Caffeine activates mouse TRPA1 channelsbut suppresses human TRPA1 channelsKatsuhiro Nagatomo and Yoshihiro Kubo1

Division of Biophysics and Neurobiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan

Communicated by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, September 30, 2008 (received for review September 7, 2008)

Caffeine has various well-characterized pharmacological effects,but in mammals there are no known plasma membrane receptorsor ion channels activated by caffeine. We observed that caffeineactivates mouse transient receptor potential A1 (TRPA1) in heter-ologous expression systems by Cai

2� imaging and electrophysio-logical analyses. These responses to caffeine were confirmed inacutely dissociated dorsal root ganglion sensory neurons from WTmice, which are known to express TRPA1, but were not seen inneurons from TRPA1 KO mice. Expression of TRPA1 was detectedimmunohistochemically in nerve fibers and bundles in the mousetongue. Moreover, WT mice, but not KO mice, showed a remark-able aversion to caffeine-containing water. These results demon-strate that mouse TRPA1 channels expressed in sensory neuronscause an aversion to drinking caffeine-containing water, suggest-ing they mediate the perception of caffeine. Finally, we observedthat caffeine does not activate human TRPA1; instead, it sup-presses its activity.

Caffeine is a xanthine derivative known to exert variouspharmacological effects, including activation of ryanodine

receptors (RyRs) leading to Ca2� release from the Ca2� store,inhibition of phosphodiesterase leading to an increase in cAMP,and blockade of adenosine receptors (1, 2). However, caffeinehas not been shown to act on mammalian plasma membranereceptors or ion channels. In Drosophila, Gr66a was recentlyidentified as a G protein-coupled receptor for caffeine (3), butits mammalian orthologue has not yet been identified.

Masuho et al. (4) reported that caffeine induces an increase inintracellular Ca2� (Cai

2�) in STC-1 cells, a cell line establishedfrom a neuroendocrine tumor in the mouse small intestine (5).Notably, that response disappeared when the cells were incu-bated with the phospholipase C (PLC) inhibitor (4). We ob-served in this study that the caffeine-induced increase in Cai

2� inSTC-1 cells disappears in the absence of extracellular Ca2�

(Cao2�), which suggests the involvement of a Ca2�-permeable

channel on the plasma membrane sensitive to PLC inhibition,rather than that of a Gq-coupled receptor.

Among the potential candidates are TRP channels, which arewidely-expressed, Ca2�-permeable channels (6) that are criti-cally involved in transducing sensory signals and are activated bya variety of stimuli (7–10). For instance, the transient receptorpotential A1 (TRPA1) channel is activated by various pungentcompounds such as isothiocyanates, allicin, cinnamaldehyde,menthol, and sanshool (11–15), and it is noteworthy that TRPA1requires basal PLC activity for functionality (13).

Caffeine gives a bitter taste to humans, and the whole nerverecordings in mice (16–18) and marmosets (16) revealed thatapplication of caffeine to the tongue leads to excitation of 2nerves involved in taste, the chorda tympani and glossopharyn-geal nerves (19). To investigate the possibility that TRPA1 playsa role in the responses of taste nerves to caffeine, we assessed theexpression of TRPA1 protein in dorsal root ganglion (DRG)sensory neurons and in the nerve fibers in the mice tongue. Wealso analyzed the response to caffeine of neurons in DRGanalogous to Trigeminal ganglia (TG). Furthermore, we ana-lyzed the preference of drinking water with or without caffeineby using WT and TRPA1 KO mice.

ResultsResponse of STC-1 Cells to Caffeine and Isolation of cDNA EncodingMouse TRPA1 (mTRPA1). We initially examined the effect ofcaffeine on Cai

2� to mouse STC-1 cells by Cai2� imaging. We

found that, as reported (4), caffeine elicited an increase in Cai2�

and that the response was abolished in the absence of Cao2� and

blocked by TRP channel blockers, Gd3� (100 �M) or rutheniumred (5 �M) [supporting information (SI) Fig. S1]. Based on thesensitivity to PLC inhibitor described in the Introduction, wespeculated that mTRPA1 channels mediate the response. Wesuccessfully detected the expression of mTRPA1 in STC-1 cellsby RT-PCR and isolated a cDNA for the entire coding region.The cloned cDNA encoded amino acid sequence identical toNM�177781 in GenBank and was used in the experimentsdescribed here.

