Caffeine Activates Mouse TRPA1 Channels but Suppresses Human TRPA1 Channels

Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels

Katsuhiro 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, Results but in mammals there are no known plasma membrane receptors Response of STC-1 Cells to Caffeine and Isolation of cDNA Encoding or ion channels activated by caffeine. We observed that caffeine Mouse TRPA1 (mTRPA1). We initially examined the effect of ϩ ϩ activates mouse transient receptor potential A1 (TRPA1) in heter- caffeine on Ca2 to mouse STC-1 cells by Ca2 imaging. We 2؉ i i ϩ ologous expression systems by Cai imaging and electrophysio- 2 found that, as reported (4), caffeine elicited an increase in Cai logical analyses. These responses to caffeine were conﬁrmed in 2ϩ and that the response was abolished in the absence of Cao and acutely dissociated dorsal root ganglion sensory neurons from WT blocked by TRP channel blockers, Gd3ϩ (100 ␮M) or ruthenium mice, which are known to express TRPA1, but were not seen in red (5 ␮M) [supporting information (SI) Fig. S1]. Based on the neurons from TRPA1 KO mice. Expression of TRPA1 was detected sensitivity to PLC inhibitor described in the Introduction, we immunohistochemically in nerve ﬁbers and bundles in the mouse speculated that mTRPA1 channels mediate the response. We tongue. Moreover, WT mice, but not KO mice, showed a remark- successfully detected the expression of mTRPA1 in STC-1 cells able aversion to caffeine-containing water. These results demon- by RT-PCR and isolated a cDNA for the entire coding region. strate that mouse TRPA1 channels expressed in sensory neurons The cloned cDNA encoded amino acid sequence identical to cause an aversion to drinking caffeine-containing water, suggest- NM࿝177781 in GenBank and was used in the experiments ing they mediate the perception of caffeine. Finally, we observed described here. that caffeine does not activate human TRPA1; instead, it suppresses its activity. Response of mTRPA1 to Caffeine in Heterologous Expression Systems. 2ϩ We next measured Cai in HEK293T cells expressing the affeine is a xanthine derivative known to exert various isolated mTRPA1 channel. We observed that caffeine (5 mM) 2ϩ Cpharmacological effects, including activation of ryanodine induced an increase in Cai in cells transfected with mTRPA1 ϩ ϩ receptors (RyRs) leading to Ca2 release from the Ca2 store, (Fig. 1A), but not in cells transfected with the empty vector (Fig. 2ϩ inhibition of phosphodiesterase leading to an increase in cAMP, 1E). As with STC-1 cells, the increase in Cai was not observed 2ϩ and blockade of adenosine receptors (1, 2). However, caffeine in the absence of Cao (Fig. 1B) and was completely blocked by has not been shown to act on mammalian plasma membrane preincubation with Gd3ϩ (Fig. 1C) or ruthenium red (Fig. 1D). receptors or ion channels. In Drosophila, Gr66a was recently Responses to various doses of caffeine are shown in Fig. 1F, and identified as a G protein-coupled receptor for caffeine (3), but the dose–response relationship is plotted in Fig. 1G. The EC50 its mammalian orthologue has not yet been identified. for caffeine was between 1 and 2.5 mM. The responses of Masuho et al. (4) reported that caffeine induces an increase in mTRPA1 channels to theophylline (5 mM) and theobromine (5 2ϩ 2ϩ intracellular Ca (Cai ) in STC-1 cells, a cell line established mM), 2 other xanthine derivatives, were also confirmed (Fig. S2). from a neuroendocrine tumor in the mouse small intestine (5). For a more quantitative analysis, we carried out electrophys- Notably, that response disappeared when the cells were incu- iological recordings by using 2-electrode voltage clamp with

