfrom the collared flycatcher Ficedula albicollis(TRPM8FA) was used to determine the structureof TRPM8 in complex with the menthol analogWS-12 and PIP2 (fig. S1 andmethods) (30). WS-12was chosen because of its higher potency andshared activation mechanism with menthol (31)(fig. S2A). To confer icilin sensitivity to TRPM8FA,we introduced an Ala805→Gly mutation (23) andused the construct (TRPM8FA_AG) for determiningthe structure of TRPM8 in complex with icilin,Ca2+, and PIP2. When applied to the inside-outpatches of human embryonic kidney (HEK)293 cells expressing TRPM8FA_AG, icilin elicitsrobust currents in the presence of Ca2+ (Fig.1E and fig. S2B). TRPM8FA_AG shows a Ca2+-dependent desensitization at high concentrationsof Ca2+ (fig. S3, A to C), similar to mammalianTRPM8 channels (4). TRPM8FA also exhibits icilin-evoked currents but to a much lesser extent thanTRPM8FA_AG, consistent with previous data (23)(Fig. 1F and fig. S2B). In contrast to icilin, WS-12elicits large outward-rectifying currents in bothTRPM8FA and TRPM8FA_AG independent ofintracellular Ca2+ (Fig. 1, E and F, and fig. S2B).Icilin shows apparently higher efficacy and po-tency thanWS-12 in TRPM8FA_AG activation (Fig.1E and fig. S2B).TRPM8FA_AG or TRPM8FA channels were pu-

rified in digitonin and incubated with short-chain PIP2 (diC8-PIP2), icilin, and Ca2+, or withdiC8-PIP2 and WS-12, respectively. The complexstructures were determined by cryo-EM (seemethods and figs. S4 and S5). The final three-dimensional (3D) reconstructions of the icilin-PIP2-Ca

2+-TRPM8FA_AG complex (henceforth, theicilin-PIP2-Ca

2+ complex) yielded two 3D classes:Themajor class (~100,000 particles), containinga strong density for icilin, PIP2, and Ca2+, was re-solved to ~3.4 Å (referred to as the “class 1 re-construction”), and the minor class (~9000particles), containing weak or no density forthe ligands, was resolved to ~4.3 Å (referred to asthe “class 2 reconstruction”) (Fig. 1A and figs. S4and S6). The final 3D reconstruction of the WS-12–PIP2–TRPM8FA complex (henceforth, theWS-12–PIP2 complex) was resolved to ~4 Å (Fig. 1Band fig. S5). The overall quality of the 3D recon-structions in the transmembrane region (fig. S7)was improved compared with that of the ligand-free TRPM8, but the transmembrane helicalsegment 6 (S6) and the pore helix (PH) are notwell resolved in the class 1 reconstruction ofthe icilin-PIP2-Ca

2+ complex. Therefore, theseregions were built as poly-alanine chains (fig.S4 and table S1).The overall structure of the homotetrameric

TRPM8 complex can be divided into three layers,in which the top layer comprises the trans-membrane domain (TMD) and the bottom twolayers form the cytoplasmic domain (CD) (Fig. 1,C and D). Each subunit contains an N-terminal

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1Department of Biochemistry, Duke University School ofMedicine, Durham, NC, 27710, USA. 2Genome Integrity andStructural Biology Laboratory, National Institute ofEnvironmental Health Sciences, National Institutes of Health,Department of Health and Human Services, ResearchTriangle Park, NC 27709, USA.*Corresponding author. Email: seok-yong.lee@duke.edu

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region composed of melastatin homology regions1 to 4 (MHR1 toMHR4), a transmembrane chan-nel region, and a C-terminal region (fig. S2, E andF). The TMD is composed of the voltage-sensor–like domain (VSLD) (S1 to S4), with the poredomain (S5, S6, and PH) arranged in a domain-swapped manner. The CD is composed of theN-terminal and C-terminal regions assembledinto the bottom two layers.