Response of mTRPA1 to Caffeine in Heterologous Expression Systems.We next measured Cai

2� in HEK293T cells expressing theisolated mTRPA1 channel. We observed that caffeine (5 mM)induced an increase in Cai

2� in cells transfected with mTRPA1(Fig. 1A), but not in cells transfected with the empty vector (Fig.1E). As with STC-1 cells, the increase in Cai

2� was not observedin the absence of Cao

2� (Fig. 1B) and was completely blocked bypreincubation with Gd3� (Fig. 1C) or ruthenium red (Fig. 1D).Responses to various doses of caffeine are shown in Fig. 1F, andthe dose–response relationship is plotted in Fig. 1G. The EC50for caffeine was between 1 and 2.5 mM. The responses ofmTRPA1 channels to theophylline (5 mM) and theobromine (5mM), 2 other xanthine derivatives, were also confirmed (Fig. S2).

For a more quantitative analysis, we carried out electrophys-iological recordings by using 2-electrode voltage clamp withXenopus oocytes (Fig. 2) and patch clamp with HEK293T cells(Fig. S3). In both experiments, Ca2� was removed from the bathsolution to avoid membrane currents evoked secondarily byincreases in Cai

2�.The currents elicited by application of caffeine or 1 of 2 known

TRPA1 agonists, allyl isothiocyanate (AITC) (11, 20–22) ormenthol (14), were recoded from Xenopus oocytes (Fig. 2).Because TRPA1 channels are known to show outward rectifi-cation (11, 21), depolarizing pulses to �60 mV were appliedrepeatedly every 2 s. Representative current traces (Fig. 2 A) andthe time courses of the current amplitudes at the end of the steppulses (Fig. 2B) are shown. Caffeine (5 mM), AITC (100 �M),and menthol (400 �M) all elicited increases in the outwardcurrent but the time courses of the responses differed substan-tially from one another (Fig. 2B). We observed clear caffeinedose and voltage dependencies of the TRPA1 channel currents(Fig. 2 C and D). The inward current was much smaller than the

Author contributions: K.N. and Y.K. designed research; K.N. performed research; K.N.analyzed data; and K.N. and Y.K. wrote the paper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: ykubo@nips.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809769105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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outward current, but was also clearly visible (Fig. 2D Inset). Nocurrent was evoked by these agonists in non-cRNA injectedoocytes (data not shown).

Similar responses were obtained by using a whole-cell patchclamp with HEK293T cells expressing mTRPA1 (Fig. S3).Because the cytoplasmic Cai

2� was chelated by 5 mM EGTA inthis experiment, the results excluded a possibility that Cai

2�

increase triggered by caffeine, but not caffeine itself, indirectlyactivates mTRPA1 channel. It is noteworthy that the inwardcurrent at hyperpolarized potentials was more clearly observedin HEK293T cells than in the oocytes (Fig. S3C).

TRPV1 and TRPM8 are known to have features in commonwith TRPA1; like TRPA1, TRPV1 is activated by alicin (12), andTRPM8 is activated by menthol and cold (13). We thereforeexamined their sensitivity to caffeine in Xenopus oocytes, butneither of them responded to 5 mM caffeine (Fig. S4).

Analysis of the Expression Patterns of mTRPA1 mRNA and Protein.mTRPA1 channels were shown to be expressed in subsets ofnociceptive neurons in the dorsal root, trigeminal, and nodoseganglia and in other sensory neurons (20, 23). Purhonen et al.(24) reported expression of mTRPA1 mRNA in both STC-1 cellsand mouse duodenal mucosa. Because it seems unlikely thatthere are mM concentrations of caffeine in the serum orcerebrospinal f luid, the most physiologically significant role ofTRPA1 channels in this regard would seem to be the perceptionof caffeine intake at the tongue and/or the gastrointestinal tract.

We examined the expression of mTRPA1 mRNA in the tongueand small intestine by RT-PCR and successfully detected it (Fig.S5A). To analyze the pattern of mTRPA1 protein expression, weraised and affinity-purified a specific rabbit anti-TRPA1 anti-body. The specificity of the antibody was first confirmed byWestern blot analysis of transfected HEK293T cells (Fig. S5B),and was further confirmed immunohistochemically using trans-fected HEK293T cells and cultured DRG neurons (Fig. S5 D andE). Collectively, the results confirm the reliability of the antibodyin the immunohistochemical analyses.