bated with the phospholipase C (PLC) inhibitor (4). We ob- BIOPHYSICS ϩ Xenopus oocytes (Fig. 2) and patch clamp with HEK293T cells 2 ϩ served in this study that the caffeine-induced increase in Cai in (Fig. S3). In both experiments, Ca2 was removed from the bath STC-1 cells disappears in the absence of extracellular Ca2ϩ ϩ solution to avoid membrane currents evoked secondarily by (Ca2 ), which suggests the involvement of a Ca2ϩ-permeable 2ϩ o increases in Cai . channel on the plasma membrane sensitive to PLC inhibition, The currents elicited by application of caffeine or 1 of 2 known rather than that of a Gq-coupled receptor. TRPA1 agonists, allyl isothiocyanate (AITC) (11, 20–22) or Among the potential candidates are TRP channels, which are menthol (14), were recoded from Xenopus oocytes (Fig. 2). 2ϩ widely-expressed, Ca -permeable channels (6) that are criti- Because TRPA1 channels are known to show outward rectifi- cally involved in transducing sensory signals and are activated by cation (11, 21), depolarizing pulses to ϩ60 mV were applied a variety of stimuli (7–10). For instance, the transient receptor repeatedly every 2 s. Representative current traces (Fig. 2A) and potential A1 (TRPA1) channel is activated by various pungent the time courses of the current amplitudes at the end of the step compounds such as isothiocyanates, allicin, cinnamaldehyde, pulses (Fig. 2B) are shown. Caffeine (5 mM), AITC (100 ␮M), menthol, and sanshool (11–15), and it is noteworthy that TRPA1 and menthol (400 ␮M) all elicited increases in the outward requires basal PLC activity for functionality (13). current but the time courses of the responses differed substan- Caffeine gives a bitter taste to humans, and the whole nerve tially from one another (Fig. 2B). We observed clear caffeine recordings in mice (16–18) and marmosets (16) revealed that dose and voltage dependencies of the TRPA1 channel currents application of caffeine to the tongue leads to excitation of 2 (Fig. 2 C and D). The inward current was much smaller than the nerves involved in taste, the chorda tympani and glossopharyngeal nerves (19). To investigate the possibility that TRPA1 plays a role in the responses of taste nerves to caffeine, we assessed the Author contributions: K.N. and Y.K. designed research; K.N. performed research; K.N. expression of TRPA1 protein in dorsal root ganglion (DRG) analyzed data; and K.N. and Y.K. wrote the paper. sensory neurons and in the nerve fibers in the mice tongue. We The authors declare no conﬂict of interest. also analyzed the response to caffeine of neurons in DRG 1To whom correspondence should be addressed. E-mail: [email protected]. analogous to Trigeminal ganglia (TG). Furthermore, we ana- This article contains supporting information online at www.pnas.org/cgi/content/full/ lyzed the preference of drinking water with or without caffeine 0809769105/DCSupplemental. by using WT and TRPA1 KO mice. © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809769105 PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 ͉ 17373–17378 Downloaded by guest on September 25, 2021 2ϩ Fig. 1. Effect of caffeine on Cai in HEK293T cells expressing mTRPA1. (A) 2ϩ 2ϩ 2ϩ Effect of caffeine (5 mM) on Cai in the presence of 2 mM Cao .Cai was monitored as the ratio of the ﬂuorescence excited by 340- and 380-nm light in 2ϩ cells loaded with fura-2. (B) The caffeine-induced increase in Cai was not 2ϩ observed in the absence of Cao .(C and D) The response to caffeine was blocked by 100 ␮MGd3ϩ (C)or5␮M ruthenium red (D). (E) Effect of caffeine in cells transfected with vector alone. (F) Time course of the responses evoked by caffeine (0.1–10 mM). The n values indicate numbers of recorded cells. (G) Dose–response relationship. Means Ϯ SEM of the peak level of the increase in ϩ Fig. 2. Caffeine-induced currents in Xenopus oocytes expressing mTRPA1. Ca2 evoked by caffeine are plotted. i (A) Current recordings were obtained under 2-electrode voltage clamp by applying step pulses to ϩ60 mV from a holding potential of Ϫ20 mV repeatedly every 2 s, before and after application of agonists. Responses to 5 mM