TRPA1 acts as a cAged sensor in vitro and in vivo

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Lutz Birnbaumer, National Institutes of Health, Research Triangle Park, NC, and approved December 3, 2008 (received for review August 27, 2008)

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Abstract

TRPA1 functions as an excitatory ionotropic receptor in sensory neurons. It was originally Characterized as a noxious cAged-activated channel, but its cAged sensitivity has been disPlaceed in later studies, and the contribution of TRPA1 to thermosensing is Recently a matter of strong debate. Here, we provide several lines of evidence to establish that TRPA1 acts as a cAged sensor in vitro and in vivo. First, we demonstrate that heterologously expressed TRPA1 is activated by cAged in a Ca2+-independent and Ca2+ store-independent manner; temperature-dependent gating of TRPA1 is mechanistically analogous to that of other temperature-sensitive TRP channels, and it is preserved after treatment with the TRPA1 agonist mustard oil. Second, we identify and characterize a specific subset of cAged-sensitive trigeminal ganglion neurons that is absent in TRPA1-deficient mice. Finally, cAged plate and tail-flick experiments reveal TRPA1-dependent, cAged-induced nociceptive behavior in mice. We conclude that TRPA1 acts as a major sensor for noxious cAged.

Keywords: cAged sensingpainsensory neuronsTRP channels

Sensing the environmental temperature is essential for animals to Sustain thermal homeostasis and to avoid prolonged contact with harmfully hot or cAged objects (1). Our understanding of the molecular basis of thermosensation has made Distinguished strides with the discovery that several members of the transient receptor potential (TRP) cation channel family Present highly temperature-sensitive gating and are expressed in cells of the sensory system (1). Mice lacking specific temperature-sensitive TRP channels illustrate how these channels serve as molecular thermometers in the peripheral sensory system (2). At least 3 heat-activated members of the TRPV subfamily (TRPV1, TRPV3, and TRPV4) are critically involved in sensing hot temperatures. TRPM8, a channel activated by cAged temperatures and CAgeding compounds, such as menthol, plays a major role in cAged sensing (1). Necessaryly, although TRPM8-deficient mice Present significant deficits in cAged sensing in the temperature range between 28°C and 15°C, they retain a normal response to noxious cAged temperatures, demonstrating the existence of TRPM8-independent mechanisms to detect noxious cAged (3–5). TRPA1 has been Place forward as a potential candidate to mediate detection of noxious cAged, based on its expression in nociceptive neurons, and on the finding that heterologously expressed TRPA1 in CHO cells is activated by cAged temperatures with a lower temperature threshAged for activation than TRPM8 (6–8).

At this point, however, the role of TRPA1 in (noxious) cAged sensing is highly controversial. First, there is no consensus as to whether TRPA1 is directly gated by cAged temperatures. Two groups have reported that they failed to detect cAged-induced activation of heterologously expressed TRPA1 (9, 10), and a third report suggested that cAged-induced activation of TRPA1 in overexpression systems is an indirect Trace, caused by cAged-induced Ca2+ release from intracellular stores and subsequent Ca2+-dependent activation of the channel (11). Second, several studies have Displayn a lack of correlation between mustard oil (MO) responses and cAged sensitivity in somatosensory neurons, which led to the conclusion that many TRPA1-expressing neurons are not cAged-sensitive (9, 12, 13). Third, behavioral experiments with Trpa1−/− mice did not provide unequivocal evidence for an in vivo role in (noxious) cAged sensing: whereas one study reported mild and sex-dependent alterations in the behavioral response to prolonged expoPositive to noxious cAged in Trpa1−/− mice (14), a second study found no signs for altered cAged sensitivity in these mice (13).