When we examined the expression of mTRPA1 in the tongue,we detected no signal in circumvallate papillae, where taste budsare known to cluster (Fig. 3 A and B). Interestingly, nervebundles of various size were clearly stained in the posterior (Fig.3 C and D), and much thinner nerve branches projecting toward

Fig. 1. Effect of caffeine on Cai2� in HEK293T cells expressing mTRPA1. (A)

Effect of caffeine (5 mM) on Cai2� in the presence of 2 mM Cao

2�. Cai2� was

monitored as the ratio of the fluorescence excited by 340- and 380-nm light incells loaded with fura-2. (B) The caffeine-induced increase in Cai

2� was notobserved in the absence of Cao

2�. (C and D) The response to caffeine wasblocked by 100 �M Gd3� (C) or 5 �M ruthenium red (D). (E) Effect of caffeinein cells transfected with vector alone. (F) Time course of the responses evokedby caffeine (0.1–10 mM). The n values indicate numbers of recorded cells. (G)Dose–response relationship. Means � SEM of the peak level of the increase inCai

2� evoked by caffeine are plotted.Fig. 2. Caffeine-induced currents in Xenopus oocytes expressing mTRPA1.(A) Current recordings were obtained under 2-electrode voltage clamp byapplying step pulses to �60 mV from a holding potential of �20 mV repeat-edly every 2 s, before and after application of agonists. Responses to 5 mMcaffeine (Top), 100 �M AITC (Middle), and 400 �M menthol (Bottom) areshown. (B) Time course of the change in peak current amplitude after appli-cation of agonists. (C and D) Current–voltage relationship for responses to theindicated concentrations of caffeine. (C) During the steady state after 30-sexposure to the indicated concentrations of caffeine, 300-ms step pulses from�80 to �80 mV were applied and then stepped back to �60 mV for 100 msevery 1 s from the holding potential of �20 mV. (D) y axis shows the currentamplitudes at the indicated membrane potentials in the presence of caffeine(0.1–10 mM). (Inset) An expanded view of the voltage range at which inwardcurrent was observed. The n values indicate numbers of recorded oocytes, andthe plots depict mean � SEM.

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the anterior part of the tongue also showed an immunofluores-cent signal (Fig. 3 E and F). These mTRPA1-positive nerve fiberswould be part of either the chorda tympani or glossopharyngealnerve. The signal completely disappeared when the antibody wasabsorbed with 5 �g/ml antigen peptide (Fig. 3 G–I).

The TRPA1 gene of the KO mice (25) has a stop codon afterthe fifth transmembrane region. The encoded truncated proteinwas confirmed to be nonfunctional, but the expression in trans-fected HEK cells could be detected by our antibody as it includedthe antigen region of the N-terminal end (Fig. S6). Therefore, theKO mice could not be used, unfortunately, as a negative control inour immunohistochemical analyses, and an ultimate proof of thespecificity of the antibody could not be obtained, although theimmunofluorescent signal was absorbed by antigen peptide inboth DRG neurons (Fig. S5F) and in the tongue (Fig. 3 G–I).

Sensitivity of DRG Neurons to Caffeine. It is of interest to knowwhether the mTRPA1 channels detected in the nerves inner-vating the mouse tongue are involved in the perception ofcaffeine. We examined by Ca2� imaging the sensitivity tocaffeine of acutely dissociated sensory neurons in DRG analo-gous to TG. Time courses of representative recordings fromDRG neurons isolated from WT mice are shown in Fig. 4A. Note