Icilin binding site within VSLD

In the class 1 reconstruction of the icilin-PIP2-Ca2+ complex, we observed a strong EM densitylocated in the cavity between the VSLD and theTRP domain (the VSLD cavity). Because theelongated density matches the size and shapeof an icilin molecule (Fig. 2A) and is presentin the half-map reconstructions (fig. S6), weassigned this EM density to icilin. From the

possible assignments of icilin (fig. S6E), we se-lected the one with the better fit to the densityand fewer steric clashes. Within the VSLD cavity,the icilin molecule is surrounded by aromaticresidues, including Tyr745 in S1 and Tyr1004 in theTRP domain, both of which have previously beenproposed to be important for menthol and icilinbinding (24). In this structure, we observed twoadditional residues in the C-terminal part of S4(termed S4b) that establish prominent side-chaininteractions with icilin. The side chains of Arg841,positioned underneath the central pyrimidinemoiety of icilin, and His844, which points towardthe center of the VSLD cavity, interact with icilin.Notably, S4b adopts a 310-helical conformation,in contrast to the a-helical conformation observedin the ligand-free TRPM8FA and the class 2 (low-occupancy) icilin-PIP2-Ca

2+ complex structures(fig. S8). The transition from a- to 310-helical

conformation results in a register shift in S4b,which enables the interactions of Arg841 andHis844 with icilin.Icilin-mediated activation of TRPM8 has been

shown todependonCa2+ (23). In the class 1 recon-struction, we identified a strong EM density peakbetween S2 and S3 and assigned it as Ca2+ on thebasis of its presence in both half-maps (fig. S6)and the analogous Ca2+ location identified inother TRPM channels (32, 33). Previous studieshave identified Asn799, Asp802, and Gly805 inTRPM8 as important for icilin-mediated activa-tion (23). Instead of directly participating in icilinbinding, Asn799 and Asp802 in S3, along withGlu782 and Gln785 in S2, participate in Ca2+ co-ordination (Fig. 2B). Despite its proximity to icilinin the VSLD cavity, Ca2+ does not directly interactwith the agonist, suggesting that binding of Ca2+

may induce a conformational change that primes

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 2 of 9

Fig. 1. Structures and functions of TRPM8 in complex with PIP2 andcooling compounds. (A and B) Cryo-EM reconstructions and (C andD) structures of the class 1 icilin-PIP2-Ca

2+ complex [(A) and (C)] andthe WS-12–PIP2 complex [(B) and (D)], viewed from the membrane planeand from the extracellular side. Ligands are highlighted in surfacerepresentations. Gray bars indicate division of channel layers. (E andF) Functional characterization of the TRPM8FA_AG (E) and TRPM8FA

(F) channels by icilin,WS-12, and Ca2+. Channel activation was elicited viaapplication of 20 mM icilin or 40 mM WS-12 in the presence or absence of12.5 mM Ca2+ to the cytoplasmic side of inside-out patches. Time courseof 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 currenttraces are shown on the right. Quantifications of current amplitudes areshown in fig. S2B.

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the site for icilin binding. In the ligand-freeTRPM8 structure, Asp802 in S3 and Arg841 in S4apparently form a salt bridge and occupy theposition where the central pyrimidine ring oficilin is located in the icilin-PIP2-Ca

2+ complexstructure. Binding of Ca2+ is associated with aslight rotation of the N-terminal part of S3, al-lowingAsn799 andAsp802 in S3 to coordinate Ca2+.This moves the Asp802 side chain away fromArg841, breaking the salt bridge; as a result, theVSLD cavity widens to accommodate icilin, and

Arg841 is positionedbelow icilin’s central pyrimidinemoiety (Fig. 2, C and D). The residue at position805,which accounts for the differential sensitivityto icilin in mammalian and avian orthologs (23),is located one helical turn above Asp802 in S3,facing the hydroxyphenyl moiety of icilin (Fig. 2,C and D). We propose that the Ala805→Gly sub-stitution confers icilin sensitivity by (i) providingflexibility for rotation of the N-terminal partof S3, which contains Asp802 and Asn799, thusleading to the assembly of the Ca2+ binding site,

and (ii) enlarging the VSLD cavity to accommo-date the hydroxyphenyl moiety of icilin (Fig. 2C).