outward current, but was also clearly visible (Fig. 2D Inset). No caffeine (Top), 100 ␮M AITC (Middle), and 400 ␮M menthol (Bottom) are current was evoked by these agonists in non-cRNA injected shown. (B) Time course of the change in peak current amplitude after appli- oocytes (data not shown). cation of agonists. (C and D) Current–voltage relationship for responses to the Similar responses were obtained by using a whole-cell patch indicated concentrations of caffeine. (C) During the steady state after 30-s clamp with HEK293T cells expressing mTRPA1 (Fig. S3). exposure to the indicated concentrations of caffeine, 300-ms step pulses from 2ϩ Ϫ80 to ϩ80 mV were applied and then stepped back to ϩ60 mV for 100 ms Because the cytoplasmic Cai was chelated by 5 mM EGTA in 2ϩ every 1 s from the holding potential of Ϫ20 mV. (D) y axis shows the current this experiment, the results excluded a possibility that Cai increase triggered by caffeine, but not caffeine itself, indirectly amplitudes at the indicated membrane potentials in the presence of caffeine activates mTRPA1 channel. It is noteworthy that the inward (0.1–10 mM). (Inset) An expanded view of the voltage range at which inward current was observed. The n values indicate numbers of recorded oocytes, and current at hyperpolarized potentials was more clearly observed the plots depict mean Ϯ SEM. in HEK293T cells than in the oocytes (Fig. S3C). TRPV1 and TRPM8 are known to have features in common with TRPA1; like TRPA1, TRPV1 is activated by alicin (12), and We examined the expression of mTRPA1 mRNA in the tongue TRPM8 is activated by menthol and cold (13). We therefore and small intestine by RT-PCR and successfully detected it (Fig. examined their sensitivity to caffeine in Xenopus oocytes, but S5A). To analyze the pattern of mTRPA1 protein expression, we neither of them responded to 5 mM caffeine (Fig. S4). raised and affinity-purified a specific rabbit anti-TRPA1 antibody. The specificity of the antibody was first confirmed by Analysis of the Expression Patterns of mTRPA1 mRNA and Protein. Western blot analysis of transfected HEK293T cells (Fig. S5B), mTRPA1 channels were shown to be expressed in subsets of and was further confirmed immunohistochemically using trans- nociceptive neurons in the dorsal root, trigeminal, and nodose fected HEK293T cells and cultured DRG neurons (Fig. S5 D and ganglia and in other sensory neurons (20, 23). Purhonen et al. E). Collectively, the results confirm the reliability of the antibody (24) reported expression of mTRPA1 mRNA in both STC-1 cells in the immunohistochemical analyses. and mouse duodenal mucosa. Because it seems unlikely that When we examined the expression of mTRPA1 in the tongue, there are mM concentrations of caffeine in the serum or we detected no signal in circumvallate papillae, where taste buds cerebrospinal fluid, the most physiologically significant role of are known to cluster (Fig. 3 A and B). Interestingly, nerve TRPA1 channels in this regard would seem to be the perception bundles of various size were clearly stained in the posterior (Fig. of caffeine intake at the tongue and/or the gastrointestinal tract. 3 C and D), and much thinner nerve branches projecting toward

17374 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809769105 Nagatomo and Kubo Downloaded by guest on September 25, 2021 A B

C D

2ϩ Fig. 4. Effects of caffeine, AITC, and capsaicin on Cai in acutely dissociated DRG neurons from WT and TRPA1 KO mice. (A) Effects of 5 mM caffeine, 100 ␮ ␮ 2ϩ M AITC, and 1 M capsaicin on Cai in acutely dissociated, fura-2-loaded F 2ϩ E DRG neurons from WT mice in the presence of 2 mM Cao . Time course of the 2ϩ evoked changes in Cai is plotted. In this experiment agonists were applied one after the other by switching solutions used for bath perfusion. (B) Time 2ϩ course of the evoked changes in Cai in acutely dissociated DRG neurons from a TRPA1 KO mouse.