that some cells responded to caffeine, AITC, and capsaicin,whereas others responded only to capsaicin. Of 63 cells respond-ing to capsaicin, 20 cells responded to both AITC and caffeine,9 cells responded only to AITC, 4 cells responded only tocaffeine, and 30 cells responded to neither. There were cells thatshowed a small response to AITC but no clear response tocaffeine (e.g., light blue in Fig. 4A), which might be caused by alow expression level of TRPA1 channels in these cells. Therewere also cells that showed a large and rapidly rising response tocaffeine but only a small response to AITC (light green in Fig.4A), which might be caused by a sort of cross-desensitization, butthe reason is not clear. With these exceptions, the responses tocaffeine and AITC were mostly correlated (20 of 29 cells or 20of 24 cells). These results go very well with earlier reportsshowing that whereas capsaicin-sensitive TRPV1 channels areexpressed in most DRG neurons, AITC-sensitive TRPA1 chan-nels are expressed in only some TRPV1-positive neurons (20,23). By contrast, when we examined the caffeine sensitivity ofDRG neurons isolated from TRPA1 KO mice (Fig. 4B), wefound that of 115 cells responding to 1 �M capsaicin noneresponded to both 100 �M AITC and 5 mM caffeine. In 17 cellsof 115, a response to caffeine that was clearly distinguishablefrom that in WT cells was observed. The amplitude was small,and the activation was slow and gradual (Fig. 4B).

Aversion to Caffeine-Containing Water in WT Mice and Its Dependencyon mTRPA1 Channels. To determine whether mice can actuallyperceive caffeine in drinking water and, if so, whether TRPA1channels truly function as the caffeine sensor, we carried out abehavioral study in which mice could choose to drink from abottle containing plain water or one containing water withcaffeine. For this test, WT and TRPA1 KO mice were kept incages with free access to food and the 2 water bottles. Theconsumption of plain water and caffeine-containing water byWT and TRPA1 KO mice were recorded and the total dailyconsumption by all of the mice, divided by the total number ofmice, is plotted in Fig. 5A. The plot of the consumption dividedby the total weight of the mice (data not shown) was highlysimilar to that of Fig. 5A. The fractions of the plain andcaffeine-containing water in the total consumption are plotted inFig. 5B. We found that WT mice showed a marked aversion tocaffeine-containing water, but the KO mice did not (Fig. 5 B andC), which suggests mTRPA1 channels transduce the aversivestimulation.

B

D

F

H I

A

C

E

G

Fig. 3. Immunohistochemical analyses of mTRPA1 expression in mousetongue. Coronal sections of mouse tongue were immunostained by using theaffinity-purified anti-mTRPA1 antibody. (A, C, and E) Expression of mTRPA1was not detected in the taste buds of the circumvallate papillae (A). Instead,it was clearly and specifically detected in bundles of thick and thin nerve fibersin the posterior (C) areas of the tongue. Expression of mTRPA1 was alsodetected in the nerve bundles and their thinner branches in the anterior areas(E). (B, D, and F) Bright-phase images of regions corresponding to those in A,C, and E, respectively; taste buds in a circumvallate papilla are circled by dottedlines (B). (G–I) Immunofluorescent signal in the middle part of the tongue (Gand H) disappeared completely in an adjacent section (I) by absorbing theantibody with the antigen peptide (I). In this absorption experiment, frozensections prepared from a mouse without paraformaldehyde perfusion wereused to decrease a background signal resulting from the antigen peptideitself.

Fig. 4. Effects of caffeine, AITC, and capsaicin on Cai2� in acutely dissociated

DRG neurons from WT and TRPA1 KO mice. (A) Effects of 5 mM caffeine, 100�M AITC, and 1 �M capsaicin on Cai

2� in acutely dissociated, fura-2-loadedDRG neurons from WT mice in the presence of 2 mM Cao

2�. Time course of theevoked changes in Cai

2� is plotted. In this experiment agonists were appliedone after the other by switching solutions used for bath perfusion. (B) Timecourse of the evoked changes in Cai

2� in acutely dissociated DRG neurons froma TRPA1 KO mouse.

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Sensitivity of Human TRPA1 (hTRPA1) to Caffeine. As a species-specific difference of the effect of a Cys-reacting chemicalcompound, CMP1 [4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide], was reported between ratTRPA1 and hTRPA1 (26), we tested whether or not the caffeinesensitivity of mTRPA1 was conserved in humans and examinedthe effects of caffeine on oocytes expressing hTRPA1 by usinga 2-electrode voltage clamp (Fig. 6). In contrast to mTRPA1

(Fig. 6 A and C), hTRPA1 responded to 100 �M AITC, but notto 5 mM caffeine (Fig. 6B). Indeed, when caffeine was appliedafter washing out AITC, the residual increase in current wassuppressed by caffeine (Fig. 6B), and this suppression wasobserved even more clearly when caffeine was chase-applied inthe presence of AITC (Fig. 6D). Similar results were alsoobserved in whole-cell patch clamp recordings from HEK293Ttransfectants expressing hTRPA1 (Fig. S7).