The binding site for the mentholanalog WS-12

We identified a strong, asymmetric, dumbbell-shaped EMdensity within the VSLD cavity of theWS-12–PIP2 complex 3D reconstruction that ispresent in both half-maps (Fig. 3A and fig. S9).We assigned this density to WS-12 because itssize and shape are consistent with those of a

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 3 of 9

Fig. 2. Structural basisof icilin recognitionand its Ca2+

dependence inTRPM8. (A andB) Close-up viewsof the VSLD cavity,showing key residuesinteracting with icilin[(A), blue sticks] andCa2+ [(B), greensphere]. S2 and theS2-S3 linker are omittedin (A) for clarity. (C andD) Viewed orthogonallyto the membrane (C) orfrom the intracellularside (D), comparison ofthe conformationalchanges in the VSLDcavity upon icilin andCa2+ binding (blue) withthe apo TRPM8 struc-ture (yellow, PDB6BPQ). The icilin mole-cule (blue) and Ca2+ ion(green) are shown asspheres. Single-letterabbreviations for theamino acid residues areas follows: A, Ala;C, Cys; D, Asp; E, Glu;F, Phe; G, Gly; H, His;I, Ile; K, Lys; L, Leu;M, Met; N, Asn;P, Pro; Q, Gln; R, Arg;S, Ser; T, Thr; V, Val;W, Trp; and Y, Tyr.

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WS-12 molecule. The density is sandwichedbetween Tyr745 in S1 and Tyr1004 in the TRP do-main, with the menthol-like moiety and themethoxyphenyl ring near Tyr745 and Tyr1004,respectively (Fig. 3A). Side chains of Arg841 inS4 and Tyr1004 and Arg1007 in the TRP domainarewithin the interacting distances of the centralamide bond in WS-12. This structure is con-sistent with previous functional studies; Tyr745,Tyr1005 (Tyr1004 in TRPM8FA), and Arg842 (Arg841

in TRPM8FA) have all been identified as impor-tant for menthol sensing (18, 24). Because WS-12is composed of a menthol-like moiety linked to amethoxyphenyl group via a central amide bond,we anticipate that menthol binding to TRPM8will be similar to that of the menthol-like moietyin WS-12 in our structure.

Comparison of the VSLD cavities from ligand-free, WS-12–bound, and icilin-bound TRPM8structures shows that residues lining the bindingpocket within the cavity, including Glu782, Arg841,His844, Tyr1004, and Arg1007, can adopt multipleconformations, enabling the cavity to fit struc-turally distinct agonist molecules (Fig. 3B). No-tably, His844 does not interact with WS-12 butdoes with icilin. We found that introduction oftheHis844→Alamutation toTRPM8FA_AG stronglysuppresses icilin-dependent activation (Fig. 3,C and D). Whereas the His844→Ala mutationprofoundly right-shifts the conductance-voltage(G-V) curve in response to icilin, it exerts noeffect on TRPM8 activation by WS-12 (Fig. 3Eand fig. S10). Previous studies have shown thatmenthol-dependent TRPM8 activation is not sen-

sitive to intracellular pH, but intracellular pHdoes affect icilin-dependent activation (29). Onthe basis of our studies, we posit that the differ-ential contribution of His844 to icilin and WS-12binding may be the cause of the differencesin pH sensitivity between menthol and icilinactivation.

Distinct location of the PIP2 binding sitein TRPM8

TRPM8FA andTRPM8FA_AG possess a pronouncedPIP2 dependence, as depletion by poly-L-lysine(34) strongly induces channel desensitization,which is rapidly reversed by exogenous appli-cation of diC8-PIP2 (Fig. 4A and fig. S2, C andD), similar to mammalian TRPM8 channels(16, 17, 19). In our class 1 reconstruction of the

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 4 of 9

Fig. 3. The binding site for menthol analogWS-12 in TRPM8. (A) WS-12 binding inthe VSLD cavity (left) and close-up view of thebinding site (right). PIP2 (red) and WS-12 (green)molecules are shown as sticks and highlightedwith surfaces in the left panel. The S2-S3 linkerand S3 are omitted in the right panel.(B) Comparison of the VSLD cavity in theapo TRPM8 (yellow),WS-12–PIP2 complex (green),and class 1 icilin-PIP2-Ca