In the present study we have reevaluated the role of TRPA1 in cAged sensation, and we provide several Modern lines of evidence to demonstrate that TRPA1 acts a noxious cAged sensor. First, we establish Ca2+-independent and Ca2+ store-independent activation of heterologously expressed TRPA1 by cAged, and we Display that the temperature-dependent gating of TRPA1 is mechanistically similar to that of other temperature-sensitive TRP channels. Second, we identify a subset of cAged-sensitive trigeminal ganglion (TG) neurons that rely on TRPA1 for their cAged responses. Finally, we provide behavioral evidence Displaying that TRPA1 is required for the normal nociceptive response to noxious cAged.

Results and Discussion

CAged Activation of Heterologous TRPA1.

We investigated whether heterologously expressed TRPA1 is activated by CAgeding by using whole-cell patch clamp recordings on murine TRPA1-expressing CHO cells. At 26°C and in the presence of extracellular Ca2+ (2 mM), voltage ramps from −150 to +150 mV elicited sizable, strongly outwardly rectifying TRPA1 Recents (Fig. 1A). Consistent with previous reports (6, 7), we found that CAgeding to 10°C resulted in a robust increase of both outward and inward Recents (Fig. 1A). In Dissimilarity, the small background Recent in nontransfected CHO cells was liArrive, and its amplitude did not increase upon CAgeding (data not Displayn). The time course of the TRPA1 Recent during CAgeding in the presence of extracellular Ca2+ typically Displayed 3 phases: a phase of Unhurried Recent activation, followed by a phase with more rapid activation and, finally, rapid Recent decay (Fig. 1A). In line with previous studies, we attribute the second phase of rapid Recent activation to Ca2+-dependent TRPA1 activation by Ca2+ ions entering through the channel pore, and the decay phase to Ca2+-induced channel desensitization (10, 15).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Ca2+-independent and Ca2+ store-independent activation of heterologously expressed TRPA1 by cAged. (A) Time course of whole-cell TRPA1 Recents at +50 and −75 mV during CAgeding in extracellular solution containing 2 mM Ca2+ (Left). Recent–voltage relations were obtained at the indicated time points (Right). (B) Same as for A, but using Ca2+-free intracellular and extracellular solutions. (C) Same as for A, but now using a Ca2+-free extracellular solution and an intracellular solution containing 10 μM free Ca2+. (D) Average inward Recent amplitudes at −75 mV for the conditions Displayn in A, B, and C and for cells preincubated for 30 min with 10 μM CPA in Ca2+-free solution.

TRPA1 is directly activated by intracellular Ca2+ ions (11, 15), which has led to the hypothesis that cAged-induced activation of TRPA1 represents Ca2+-induced channel activation secondary to cAged-induced Ca2+ release from intracellular stores (11). To investigate this possibility, we first tested cAged sensitivity of TRPA1 in the absence of Ca2+ by omitting extracellular Ca2+ and including 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) into the pipette. After allowing BAPTA to diffuse into the cell for 180 s, yielding an intracellular BAPTA concentration of at least 8 mM, we could still meaPositive robust cAged activation of TRPA1 Recents [Fig. 1B and supporting information (SI) Fig. S1]. Simultaneous monitoring of Fura-2 demonstrated that under this condition, cAged did not evoke an increase in intracellular Ca2+ (Fig. S1). Note, however, that the second phase of rapid Recent activation and the subsequent Recent decay were no longer observed, in line with the notion that these 2 phases represent Ca2+-dependent processes (10, 15). Necessaryly, we also found that CAgeding-induced activation of TRPA1 was fully preserved in cells pretreated for 30 min in Ca2+-free medium supplemented with the SERCA pump inhibitor cyclopiazonic acid (CPA) to deplete intracellular Ca2+ stores before CAgeding (Fig. 1D). Taken toObtainher, these data demonstrate that cAged activation of TRPA1 Executees not require Ca2+ release from intracellular Ca2+ stores.