DiscussionmTRPA1 Is a Novel Mediator of the Pharmacological Effect of Caffeine.Caffeine is well known to stimulate RyRs and Ca2� release fromintracellular Ca2� stores, especially in the skeletal muscle. Forthe following reasons, however, we believe that the caffeine-induced increases in Cai

2� in the present study are not caused byCa2� release, but are caused by Ca2� inf lux through themTRPA1 channel (1). In fura-2-loaded HEK293T cells, caf-feine-induced increases in Cai

2� were observed only in cellstransfected with mTRPA1; they were not seen in vector-transfected cells in the time span of recording (Fig. 1E) (2).Caffeine-induced increases in Cai

2� were abolished by removingCao

2� just before the experiments, so as not to deplete the Ca2�

store (Fig. 1B) (3). In primary cultures of dissociated DRGneurons, caffeine-induced increases in Cai

2� were observed innot all but only a fraction of the neurons that responded tocapsaicin (Fig. 4A) (4). The rapid caffeine-induced increases inCai

2� were not observed in DRG neurons from TRPA1 KO mice(Fig. 4B). The small, slow responses observed in some cells (Fig.4B) might have been caused by other effects of caffeine, e.g., onRyRs.

We are also convinced that the effect of caffeine on TRPA1channel is direct, and it is not mediated by second messengerssuch as Cai

2� induced by caffeine for the following reasons (1).Caffeine-evoked increases in TRPA1 current were observed inHEK293T cells under whole-cell patch clamp with pipettesolution containing 5 mM EGTA to chelate Ca2� (Fig. S3 A andC) (2). Caffeine-evoked increases in TRPA1 current were ob-served in oocytes even after thapsigargin treatment to depleteCa2� store, which abolished Gq-coupled m1 receptor-mediatedactivation of Ca2�–Cl� current completely (Fig. S8) (3). Theeffects of caffeine on mTRPA1 and hTRPA1 expressed inoocytes and HEK293T cells were qualitatively different (Fig. 6B and D and Fig. S7). Collectively, the findings outlined aboveindicate that mTRPA1 is a caffeine receptor expressed on theplasma membrane that mediates a novel pharmacological effectof caffeine.

Significance of the Caffeine-Sensing Function of mTRPA1. Our im-munohistochemical analysis showed that TRPA1 protein is notexpressed in circumvallate papillae, where taste buds are clus-tered (Fig. 3A), which suggests that the caffeine-sensing functionof TRPA1 is different from conventional taste sensing. Support-ing the notion that TRPA1 plays a role in caffeine sensing are thefollowing observations (1). Expression of TRPA1 protein wasobserved in dissociated DRG neurons (Fig. S5E) analogous toTG neurons and in the nerve fibers and bundles in the tongue(Fig. 3 C and E) (2). Responses to caffeine were observed indissociated DRG neurons from WT mice (Fig. 4A), but not inthose from TRPA1 KO mice (Fig. 4B) (3). WT mice, but not KOmice, showed a clear aversion to caffeine-containing water (Fig.5). We suggest that caffeine intake is perceived through theactivity of mTRPA1-positive sensory neurons projecting fromthe TG or DRG to the tongue or small intestine. Becausecaffeine is membrane-permeable, expression of a receptor on acontact surface, such as a taste bud, may not be required for itsperception. Instead, caffeine may stimulate innervating nerveterminals in the tongue directly in a similar way that applicationof methyl p-hydroxybenzoate, another TRPA1 agonist, to the

Fig. 5. Behavioral analysis of the preferences of WT and TRPA1 KO mice forwater with or without caffeine. (A) Consumption of plain (filled symbols) andcaffeine-containing (open symbols) water by WT (circles) and TRPA1 KO(squares) mice. The total daily consumption of all of the mice divided by thenumber of mice (WT: n � 8; KO: n � 10) is plotted. (B) The fractions of plainor caffeine-containing water in the total consumption are plotted. The sym-bols are the same as in A. WT mice showed a clear aversion to caffeine-containing water, whereas KO mice did not. (C) The mean and SEM of thevalues from 4 consecutive days in B are plotted. The difference was statisticallysignificant in WT mice (P � 0.012), but not KO mice (P � 0.096).