G H I that some cells responded to caffeine, AITC, and capsaicin, whereas others responded only to capsaicin. Of 63 cells responding to capsaicin, 20 cells responded to both AITC and caffeine, 9 cells responded only to AITC, 4 cells responded only to caffeine, and 30 cells responded to neither. There were cells that showed a small response to AITC but no clear response to caffeine (e.g., light blue in Fig. 4A), which might be caused by a Fig. 3. Immunohistochemical analyses of mTRPA1 expression in mouse low expression level of TRPA1 channels in these cells. There tongue. Coronal sections of mouse tongue were immunostained by using the were also cells that showed a large and rapidly rising response to affinity-purified anti-mTRPA1 antibody. (A, C, and E) Expression of mTRPA1 caffeine but only a small response to AITC (light green in Fig. was not detected in the taste buds of the circumvallate papillae (A). Instead, 4A), which might be caused by a sort of cross-desensitization, but it was clearly and specifically detected in bundles of thick and thin nerve fibers in the posterior (C) areas of the tongue. Expression of mTRPA1 was also the reason is not clear. With these exceptions, the responses to detected in the nerve bundles and their thinner branches in the anterior areas caffeine and AITC were mostly correlated (20 of 29 cells or 20 (E). (B, D, and F) Bright-phase images of regions corresponding to those in A, of 24 cells). These results go very well with earlier reports C, and E, respectively; taste buds in a circumvallate papilla are circled by dotted showing that whereas capsaicin-sensitive TRPV1 channels are lines (B). (G–I) Immunofluorescent signal in the middle part of the tongue (G expressed in most DRG neurons, AITC-sensitive TRPA1 chan- and H) disappeared completely in an adjacent section (I) by absorbing the nels are expressed in only some TRPV1-positive neurons (20, antibody with the antigen peptide (I). In this absorption experiment, frozen sections prepared from a mouse without paraformaldehyde perfusion were 23). By contrast, when we examined the caffeine sensitivity of used to decrease a background signal resulting from the antigen peptide DRG neurons isolated from TRPA1 KO mice (Fig. 4B), we ␮

itself. found that of 115 cells responding to 1 M capsaicin none BIOPHYSICS responded to both 100 ␮M AITC and 5 mM caffeine. In 17 cells of 115, a response to caffeine that was clearly distinguishable the anterior part of the tongue also showed an immunofluores- from that in WT cells was observed. The amplitude was small, cent signal (Fig. 3 E and F). These mTRPA1-positive nerve fibers and the activation was slow and gradual (Fig. 4B). would be part of either the chorda tympani or glossopharyngeal nerve. The signal completely disappeared when the antibody was ␮ Aversion to Caffeine-Containing Water in WT Mice and Its Dependency absorbed with 5 g/ml antigen peptide (Fig. 3 G–I). on mTRPA1 Channels. To determine whether mice can actually The TRPA1 gene of the KO mice (25) has a stop codon after perceive caffeine in drinking water and, if so, whether TRPA1 the fifth transmembrane region. The encoded truncated protein channels truly function as the caffeine sensor, we carried out a was confirmed to be nonfunctional, but the expression in trans- behavioral study in which mice could choose to drink from a fected HEK cells could be detected by our antibody as it included bottle containing plain water or one containing water with the antigen region of the N-terminal end (Fig. S6). Therefore, the KO mice could not be used, unfortunately, as a negative control in caffeine. For this test, WT and TRPA1 KO mice were kept in our immunohistochemical analyses, and an ultimate proof of the cages with free access to food and the 2 water bottles. The specificity of the antibody could not be obtained, although the consumption of plain water and caffeine-containing water by immunofluorescent signal was absorbed by antigen peptide in WT and TRPA1 KO mice were recorded and the total daily both DRG neurons (Fig. S5F) and in the tongue (Fig. 3 G–I). consumption by all of the mice, divided by the total number of mice, is plotted in Fig. 5A. The plot of the consumption divided Sensitivity of DRG Neurons to Caffeine. It is of interest to know by the total weight of the mice (data not shown) was highly whether the mTRPA1 channels detected in the nerves inner- similar to that of Fig. 5A. The fractions of the plain and vating the mouse tongue are involved in the perception of caffeine-containing water in the total consumption are plotted in caffeine. We examined by Ca2ϩ imaging the sensitivity to Fig. 5B. We found that WT mice showed a marked aversion to caffeine of acutely dissociated sensory neurons in DRG analo- caffeine-containing water, but the KO mice did not (Fig. 5 B and gous to TG. Time courses of representative recordings from C), which suggests mTRPA1 channels transduce the aversive DRG neurons isolated from WT mice are shown in Fig. 4A. Note stimulation.