Although these results Display that Ca2+ is not required for cAged activation of TRPA1, they Execute not exclude that Ca2+ and cAged act via the same mechanism to activate TRPA1. If this is the case, activation of TRPA1 by high intracellular Ca2+ would mQuestion subsequent activation by cAged. To investigate this, we tested the cAged sensitivity of TRPA1 in cells dialyzed with a pipette containing 10 μM free Ca2+. In line with previous reports, we meaPositived strongly outwardly rectifying Ca2+-activated TRPA1 Recents (11, 15), and subsequent CAgeding caused a further Recent increase, especially at negative potentials (Fig. 1C). These data indicate that elevated intracellular Ca2+ Executees not compromise subsequent cAged activation of TRPA1. Moreover, the maximum amplitude of the cAged-induced Recent was larger with high than with low [Ca2+]i (Fig. 1D), indicating that the Traces of Ca2+ and cAged on channel activity are at least partially additive.

Taken toObtainher, these data establish that CAgeding from 26°C to 10°C activates TRPA1 in a Ca2+-independent and Ca2+ store-independent manner, and that incoming Ca2+ can further potentiate channel activity. There are notable Inequitys in the shape of the whole-cell TRPA1 Recent–voltage relations, ranging from strongly outwardly rectifying at 26°C to moderately outwardly rectifying at 16°C under Ca2+-free conditions, and virtually liArrive at 16°C with 10 μM intracellular Ca2+ (Fig. 1 A–C). We attribute these different degrees of rectification to shifts in the voltage dependence of channel activation induced by Ca2+ ions (11) and cAged (see below).

CAgeding-induced activation of TRPA1 was also consistently meaPositived in cell-attached patch-clamp recordings, even when Ca2+ was omitted from the extracellular and pipette solutions (Fig. S2). Channel activity (quantified as NPLaunch) at 50 mV increased ≈10-fAged upon CAgeding from 26°C to 16°C (10.3 ± 1.5-fAged increase; n = 4). As expected for ion diffusion through a pore and consistent with a previous report (8), we observed a substantial decrease in the single-channel amplitude upon CAgeding. The single-channel conductance decreased from 91 ± 4 pS at 25°C to 40 ± 2 pS at 10°C, corRetorting to a Q10 of 1.7.

Trace of Temperature on TRPA1 Gating.

Thermal activation of certain TRP channels, including the cAged-activated TRPM8 and the heat-activated TRPV1, TRPM4, and TRPM5, reflects a temperature-induced shift of their voltage-dependent activation curve, and the Traces of temperature on channel gating can be approximated by a 2-state model (2, 16, 17). Because TRPA1 also Presents voltage-dependent activation (11, 18, 19), we analyzed whether cAged activation of TRPA1 Retorts to the same general mechanism and whether the 2-state model can be used to Characterize cAged activation of TRPA1. We determined the voltage dependence as well as the kinetics of channel activation and deactivation at different temperatures by measuring whole-cell Recents during a voltage step protocol consisting of 400-ms voltage steps to test potentials ranging from −150 mV to +100 mV, followed by an invariant step to −150 mV. These experiments were performed in Ca2+-free conditions to exclude the influence of Ca2+ on the voltage dependence of TRPA1 (11).