Fig. 6. Current recordings from Xenopus oocytes expressing hTRPA1. Usinga 2-electrode voltage clamp, 200-ms ramp pulses from �100 to �100 mV wereapplied every 5 s from a holding potential of �60 mV to oocytes expressingmTRPA1 (A and C) or hTRPA1 (B and D). Agonists were applied at the timesindicated by the bars. (A and C) Responses of mTRPA1 to both caffeine andAITC were confirmed; C is an expanded view of the boxed region in A. (B andD) The response of hTRPA1 to AITC was confirmed, but a decrease in thecurrent amplitude was observed upon application of caffeine. The suppres-sion was more clearly observed when basal channel current was increased byapplication and washout of AITC (B; expanded in D) or in the presence of AITC(B). Recordings similar to A and B were obtained from 3 cells, and represen-tative data are shown.

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skin stimulates innervating nerve terminals (27). In addition, thefact that mTRPA1 is activated by AITC, a pungent substance,suggests caffeine may be perceived as a pungent stimulus in themice, rather than merely a bitter one, which might explain whythey showed a remarkable aversion to caffeine-containing water.

Species Differences of the Effect of Caffeine. TRP channel ortho-logues from different species show remarkable functional dif-ferences; for example, whereas mammalian TRPV1 channels areactivated by capsaicin, those from chick are completely insen-sitive to capsaicin (28). A species-specific difference of the effectof a thioaminal-containing chemical compound, CMP1, was alsorecently reported between rat TRPA1 (activation) and hTRPA1(blockade) (26). Therefore, it is of high interest to study theeffect of caffeine on the TRPA1 channel of other species. Weobserved that the responses of mTRPA1 and hTRPA1 tocaffeine are qualitatively different from one another (Fig. 6, Fig.S3, and Fig. S7). Humans sense a bitter taste when they takecaffeine. Although it is possible that this bitter taste reflects thesuppression of hTRPA1 channels, it does not seem likely to bea mechanism of caffeine taste because such suppression wouldresult in a decrease in synaptic transmission. Another and morelikely possibility is that humans express an as-yet-unidentifiedT2R receptor (29, 30) or a molecule related to Drosophila Gr66a(3) as a caffeine receptor.

Materials and MethodsExperimental Animals. Xenopus laevis, WT mice of the C57BL/6 strain, andTRPA1 KO mice of the same strain were used in this study. KO mice weregenerously provided by David Julius (University of California, San Francisco).All animal experiments described below conformed to the institutional guide-lines of and were approved by the Animal Experiment Committee of NationalInstitute for Physiological Sciences.

Cell Culture. STC-1 and HEK293T cells were maintained as described (31) (seealso SI Text). Transfection of cDNA was carried out with Lipofectamine 2000(Invitrogen). To establish primary cultures of DRG neurons, 4- to 20-week-oldC57BL/6 mice were deeply anesthetized with pentobarbital and then killed bydecapitation, after which the DRG were mechanically isolated. The isolatedganglia were dissociated and cultured as described (32).

Molecular Biology. Isolation of poly(A)� RNA and reverse transcription werecarried out with a FastTrack 2.0 mRNA Isolation Kit (Invitrogen) and Super-Script II Reverse Transcriptase (Invitrogen). A cDNA fragment covering theentire coding region of mTRPA1 was amplified by KOD Plus polymerase(Toyobo). cDNAs encoding rat TRPM8 and rat TRPV1 were provided by DavidJulius, hTRPA1 cDNA was provided by Ardem Patapoutian (Scripps ResearchInstitute, La Jolla, CA), and porcine m1 receptor cDNA was provided by TaiKubo (National Institute of Advanced Industrial Science and Technology,Tokyo). cRNA for oocyte injection was transcribed in vitro with a mMESSAGEmMACHINE transcription kit (Ambion).