Nagatomo and Kubo PNAS ͉ November 11, 2008 ͉ vol. 105 ͉ no. 45 ͉ 17375 Downloaded by guest on September 25, 2021 (Fig. 6 A and C), hTRPA1 responded to 100 ␮M AITC, but not to 5 mM caffeine (Fig. 6B). Indeed, when caffeine was applied after washing out AITC, the residual increase in current was suppressed by caffeine (Fig. 6B), and this suppression was observed even more clearly when caffeine was chase-applied in the presence of AITC (Fig. 6D). Similar results were also observed in whole-cell patch clamp recordings from HEK293T transfectants expressing hTRPA1 (Fig. S7). Discussion mTRPA1 Is a Novel Mediator of the Pharmacological Effect of Caffeine. Caffeine is well known to stimulate RyRs and Ca2ϩ release from Fig. 5. Behavioral analysis of the preferences of WT and TRPA1 KO mice for intracellular Ca2ϩ stores, especially in the skeletal muscle. For water with or without caffeine. (A) Consumption of plain (ﬁlled symbols) and the following reasons, however, we believe that the caffeine- caffeine-containing (open symbols) water by WT (circles) and TRPA1 KO 2ϩ induced increases in Cai in the present study are not caused by (squares) mice. The total daily consumption of all of the mice divided by the 2ϩ 2ϩ number of mice (WT: n ϭ 8; KO: n ϭ 10) is plotted. (B) The fractions of plain Ca release, but are caused by Ca influx through the or caffeine-containing water in the total consumption are plotted. The sym- mTRPA1 channel (1). In fura-2-loaded HEK293T cells, caf- 2ϩ bols are the same as in A. WT mice showed a clear aversion to caffeine- feine-induced increases in Cai were observed only in cells containing water, whereas KO mice did not. (C) The mean and SEM of the transfected with mTRPA1; they were not seen in vector- values from 4 consecutive days in B are plotted. The difference was statistically transfected cells in the time span of recording (Fig. 1E) (2). signiﬁcant in WT mice (P ϭ 0.012), but not KO mice (P ϭ 0.096). 2ϩ Caffeine-induced increases in Cai were abolished by removing 2ϩ 2ϩ Cao just before the experiments, so as not to deplete the Ca store (Fig. 1B) (3). In primary cultures of dissociated DRG 2ϩ Sensitivity of Human TRPA1 (hTRPA1) to Caffeine. As a species- neurons, caffeine-induced increases in Cai were observed in specific difference of the effect of a Cys-reacting chemical not all but only a fraction of the neurons that responded to compound, CMP1 [4-methyl-N-[2,2,2-trichloro-1-(4-nitro- capsaicin (Fig. 4A) (4). The rapid caffeine-induced increases in 2ϩ phenylsulfanyl)-ethyl]-benzamide], was reported between rat Cai were not observed in DRG neurons from TRPA1 KO mice TRPA1 and hTRPA1 (26), we tested whether or not the caffeine (Fig. 4B). The small, slow responses observed in some cells (Fig. sensitivity of mTRPA1 was conserved in humans and examined 4B) might have been caused by other effects of caffeine, e.g., on the effects of caffeine on oocytes expressing hTRPA1 by using RyRs. a 2-electrode voltage clamp (Fig. 6). In contrast to mTRPA1 We are also convinced that the effect of caffeine on TRPA1 channel is direct, and it is not mediated by second messengers 2ϩ such as Cai induced by caffeine for the following reasons (1). Caffeine-evoked increases in TRPA1 current were observed in HEK293T cells under whole-cell patch clamp with pipette solution containing 5 mM EGTA to chelate Ca2ϩ (Fig. S3 A and C) (2). Caffeine-evoked increases in TRPA1 current were observed in oocytes even after thapsigargin treatment to deplete Ca2ϩ store, which abolished Gq-coupled m1 receptor-mediated activation of Ca2ϩ–ClϪ current completely (Fig. S8) (3). The effects of caffeine on mTRPA1 and hTRPA1 expressed in oocytes and HEK293T cells were qualitatively different (Fig. 6 B and D and Fig. S7). Collectively, the findings outlined above indicate that mTRPA1 is a caffeine receptor expressed on the plasma membrane that mediates a novel pharmacological effect of caffeine.