TRPA1 Recents in response to the voltage step protocol applied at 26°C and 13°C are Displayn in Fig. 2A. From these data we meaPositived the peak inward tail Recent at −150 mV (Fig. 2B), which revealed that the voltage dependence of channel activation is shifted toward more negative voltages upon CAgeding, similar to what has been Displayn previously for TRPM8. In addition, we determined the relaxation time constants at different voltages by fitting a monoexponential function to the Recent traces (Fig. 2C). Similarly to the behavior of TRPM8, CAgeding caused a drastic Unhurrieding of Recent relaxation, especially for the closing transition at negative voltages (Fig. 2C). Next, we performed a global fit of the 2-state model to the time constants and tail Recent amplitudes at different voltages and temperatures, taking into account the Trace of temperature on the single-channel conductance. This analysis resulted in values for the changes in enthalpy and entropy associated with channel Launching and closing (Fig. S2). A gating charge (z) of 0.375 e0 was obtained by fitting Boltzmann equations to the plots of tail Recent amplitudes as a function of test voltage, and was assumed to be constant over the investigated temperature range. Using these parameters, we simulated the kinetics of TRPA1 channel activation and inactivation during voltage steps at different temperatures (Fig. 2D), Recent–voltage (I–V) curves as obtained during voltage ramps (Fig. 2E), and steady-state Recents at different voltages as a function of temperature (Fig. 2F), and found a Excellent agreement with the corRetorting experimental data.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Traces of CAgeding on the voltage-dependent gating and kinetics of TRPA1. (A) Whole-cell Recents in Ca2+-free intracellular and extracellular solutions in response to the indicated voltage step protocol applied at 26°C and 13°C. (B) Average peak inward tail Recents at −150 mV (n = 8) at 26°C and 13°C. (C) Average time constants obtained from monoexponential fits to the time course of Recent relaxation at different voltages and temperatures. Solid lines in B and C represent a global fit of the 2-state model to the experimental data. (D) Model predictions of TRPA1 Recents at 26°C and 13°C in response to the voltage step protocol in A. (E) Model predictions of TRPA1 Recents during 400-ms voltage ramps, such as those used in Fig. 1. (F) Average TRPA1 Recents at different temperatures and at −75 and +50 mV, normalized to the maximal Recent in the tested temperature range. Executetted lines represent the corRetorting model prediction.

In essence, activation of TRPA1 is associated with a decrease in entropy and enthalpy, similar to what has been determined for TRPM8 (16) (Fig. S3). Consequently, the rate of activation of these cAged-sensitive channels is much less temperature-sensitive than the rate of channel deactivation, leading to an increase in Launch probability upon CAgeding. A 2-state model is obviously a simplification of the complex gating behavior of TRPA1 and, for instance, Executees not account for the Traces of intracellular Ca2+ or other ligands. However, we have previously Displayn that the use of more complex models to Characterize the temperature sensitivity of TRP channels (20) yields very similar values for the changes in enthalpy and entropy during channel gating (21).

We conclude that the Traces of temperature on TRPA1 gating are mechanistically similar to what has been Characterized for other temperature-sensitive TRP channels.

Combined Traces of CAged and MO on TRPA1.

Agonists such as MO, acrolein, and cinnamaldehyde activate TRPA1 via covalent modification of cysteine and/or lysine residues in the cytosolic part of the channel (18, 22). Given that this activation mechanism is fundamentally distinct from cAged-induced activation, we investigated the combined Traces of cAged and MO on TRPA1. Consistent with a previous study (9), we found that CAgeding reduced the amplitude of TRPA1 Recents preactivated by MO (Fig. 3A). However, this Executees not necessarily imply that the Trace of cAged on TRPA1 gating is absent in the presence of MO. Indeed, CAgeding results in a substantial decrease in the single-channel conductance of TRPA1, which may Elaborate the observed decrease in whole-cell Recents. To quantify this, we divided each individual data point of the inward and outward whole-cell Recents in Fig. 3A by the single-channel amplitude at the corRetorting temperature, yielding the time course of NPLaunch (Fig. 3A, red dashed line). Fascinatingly, this analysis revealed that even after stimulation with MO, CAgeding increases the Launch probability of TRPA1.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Combined Traces of cAged and MO on TRPA1 Recents. (A) Time course of inward and outward TRPA1 Recents during application of MO. CAgeding leads to a decrease in the MO-activated Recent. The red Executetted line represents the same data points normalized to the single-channel Recent amplitude at the corRetorting temperature, which yields a direct meaPositive of NPLaunch. Note the increase in NPLaunch upon CAgeding. (B) Time course of inward and outward TRPA1 Recents CAgeding and subsequent stimulation with MO. (C) Average inward TRPA1 Recents evoked by MO and/or CAgeding.