Functional Analysis. Caffeine was purchased from Kanto Chemical; AITC wasfrom Tokyo Kasei; and theophylline, theobromine, menthol, and capsaicinwere from Sigma. Stock solutions in water (caffeine, theophylline) or DMSO(AITC, menthol, capsaicin) were stored at �20 °C and diluted in the recording

solution just before use. Theobromine was dissolved in recording solutionadjusted to an alkaline pH (pH 10.9) just before use.

To image Cai2�, STC-1 cells, HEK293T cells, or dissociated DRG neurons on

coverslips were incubated in culture medium containing 8 �M fura-2 AM(Molecular Probes) for 1 h at 37 °C under 5% CO2. The cells were then rinsedand incubated for up to 1 h at room temperature in the bath solution (140 mMNaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM Hepes, pH 7.4)supplemented with 10 mM glucose. Cai

2� was monitored by ratiometric mea-surements as described (33).

Xenopus oocytes were surgically isolated from frogs anesthetized in watercontaining 0.15% tricaine. Injection of cRNA into oocytes and 2-electrodevoltage-clamp recording were carried out as described (34). The bath solutioncontained 96 mM NaCl, 2 mM KCl, 3 mM MgCl2, and 5 mM Hepes (pH 7.4), withno added Ca2�.

Methods of patch clamp experiments are in SI Text.

Immunochemical Analyses. Custom-made antiserum was prepared by Operon.Briefly, a peptide corresponding to the amino acid sequence of the N-terminalend of mTRPA1 [MKRGLRRILLPEERKEVQG(C)] was synthesized, conjugatedwith keyhole limpet hemocyanin, and used to raise antiserum in a rabbit. Theanti-mTRPA1 antibody was then affinity-purified by using the antigen pep-tide. In some experiments, anti-mTRPA1 antibody was preincubated with theantigen peptide (5 �g/ml) to confirm the specificity of the immunostaining.Alexa-conjugated goat anti-rabbit IgG (Alexa Fluor 488) (Invitrogen) was usedas a secondary antibody.

Immunocytochemical analyses were carried out as described (31). WT micewere deeply anesthetized with pentobarbital, after which PBS containing 4%paraformaldehyde was perfused for fixation by cardiac injection. After isola-tion, the tongues were soaked in PBS containing 10%, 20%, and then 30%sucrose for several hours each, then embedded in OCT compound (SakuraFinetech). For analysis of the DRG, the tissues were removed from micewithout perfusion of fixation solution. They were then treated with PBScontaining 4% paraformaldehyde for 15 min at room temperature, rinsedwith PBS, and embedded in OCT compound. The frozen sections were pre-pared and immunostained as described (35).

Behavioral Analyses. The 2-bottle preference test was carried out as follows.Eight WT and 10 KO mice (7–8 weeks old) were each kept in 4 cages (WT: 2males, 2 males, 2 females, 2 females; KO: 3 males, 2 males, 3 females, 2 females)with free access to food and water. After a 7-day control period, during whichthe mice became accustomed to the 2 water bottles in each cage, the bottleswere exchanged for a bottle containing only water and one containing waterwith 5 mM caffeine. The positions of the bottles were then swapped every 24 hto avoid an effect of bottle position on intake volume. The consumed volumesof plain and caffeine-containing water in each cage were measured daily for4 days, as was the total weight of the mice in each cage.

Statistical Analyses. The data are shown as mean � SEM, with n indicating thenumber of samples. Differences between means were analyzed by usingStudent’s unpaired t test. In Fig. 5D, differences among means were analyzedby using Dunnett’s test. Values of P � 0.05 were considered significant.

ACKNOWLEDGMENTS. We thank Dr. D. Julius for rat TRPV1 cDNA, rat TRPM8cDNA, and TRPA1 KO mice; Dr. A. Patapoutian for hTRPA1 cDNA; Dr. T. Kubofor porcine m1 receptor cDNA; Drs. M. Tominaga, K. Shibasaki, and S. Furuyafor valuable suggestions and technical advice; and Dr. O. Saitoh for discussion.This work was supported by research grants from the Ministry of Education,Science, Sports, Culture, and Technology of Japan and the Japan Society forthe Promotion of Science (to Y.K.). Y.K. is also supported by Solution OrientedResearch for Science and Technology, Japan Science and Technology Agency.

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