Significance of the Caffeine-Sensing Function of mTRPA1. Our immunohistochemical analysis showed that TRPA1 protein is not expressed in circumvallate papillae, where taste buds are clus- tered (Fig. 3A), which suggests that the caffeine-sensing function of TRPA1 is different from conventional taste sensing. Support- ing the notion that TRPA1 plays a role in caffeine sensing are the following observations (1). Expression of TRPA1 protein was observed in dissociated DRG neurons (Fig. S5E) analogous to TG neurons and in the nerve fibers and bundles in the tongue (Fig. 3 C and E) (2). Responses to caffeine were observed in Fig. 6. Current recordings from Xenopus oocytes expressing hTRPA1. Using dissociated DRG neurons from WT mice (Fig. 4A), but not in a 2-electrode voltage clamp, 200-ms ramp pulses from Ϫ100 to ϩ100 mV were those from TRPA1 KO mice (Fig. 4B) (3). WT mice, but not KO applied every 5 s from a holding potential of Ϫ60 mV to oocytes expressing mice, showed a clear aversion to caffeine-containing water (Fig. mTRPA1 (A and C) or hTRPA1 (B and D). Agonists were applied at the times 5). We suggest that caffeine intake is perceived through the indicated by the bars. (A and C) Responses of mTRPA1 to both caffeine and activity of mTRPA1-positive sensory neurons projecting from AITC were conﬁrmed; C is an expanded view of the boxed region in A.(B and the TG or DRG to the tongue or small intestine. Because D) The response of hTRPA1 to AITC was conﬁrmed, but a decrease in the current amplitude was observed upon application of caffeine. The suppres- caffeine is membrane-permeable, expression of a receptor on a sion was more clearly observed when basal channel current was increased by contact surface, such as a taste bud, may not be required for its application and washout of AITC (B; expanded in D) or in the presence of AITC perception. Instead, caffeine may stimulate innervating nerve (B). Recordings similar to A and B were obtained from 3 cells, and represen- terminals in the tongue directly in a similar way that application tative data are shown. of methyl p-hydroxybenzoate, another TRPA1 agonist, to the

17376 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809769105 Nagatomo and Kubo Downloaded by guest on September 25, 2021 skin stimulates innervating nerve terminals (27). In addition, the solution just before use. Theobromine was dissolved in recording solution fact that mTRPA1 is activated by AITC, a pungent substance, adjusted to an alkaline pH (pH 10.9) just before use. 2ϩ suggests caffeine may be perceived as a pungent stimulus in the To image Cai , STC-1 cells, HEK293T cells, or dissociated DRG neurons on ␮ mice, rather than merely a bitter one, which might explain why coverslips were incubated in culture medium containing 8 M fura-2 AM (Molecular Probes) for1hat37°Cunder 5% CO2. The cells were then rinsed they showed a remarkable aversion to caffeine-containing water. and incubated for up to1hatroom temperature in the bath solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM Hepes, pH 7.4) 2ϩ Species Differences of the Effect of Caffeine. TRP channel ortho- supplemented with 10 mM glucose. Cai was monitored by ratiometric mea- logues from different species show remarkable functional dif- surements as described (33). ferences; for example, whereas mammalian TRPV1 channels are Xenopus oocytes were surgically isolated from frogs anesthetized in water activated by capsaicin, those from chick are completely insen- containing 0.15% tricaine. Injection of cRNA into oocytes and 2-electrode sitive to capsaicin (28). A species-specific difference of the effect voltage-clamp recording were carried out as described (34). The bath solution contained 96 mM NaCl, 2 mM KCl, 3 mM MgCl2, and 5 mM Hepes (pH 7.4), with of a thioaminal-containing chemical compound, CMP1, was also 2ϩ recently reported between rat TRPA1 (activation) and hTRPA1 no added Ca . Methods of patch clamp experiments are in SI Text. (blockade) (26). Therefore, it is of high interest to study the effect of caffeine on the TRPA1 channel of other species. We Immunochemical Analyses. Custom-made antiserum was prepared by Operon. observed that the responses of mTRPA1 and hTRPA1 to Briefly, a peptide corresponding to the amino acid sequence of the N-terminal caffeine are qualitatively different from one another (Fig. 6, Fig. end of mTRPA1 [MKRGLRRILLPEERKEVQG(C)] was synthesized, conjugated S3, and Fig. S7). Humans sense a bitter taste when they take with keyhole limpet hemocyanin, and used to raise antiserum in a rabbit. The caffeine. Although it is possible that this bitter taste reflects the anti-mTRPA1 antibody was then affinity-purified by using the antigen pep- suppression of hTRPA1 channels, it does not seem likely to be tide. In some