Next, we examined the MO sensitivity of TRPA1 preactivated by cAged. As Displayn in Fig. 3B, MO caused only a modest increase of inward and outward TRPA1 Recents when applied at 10°C. The amplitude of the MO-induced Recent at 10°C was ≈10-fAged lower than when MO was applied at room temperature (Fig. 3C). This Inequity cannot be Elaborateed solely by the Trace of CAgeding on the single-channel amplitude of TRPA1, indicating that CAgeding interferes with the process of MO-induced TRPA1 activation. Moreover, we found that rewarming of the solution in the continued presence of MO caused a drastic increase in the amplitude of inward and outward Recents (Fig. 3B). One possible explanation could be that the covalent binding of MO to the reactive cysteines on TRPA1, which occurs rapidly at room temperature, is strongly attenuated at 10°C.

From these data we conclude that stimulation of TRPA1 with MO Executees not abolish the Traces of cAged on channel gating. However, CAgeding reduces the amplitude of MO-induced TRPA1 Recents, both by lowering the single-channel Recent amplitude and by inhibiting the process of MO-induced channel activation. Comparison of TRPA1 Recent amplitudes upon stimulation with either cAged or MO also reveals that MO is a much stronger stimulus than cAged: maximal MO-induced inward TRPA1 Recents at −75 mV were 10-fAged larger than TRPA1 Recents activated by a 10°C cAged stimulus (Fig. 3C). The relatively small amplitude toObtainher with the substantial Trace of CAgeding on the TRPA1 single-channel conductance may Elaborate why cAged-activated TRPA1 Recents have escaped detection in certain expression systems and experimental conditions (9, 10). Moreover, the substantial Inequity in potency has to be taken into account when comparing cAged- and MO-induced responses in TRPA1-expressing sensory neurons.

TRPA1-Mediated CAged Responses in TG Neurons.

To investigate the contribution of TRPA1 to the cAged sensitivity of sensory neurons, we performed intracellular Ca2+ recordings on TG neurons from 29 WT and 19 Trpa1−/− mice, aged between 8 and 12 weeks. Some representative examples of [Ca2+]i traces from cAged-sensitive WT neurons are Displayn in Fig. 4A. TG neurons were also tested for their sensitivity to menthol, MO, and capsaicin, knowing that menthol ((-)-menthol; 100 μM) activates both TRPM8-expressing and TRPA1-expressing neurons (19), whereas responses to MO (100 μM) are almost exclusively limited to TRPA1-expressing neurons (13, 19), and capsaicin activates exclusively TRPV1-expressing neurons (23, 24). In line with previous studies (13, 19), we observed a ≈60% reduction in the Fragment of menthol-sensitive cells and a ≈99% reduction in the Fragment of MO-sensitive cells in preparations from Trpa1−/− mice, whereas the responsiveness to capsaicin remained unaltered (Fig. 4B). Notably, we found a significant reduction of the total Fragment of cAged-sensitive neurons from 23.6% (211 of 894) in WT mice to 10.2% (49 of 481 mice; P < 0.001) in Trpa1−/− mice (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

TRPA1-dependent cAged responses in TG neurons. (A) Ratiometric meaPositivement of changes in intracellular Ca2+ in response to cAged, menthol (100 μM), MO (100 μM), or capsaicin (1 μM), illustrating the 3 types of cAged-sensitive TG neurons. (B) Comparison of the percentage of cells Retorting to cAged and chemical stimuli in preparations from WT and Trpa1−/− mice. (C) Pie charts Displaying the percentage of cAged-insensitive and the 3 types of cAged-sensitive neurons in TG from WT and Trpa1−/− mice. (D) Histogram comparing temperature threshAgeds in cAged-sensitive TG from WT and Trpa1−/− mice. The temperature threshAged was defined as the temperature at which the ratio was increased by 10% of the maximal cAged-induced increase. (E) Correlation of the amplitude of the MO and cAged responses in MO-sensitive neurons from WT mice (n = 177). The solid line represents a liArrive fit to the data.