2+ complex (blue)structures. Agonists are shown as spheres.The green sphere in the right panel representsCa2+. Gray surfaces represent the binding pocketshaped by residues lining the VSLD cavity. TheS2-S3 linker and S3 are omitted. (C) Representa-tive recordings showing differential channelactivation by icilin and WS-12 in TRPM8FA_AG andTRPM8FA_AG with the His844→Ala (H844A)mutation in response to 20 mM icilin or 40 mMWS-12 in the presence of 12.5 mM Ca2+. Themembrane was held at +60 mV. (D) Icilin- andWS-12–evoked current ratio from recordingsin (C). A two-tailed unpaired Student’s t test wasused for the comparison; ****P < 0.0001.(E) Conductance-voltage (G-V) relationship ofTRPM8FA_AG and TRPM8FA_AG H844A in responseto 20 mM icilin (left) or 40 mM WS-12 (right), asmeasured by their normalized peak tail currents(see methods and fig. S10 for details). Numbers ofindividual recordings are noted in parentheses.Error bars indicate SEM.

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icilin-PIP2-Ca2+ complex, we observed a strong

nonprotein density located at the interface be-tween the TMD and the top layer of the CD ring(Fig. 4B). We assigned this density to PIP2, whichis present in both half-maps (fig. S6), because theshape of the density is consistent with PIP2 andis absent in the reconstruction of the ligand-freeTRPM8FA (30). The inositol 1,4,5-trisphosphatehead group of the PIP2 molecule is positioned inthe interfacial region (the interfacial cavity) formedby the pre-S1 domain, the junction between S4 andS5, the TRP domain, and the MHR4 from theadjacent subunit (Fig. 4B), with the acyl chainsextended upward into the putative membraneregion. Our structure reveals that basic aminoacid residues from different subdomains—Lys605

from the neighboring MHR4 domain (MHR4′),Arg688 from the pre-S1 domain, Arg850 from thejunction between S4 and S5, and Arg997 from theTRP domain—cluster at the interfacial cavity andinteract with PIP2. Specifically, Lys

605 and Arg850

interact with both the C-4 and the C-5 phosphategroups, whereas Arg688 and Arg997 form electro-static interactions with the C-4 phosphate moietyof PIP2 (Fig. 4C). Among these residues, onlyArg997 has previously been implicated in PIP2binding (17). Consistent with our structure, neu-tralizing mutations of residues Lys605, Arg850,and Arg997 via Gln substitutions impaired chan-nel activation, as evidenced by the rightwardshift in theG-V curves (Fig. 4D and fig. S11, A andB). As PIP2 binding and membrane depolariza-

tion have been demonstrated to be coupled forchannel gating (17), the large increase in thevoltage required for channel opening in thesePIP2 binding mutations is consistent with re-duced binding of PIP2. Therefore, our structuraldata in combination with the functional studiesidentify the PIP2 binding site in TRPM8.Our TRPM8 complex structure reveals an un-

foreseen binding site for PIP2 previously notobserved in other TRP channels (Fig. 4E andfig. S12). Phosphatidylinositol lipids have beenshown to bind to a cleft formed by S3, S4, andthe S4-S5 linker in TRPV1 (Fig. 4E) and TRPV5(35, 36). In marked contrast, PIP2 binds to theTRPM8 channel on the opposite side of S4and S5, facing the interfacial cavity formed

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 5 of 9

Fig. 4. PIP2 binding in TRPM8. (A) Functional characterization ofTRPM8FA_AG by icilin and PIP2. 20 mM icilin in the presence of 12.5 mM Ca2+

was used to activate TRPM8FA_AG channels using inside-out patches.Membrane PIP2 was depleted by poly-L-lysine (PLL, 50 mg/ml) in the absenceof icilin and Ca2+. 40 mM 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 theleft, and representative current traces are shown on the right. Quantificationsof current amplitudes are in fig. S2D. (B) Global and close-up views (inset)of the PIP2 binding site in the class 1 icilin-PIP2-Ca

2+ complex structure. PIP2

and icilin molecules are shown as sticks and surfaces. Gray bars indicatedivision of channel layers. (C) Key residues in the interfacial cavity thatinteract 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 mM icilin and12.5 mM Ca2+, as measured by their normalized peak tail currents (seemethods and fig. S11A for details). Numbers of individual recordings arenoted in parentheses. Error bars indicate SEM. (E) Comparison of the PIP2

binding site in TRPM8 (left) and the phosphatidylinositol binding site inTRPV1 (right, PDB 5IRZ). Lipid molecules are shown as red sticks.