experiments, anti-mTRPA1 antibody was preincubated with the a mechanism of caffeine taste because such suppression would antigen peptide (5 ␮g/ml) to confirm the specificity of the immunostaining. result in a decrease in synaptic transmission. Another and more Alexa-conjugated goat anti-rabbit IgG (Alexa Fluor 488) (Invitrogen) was used as a secondary antibody. likely possibility is that humans express an as-yet-unidentified Immunocytochemical analyses were carried out as described (31). WT mice T2R receptor (29, 30) or a molecule related to Drosophila Gr66a were deeply anesthetized with pentobarbital, after which PBS containing 4% (3) as a caffeine receptor. paraformaldehyde was perfused for fixation by cardiac injection. After isolation, the tongues were soaked in PBS containing 10%, 20%, and then 30% Materials and Methods sucrose for several hours each, then embedded in OCT compound (Sakura Experimental Animals. Xenopus laevis, WT mice of the C57BL/6 strain, and Finetech). For analysis of the DRG, the tissues were removed from mice TRPA1 KO mice of the same strain were used in this study. KO mice were without perfusion of fixation solution. They were then treated with PBS generously provided by David Julius (University of California, San Francisco). containing 4% paraformaldehyde for 15 min at room temperature, rinsed All animal experiments described below conformed to the institutional guide- with PBS, and embedded in OCT compound. The frozen sections were pre- lines of and were approved by the Animal Experiment Committee of National pared and immunostained as described (35). Institute for Physiological Sciences. Behavioral Analyses. The 2-bottle preference test was carried out as follows. Cell Culture. STC-1 and HEK293T cells were maintained as described (31) (see Eight WT and 10 KO mice (7–8 weeks old) were each kept in 4 cages (WT: 2 also SI Text). Transfection of cDNA was carried out with Lipofectamine 2000 males, 2 males, 2 females, 2 females; KO: 3 males, 2 males, 3 females, 2 females) (Invitrogen). To establish primary cultures of DRG neurons, 4- to 20-week-old with free access to food and water. After a 7-day control period, during which C57BL/6 mice were deeply anesthetized with pentobarbital and then killed by the mice became accustomed to the 2 water bottles in each cage, the bottles decapitation, after which the DRG were mechanically isolated. The isolated were exchanged for a bottle containing only water and one containing water ganglia were dissociated and cultured as described (32). with 5 mM caffeine. The positions of the bottles were then swapped every 24 h to avoid an effect of bottle position on intake volume. The consumed volumes Molecular Biology. Isolation of poly(A)ϩ RNA and reverse transcription were of plain and caffeine-containing water in each cage were measured daily for carried out with a FastTrack 2.0 mRNA Isolation Kit (Invitrogen) and Super- 4 days, as was the total weight of the mice in each cage. Script II Reverse Transcriptase (Invitrogen). A cDNA fragment covering the BIOPHYSICS entire coding region of mTRPA1 was amplified by KOD Plus polymerase Statistical Analyses. The data are shown as mean Ϯ SEM, with n indicating the (Toyobo). cDNAs encoding rat TRPM8 and rat TRPV1 were provided by David number of samples. Differences between means were analyzed by using Julius, hTRPA1 cDNA was provided by Ardem Patapoutian (Scripps Research Student’s unpaired t test. In Fig. 5D, differences among means were analyzed Institute, La Jolla, CA), and porcine m1 receptor cDNA was provided by Tai by using Dunnett’s test. Values of P Ͻ 0.05 were considered significant. Kubo (National Institute of Advanced Industrial Science and Technology, Tokyo). cRNA for oocyte injection was transcribed in vitro with a mMESSAGE ACKNOWLEDGMENTS. We thank Dr. D. Julius for rat TRPV1 cDNA, rat TRPM8 mMACHINE transcription kit (Ambion). cDNA, and TRPA1 KO mice; Dr. A. Patapoutian for hTRPA1 cDNA; Dr. T. Kubo for porcine m1 receptor cDNA; Drs. M. Tominaga, K. Shibasaki, and S. Furuya for valuable suggestions and technical advice; and Dr. O. Saitoh for discussion. Functional Analysis. Caffeine was purchased from Kanto Chemical; AITC was This work was supported by research grants from the Ministry of Education, from Tokyo Kasei; and theophylline, theobromine, menthol, and capsaicin Science, Sports, Culture, and Technology of Japan and the Japan Society for were from Sigma. Stock solutions in water (caffeine, theophylline) or DMSO the Promotion of Science (to Y.K.). Y.K. is also supported by Solution Oriented (AITC, menthol, capsaicin) were stored at Ϫ20 °C and diluted in the recording Research for Science and Technology, Japan Science and Technology Agency.

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