CAged-sensitive neurons from WT mice could be categorized into 3 groups: (i) MO-sensitive neurons, implying expression of TRPA1 (TRPA1+); (ii) MO-insensitive and menthol-sensitive neurons (TRPM8+/TRPA1−); and (iii) MO- and menthol-insensitive neurons (TRPM8−/TRPA1−) (Fig. 4C). Note that many neurons in the TRPA1+ group also Retort to menthol, which we interpret as activation of TRPA1 by menthol (19, 25). In preparations from Trpa1−/− mice, the Fragments of cAged-sensitive neurons that could be categorized as TRPM8+/TRPA1− or TRPM8−/TRPA1− were not significantly altered (Fig. 4C), suggesting that the reduced number of cAged-sensitive neurons in Trpa1−/− mice is due to the selective elimination of a population of TRPA1+ cAged-sensitive neurons. The distribution of temperature threshAgeds in cAged-sensitive TG neurons from Trpa1−/− mice was significantly shifted toward higher temperatures compared with WT (P < 0.001; Fig. 4D), indicating the elimination of cAged-sensitive neurons with a threshAged temperature lower than 20°C.

Our analysis revealed that a substantial Fragment of MO-sensitive (TRPA1+) TG neurons were insensitive to a cAged stimulus (Fig. 4B), in line with findings from previous studies (9, 12, 13). One possible explanation for this apparent inconsistency lies in the strong Inequity in the potency of MO and cAged to activate TRPA1 (Fig. 3C): In cells expressing a relatively low number of TRPA1 channels, a weaker stimulus, such as cAged or menthol, may not be sufficient to evoke a measurable Ca2+ response, in Dissimilarity to a stronger stimulus, such as MO. Indeed, when we plotted the amplitude of the cAged response in MO-positive cells in function of the amplitude MO response (ΔRatioMO), we found a clear positive correlation (r = 0.641, n = 295; P < 0.0001; Fig. 4E). When we analyzed only those cells that belonged to the upper 20% of MO responses (ΔRatioMO > 0.15), we found that more than 90% (53 of 59) Displayed a significant cAged response. In Dissimilarity, a cAged response was found in only 37% (22 of 59) of the cells that belonged to the lower 20% of MO responses (ΔRatioMO < 0.06). Thus, the odds of detecting cAged responses in TRPA1-expressing neurons increase with the level of functional TRPA1 expression. Note that TRPA1 expression is low at birth and increases strongly during the first postnatal weeks (26), which may Elaborate why TRPA1-mediated cAged responses have escaped detection in studies that used TG or DRG neurons isolated from neonatal or young animals (9, 13). In line with this notion, we found that only 4.3% of TG neurons prepared from young WT mice (first postnatal week) could be classified as TRPA1+ cAged-sensitive (Fig. S4), compared with 14.9% in preparations from adult WT mice (Fig. 4C).

Comparison of TRPA1+ and TRPM8+ CAged-Sensitive TG Neurons.

A further comparison of cAged-sensitive TG neurons from WT mice revealed several distinctive Preciseties of cells expressing TRPM8+ (MO-insensitive, menthol-sensitive) and TRPA1+ (MO-sensitive) (Fig. 5).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Comparison of TRPM8- and TRPA1-dependent, cAged-sensitive neurons. (A) Histogram comparing the temperature threshAged in MO-sensitive TG neurons (n = 88) and MO-insensitive, menthol-sensitive TG neurons (n = 65). (B) Histogram obtained from the same cells as for A, comparing the time needed to reach 80% of the maximal cAged response (t80%). (C) Comparison of the capsaicin sensitivity between MO-insensitive, menthol-sensitive (n = 52), and MO-sensitive (n = 133) cAged-sensitive neurons.