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by multiple subdomains, including the pre-S1 andMHR4 domains, both of which are absent inTRPV channels. Our comparison of TRPM8 withthe recently published TRPM4 and TRPM2 struc-tures (33, 37) suggests that the observed PIP2site may not be conserved throughout TRPMchannels. In TRPM8, the pre-S1 domain pre-ceding the TMD makes extensive interactionswith the MHR4 of the CD at the membrane in-terface, which forms a large part of the PIP2binding site (fig. S12A). By contrast, these inter-actions are absent in TRPM4 and TRPM2, andthe amino acids involved in PIP2 binding are notconserved in these channels (fig. S12, B and C).Consistent with this structural observation, mu-tation of the PIP2-interacting Lys

605 in MHR4 toGln severely impairs the TRPM8 channel open-ing by icilin and WS-12 (Fig. 4D and fig. S11).Notably, among mutations of the PIP2 bind-ing residues, Lys605→Gln exhibits the largestrightward shifts in the G-V curves, further in-dicating the importance of the PIP2-mediatedinteractions between CD and TMD for TRPM8channel gating.

Two conformations of the interfacialcavity capture distinct PIP2

binding modesNotably, structures of the ligand-free TRPM8FA,the WS-12–PIP2 complex, and the class 2 low-occupancy icilin-PIP2-Ca

2+ complex possess sim-ilar interfacial cavity conformations, which arewider than that of the class 1 icilin-PIP2-Ca

2+

complex structure (Fig. 5, A to E). The PIP2density is also present in the 3D reconstructionof the WS-12–PIP2 complex (fig. S9). However,owing to the wider interfacial cavity in the WS-12–PIP2 complex structure, PIP2 fits less snuglyin its binding site than it does in the class 1 icilin-PIP2-Ca

2+ complex structure (Fig. 5, F and G).Structural analyses show that the difference inthe size of the interfacial cavity stems from sec-ondary structure rearrangements in S4b. In thestructures of ligand-free TRPM8FA, the WS-12–PIP2 complex, and the class 2 icilin-PIP2-Ca

2+

complex, the S4 is entirely a-helical, which posi-tions Arg850 away from the PIP2 binding cavity.By contrast, in the class 1 high-occupancy icilin-PIP2-Ca

2+ complex, S4b adopts a 310-helical con-

formation, which leads to a register change andrepositions Arg850 toward the PIP2 binding cav-ity. This secondary structure change is accom-panied by an upward tilt of the TRP domain andmovement of S5 toward the interfacial cavity.Therefore, two distinct arrangements of the inter-facial cavity provide different PIP2 bindingmodes(Fig. 5).We suggest that the class 1 icilin-PIP2-Ca

2+

complex represents a conformation of TRPM8with fully engaged PIP2 because (i) Arg

850 formsadditional interactions with PIP2, (ii) PIP2 fitsmore snugly in the binding pocket, and (iii) theligand-free and the class 2 low-occupancy icilin-PIP2-Ca

2+ structures adopt a distinct, wider inter-facial cavity conformation.

Allosteric coupling between agonistsand PIP2

Our studies illustrate a structural basis of cou-pling between PIP2 and cooling agonists, espe-cially icilin (Fig. 6). In the class 1 icilin-PIP2-Ca

2+

complex structure, PIP2 bindsmore optimally tothe interfacial cavity because S4b adopts a 310-helical configuration, S5 is bent, and the TRP

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 6 of 9

Fig. 5. Comparison of PIP2 binding in TRPM8 complex structures. (A to D) Side-by-side comparison of the PIP2 binding site in the ligand-freeTRPM8 [(A), yellow], WS-12–PIP2 complex [(B), green], class 2 icilin-PIP2-Ca

2+ 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 moleculesare 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-Ca

2+

complex (G) structures. PIP2 molecules are shown as spheres.

RESEARCH | RESEARCH ARTICLE

domain moves closer to PIP2 (Figs. 2A and 6A).Likewise, icilin binding prefers a 310 configura-tion of S4b in which Arg841 and His844 rotatetoward the center of the VSLD cavity. Therefore,by binding to a different region of S4b, PIP2 en-hances the conformational change to accommo-date icilin binding; conversely, icilin binding toS4b and the TRP domain induces the structuralrearrangements favorable for PIP2 binding. Bycontrast, PIP2 is not fully engaged in the WS-12–PIP2 complex structure (Fig. 5F). We postulatethat allosteric coupling of WS-12 with PIP2 likelyproceeds via a similar mechanism, indicatingthat theWS-12–PIP2 complex structure representsa state in which the allosteric coupling betweenPIP2 and agonist is not yet established. The dif-ference in the conformations of the interfacialcavity between theWS-12–PIP2 complex and theclass 1 icilin-PIP2-Ca

2+ complex likely originatesfrom the higher potency and efficacy of icilincompared with WS-12 (Figs. 1E and 4D andfig. S11G).

Structural rearrangement in the poreupon ligand binding

In TRPM8, the binding of PIP2 along with icilinand Ca2+ or WS-12 triggers global conformation-

al changes in the TMD that are propagated to theCD (fig. S13A). In contrast to the VSLD of TRPV1,which remains stationary during channel activa-tion (35), the binding of cooling agonists andPIP2 to TRPM8 results in a rigid body rotation ofthe VSLD away from the pore domain as well asconformational changes in PH and S6 (Fig. 7, Aand B, and fig. S13, B and C). Compared to theapo structure, the ligand-induced conformationalchanges appeared to be most pronounced in theclass 1 icilin-PIP2-Ca

2+ complex, so we focus ouranalysis on the structural rearrangements in-duced by icilin, Ca2+, and PIP2. In the ligand-free TRPM8 structure, all transmembrane helicesare straight and a-helical (30). S6 in the poredomain is restrained by its apparently tight in-teractions with VSLD (Trp798 in S3); thus, thepore is locked in a closed conformation (Fig. 7, Cand D). In contrast, icilin, Ca2+, and PIP2 bindingin the VSLD domain triggers an a- to 310-helicaltransition in S4b (Fig. 2C), bending of S5 [analo-gous to the S4-S5 linker bend in other TRPchannels (35, 38)] (Fig. 7B), andmovement of theTRP domain. These rearrangements generatemore-extensive interactions between the TRPdomain and S5 (fig. S13E) and, more impor-tantly, disrupt the interactions between VSLD

(Trp798 in S3) and S6, thereby enabling the re-arrangements of S6 and the PH for channelgating (Fig. 7, C and D). As a result, substantialtilt and bending occur in the pore helix and S6in the class 1 icilin-PIP2-Ca

2+ complex structure;both S6 and PH in the icilin- and Ca2+-boundTRPM8 display curved conformations comparedwith the ligand-free TRPM8 structure, suggest-ing a potential presence of p-helical turns inthese helices (Fig. 7E). The curved S6 in the icilin-PIP2-Ca

2+–bound structure is reminiscent of thep helix containing S6 of the sensitized but non-conductive conformation of the human TRPV3(39), and similar PH arrangements due to bend-ing in S5 have been observed upon resiniferatoxinbinding in TRPV2 (38). It is noteworthy that bothPH and S6 in the class 1 reconstruction are poorlyresolved whereas robust density is present for S5,indicating the mobile nature of these regions inthe icilin-PIP2-Ca

2+ complex. By contrast, in boththe class 2 icilin-PIP2-Ca

2+ complex and WS-12–PIP2 complex structures, slight rotation of theVSLD and slight widening of the S6 gate wereobserved, but S5 maintains a straight conforma-tion (Fig. 7E and fig. S13, B and C).Because the open probability of the channel at

0 mV is low (~0.2 to 0.5), even in the saturating

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 7 of 9

Fig. 6. Allosteric couplingbetween agonists and PIP2.(A) Aligned at TMD, comparisonbetween the ligand-free TRPM8(yellow) and the class 1 icilin-PIP2-Ca2+ complex (blue) structuresshows that the binding of PIP2 andicilin (in sticks and transparentsurfaces colored red and blue,respectively) induces secondarystructure rearrangements in S4band the S4-S5 junction. (B) Side-by-side comparison illustrating theallosteric coupling between PIP2

and icilin.