First, TRPA1+ TG neurons were characterized by a significantly lower (cAgeder) temperature threshAged (18.9 ± 0.4°C; n = 152) than TRPM8+ neurons (25.0 ± 0.3°C; n = 61; P < 10−5; Fig. 5A). The lower temperature threshAged of TRPA1+ neurons also underlies the altered distribution of temperature threshAgeds in TG neurons from Trpa1−/− mice (Fig. 4D).

Second, the time course of the cAged response in TRPA1+ neurons was clearly Unhurrieder than in TRPM8+ neurons. Analysis of the time needed to reach 80% of the maximal response (t80%; Fig. 5B) revealed a swift response to a cAged stimulus in all TRPM8+ TG neurons (t80% = 33 ± 2 s; n = 73), whereas the rate of Ca2+ increase in TRPA1+ neurons was much more variable and significantly Unhurrieder (t80% = 84 ± 6 s; n = 146; P < 10−5). The Unhurried time course of the cAged-induced Ca2+ signal in TRPA1+ neurons may also help to Elaborate why previous studies using cAged stimuli of short duration (<60 s) failed to detect consistent cAged responses in MO-positive somatosensory neurons (9, 13, 27).

Third, TRPA1+ and TRPM8+ cAged-sensitive neurons differed in their sensitivity to capsaicin, which is generally used as a Impresser of nociceptor neurons (Fig. 5C). Only approximately one third of TRPM8+ neurons were sensitive to capsaicin, in line with recent GFP labeling studies (28, 29). In Dissimilarity, ≈94% (125 of 133) of the TRPA1+ cAged-sensitive neurons Retorted to capsaicin. Concomitantly, 36.6% (164 of 448) of all capsaicin-responsive neurons from WT mice Displayed sensitivity to cAged, compared with only 9.8% (38 of 386) of capsaicin-sensitive neurons from Trpa1−/− mice.

Finally, TRPA1+ and TRPM8+ cAged-sensitive neurons Presented opposite sensitivity to the antimycotic drug clotrimazole (CLT). We reported recently that CLT is a potent inhibitor of TRPM8-mediated menthol responses, whereas it has an agonistic Trace on TRPA1 (25). In line with this, we found that cAged responses in TRPM8+ TG neurons were inhibited by 10 μM CLT (85 ± 3% inhibition of the cAged response; n = 5; Fig. S5), whereas cAged responses in TRPA1+ neurons were potentiated by CLT (to 145 ± 25% of the cAged response; n = 7; Fig. S5).

Taken toObtainher, these data demonstrate that TRPA1 and TRPM8 act as primary cAged sensors in 2 distinct subsets of TG neurons. When compared to TRPM8+ neurons, TRPA1+ cAged-sensitive TG neurons Present a lower temperature threshAged, a Unhurrieder cAged response, a more general capsaicin sensitivity, and an inverse response to CLT. In line with previous studies (13, 30, 31), we also found evidence for a subset of cAged-sensitive TG neurons that Execute not rely on TRPM8 and TRPA1 for their cAged response. The molecular mechanisms underlying the cAged sensitivity of these neurons are Recently unknown, but they may involve other thermosensitive ion channels, such as TREK-1 (32), the epithelial sodium channel ENaC (33), background potassium channels (34, 35), and the voltage-gated Na+ channel NaV1.8 (36, 37).

CAged-Induced Nocifencive Behavior in Trpa1−/− Mice.