RESEARCH | RESEARCH ARTICLE

concentrations of ligands, and is further low-ered by high concentrations of Ca2+ (Fig. 4D andfigs. S3, A to C, and S11G), multiple conforma-tional states exist in the cryo-EM samples. Inspite of the considerable conformational differ-ences with respect to the published ligand-freeTRPM8FA structure, both the WS-12–PIP2–boundand icilin-PIP2-Ca

2+–bound complex structuresapparently adopt nonconducting states, suggest-ing that our cryo-EM reconstructions have cap-tured conformations that either precede or followthe open state (Fig. 7E). We speculate that theclass 1 icilin-PIP2-Ca

2+ complex structure reflectsa sensitized state of the channel because PIP2 isfully engaged in the interfacial cavity, S5 formsmore extensive interactions with the TRP do-main (fig. S13E), and the bent S6 conformationis analogous to that of TRPV3 in its sensitized-but-closed state (39). However, it is also possiblethat our class 1 icilin-PIP2-Ca

2+ complex repre-sents a desensitized state that could be popu-lated by a high concentration of Ca2+ introduced

in the cryo-EM sample. By contrast, we specu-late that the WS-12–PIP2 complex structureadopts a presensitized state because PIP2 isnot fully engaged in the interfacial cavity andno substantial conformational changes in S5are observed when compared with the ligand-free TRPM8FA. Although WS-12 and icilin do notbind to TRPM8 in the same manner, we pos-tulate that they might use similar downstreammechanisms of channel activation once PIP2 isfully engaged, and thus the channel is sensitized.However, we cannot rule out the possibility thatWS-12 and icilin have distinct mechanisms ofactivation.

Discussion

Our structural analyses revealed that TRPM8adopts a sophisticated design principle, and itsmechanisms of coupled agonist and PIP2 sensingstand in stark contrast to those of TRPV1. First,unlike TRPV1 and other TRP channels (33, 35),the natural ligand-binding site is located within

the VSLD cavity in TRPM8 channel. Ligand bind-ing in the VSLD cavity offers an opportunity todirectly engage and reposition the TRP domainto open the S6 gate. Moreover, the VSLD cavityin TRPM8 can adjust its shape to accommodatecooling compounds that are chemically distinct.This conformational flexibility of the VSLD cavityholds potential for the development of analgesiccompounds to target TRPM8. Second, the PIP2binding site in TRPM8 is strategically positionedin the interfacial cavity at the nexus of key sub-domains, including the VSLD. Therefore, PIP2 caneffectively control conformational transitionsassociated with gating and enhance agonistbinding. By contrast, in TRPV1, the location ofphosphatidylinositol lipids overlaps the agonist-binding pocket, enabling phosphatidylinositollipids to serve as competitive vanilloid antago-nists (35). Therefore, we propose that the designof distinct but nearby binding sites for agonistand PIP2 in TRPM8 functions to exploit theirstructural allostery.

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 8 of 9

Fig. 7. Structural rearrangements in response to the binding of PIP2

and agonists. (A) As viewed from the intracellular side, a comparisonof the ligand-free TRPM8 (yellow) and the class 1 icilin-PIP2-Ca

2+ complex(blue) structures reveals a large rotation of VSLD. (B and C) As viewedfrom the membrane (B) and from the extracellular side (C), overlay of theTMD in the ligand-free TRPM8 (yellow) and the class 1 icilin-PIP2-Ca

2+

complex structure (blue). Arrows indicate the movement of VSLD, PH, andthe neighboring S6 (S6′). (D) Close-up views of the region highlighted by

the dashed square in (C), comparing differences in the interaction networkbetween VSLD (Trp798 in S3) and the pore (S6′) in the two structures.(E) Comparison of S6 helices and PH in TRPM8 apo (yellow) and complexstructures (WS-12, green; class 2 icilin-PIP2-Ca

2+ complex, purple; class1 icilin-PIP2-Ca

2+ complex structure, blue). Residues at the narrowest pointat the S6 gate are shown in orange spheres, with diagonal distancesindicated in angstroms. S6 and PH in the class 1 icilin-PIP2-Ca

2+ complexstructure were modeled as poly-alanine.

RESEARCH | RESEARCH ARTICLE

Members of the TRPM family have shown dif-ferent levels of PIP2 dependence on channelactivation (40). Our structural analyses indicatethat the PIP2 site in TRPM8 is likely distinct fromthat in TRPM2 and TRPM4, as the latter twochannels do not have the same quaternary struc-ture arrangement at the interface between TMDand CD as TRPM8. It is tempting to speculatethat this quaternary structure arrangement inTRPM8, which enables PIP2 binding and facili-tates the conformational change required formenthol binding, is key to understanding the lackofmenthol sensitivity in other TRPMchannels (41).

Materials and methods summary

TRPM8FA and TRPM8FA_AG channels were ex-pressed in HEK293S GnTI− cells and purifiedas described previously (30), with slight mod-ifications. For cryo-EM study, purified TRPM8FAwas incubated with WS-12 and PIP2, whereasTRPM8FA_AGwas incubatedwith icilin, PIP2, andCa2+ before vitrification. Data were collected on aTitan Krios electron microscope and processedwith RELION 3.0 using standard procedures.Point mutations were introduced at the bindingsite for PIP2, icilin, andWS-12, and the effects ofthe mutations were examined by inside-out patchelectrophysiology.

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ACKNOWLEDGMENTS

Cryo-EM data were collected at the Shared MaterialsInstrumentation Facility at Duke University as part of theMolecular Microscopy Consortium. We thank G. Landerand M. Wu at TSRI for help with preliminary studies andcryo-EM training for Y.Y. Cryo-EM image quality was monitoredon the fly during data collection using routines developedby A. Bartesaghi. We thank L. Zubcevic and W. Borschel forcritical manuscript reading and K. Yokoyama for help with icilinmodeling. Funding: This work was supported by the NationalInstitutes of Health (R35NS097241 to S.-Y.L. and DP2GM126898to H.Y.) and by the National Institute of Health IntramuralResearch Program, U.S. National Institutes of EnvironmentalHealth Sciences (ZIC ES103326 to M.J.B.). Authorcontributions: Y.Y. conducted all biochemical preparation,cryo-EM experiments, single-particle 3D reconstruction, andmodel building under the guidance of S.-Y.L. S.C.L. carriedout all electrophysiological recordings under the guidance ofH.Y. A.L.H. collected cryo-EM data and helped with cryo-EMsample screening under the guidance of M.J.B. S.-Y.L., Y.Y.,S.C.L., and H.Y. wrote the paper. Competing interests: Theauthors declare no competing interests. Data and materialsavailability: For the class 1 and class 2 icilin-PIP2-Ca

2+ complex,and the WS-12–PIP2 complex, the coordinates have beendeposited in the Protein Data Bank with IDs 6NR3, 6NR4, and6NR2, respectively; and the cryo-EM density maps have beendeposited in the Electron Microscopy Data Bank with IDsEMD-0488, EMD-0489, and EMD-0487, respectively.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/363/6430/eaav9334/suppl/DC1Materials and MethodsFigs. S1 to S13Table S1References (42–49)

2 November 2018; accepted 27 January 2019Published online 7 February 201910.1126/science.aav9334

Yin et al., Science 363, eaav9334 (2019) 1 March 2019 9 of 9

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Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channelYing Yin, Son C. Le, Allen L. Hsu, Mario J. Borgnia, Huanghe Yang and Seok-Yong Lee

originally published online February 7, 2019DOI: 10.1126/science.aav9334 (6430), eaav9334.363Science 

, this issue p. eaav9334Sciencedistinct from that used by other TRP channels.agents allosterically enhance each other and activate the channel opening. Thus, the activation mechanism of TRPM8 is binding site in TRPM8 is completely different from PIP2 sites in other TRP channels. The binding of PIP2 and coolingphosphatidylinositol-4,5-bisphosphate (PIP2), and calcium. Structural and functional analyses showed that the PIP2

electron microscopy structures of TRPM8 with cooling agents, membrane lipid− present cryoet al.channel. Yin In humans, cold is primarily sensed by transient receptor potential melastatin member 8 (TRPM8), a calcium

Cool mechanism for sensing cool

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