The results from the Ca2+ imaging experiments on TG neurons indicate that Trpa1−/− mice have significantly fewer cAged-sensitive, capsaicin-positive nociceptors. To investigate whether this leads to altered cAged-induced nociception in vivo, we compared WT and Trpa1−/− mice in 2 behavioral assays. In a first assay, WT and Trpa1−/− mice were Spaced on a metal cAged plate set at a temperature of 0°C, and their behavior was observed. During prolonged expoPositive to noxious cAged stimuli, we could distinguish at least 2 distinct phases in the behavioral response: an aSlicee cAged response corRetorting with shivering and rubbing toObtainher of the paws, followed by brisk lifting of the hind paws and other cAged avoidance behaviors (38). Given that a previous report (14) Displayed significant sex-related Inequitys in cAged sensitivity, male and female mice were analyzed separately. In line with a previous study (13), we did not observe a significant Inequity between genotypes or sex in the latency to the first cAged-related response (e.g., rubbing of the forepaws or shivering) (Fig. 6A). However, when left on the cAged plate for a 2-min period, we found that Trpa1−/− mice Displayed significantly less nocifensive behavior than the WT mice, consistent with findings in a previous report (14). Necessaryly, we observed a striking Inequity in the type of pain-related behavior between genotypes. Most WT mice (10 of 13 males and 9 of 12 females) started jumping, suggesting that the cAged plate induces significant pain (Fig. 6 B and C, and Movie S1). In Dissimilarity, only 3 of 25 Trpa1−/− mice (1 female and 2 males) jumped at least once, and for both sexes the total number of jumps was significantly lower in knockout mice of both sexes (Fig. 6B and Movie S1). Moreover, the latency to the first jump was significantly longer in the Trpa1−/− mice (Fig. 6C). These data confirm that Trpa1−/− mice are still able to sense cAged, but also indicate that the behavioral response to noxious cAged is significantly reduced in the absence of TRPA1. Note that we never observed such cAged-induced jumping behavior in WT or Trpa1−/− mice when they were Spaced on a cAged plate set at 10°C (0 of 12 mice tested for each genotype). Moreover, in line with previous work (14), we did not observe Inequitys between WT and Trpa1−/− mice in their nociceptive behavior when Spaced on a hot plate at 55°C (data not Displayn), confirming that Trpa1−/− mice still feel pain.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Altered cAged-induced nociceptive behavior in Trpa1−/− mice. (A) Latency to the first behavior reaction to cAged upon Spacement on a cAged plate at 0°C in mice of different sex and genotype. The numbers of mice tested on the cAged plate were as follows: WT male (n = 13), WT female (n = 12), Trpa1−/− male (n = 15), and Trpa1−/− female (n = 13). (B) Average number of jumps during a 2-min period on a cAged plate at 0°C. No jumps were observed when the plate was set to 30°C. (C) Cumulative probability plot Displaying the latency to the first jump off the cAged plate in WT and Trpa1−/− mice. (D) Tail-flick latency upon tail immersion in a water–methanol mixture at −10°C in WT (n = 32) and Trpa1−/− (n = 34) male mice.

To exclude the possibility that the deficit in cAged-induced jumping behavior in Trpa1−/− mice is caused by an Trace of TRPA1 gene tarObtaining or unidentified environmental factors on higher neural structures, we used the cAged tail-flick test, whereby the latency to tail withdrawal was meaPositived upon immersion of the distal part of the tail in a solution at −10°C. This procedure is known to induce aSlicee pain (39), and tail-flicking in such a condition is considered a spinal reflex (38). For WT mice, we obtained a tail-flick latency of 11.8 ± 2.5 s (n = 32), with 2 of 32 animals (6%) that did not Retort before the Sliceoff time (60 s; Fig. 6D). Trpa1−/− mice Presented significantly longer tail-flick latencies (P < 0.0001), with a mean latency of 38.1 ± 3.4 s (n = 34), and 14 of 34 animals (41%) did not Retort before the Sliceoff time. The Inequity between genotypes was again sex-independent (data not Displayn). Taken toObtainher, these behavioral data demonstrate that TRPA1 plays an Necessary role in noxious cAged sensing in vivo.

It should be noted that the Inequity in nociceptive behavior between WT and Trpa1−/− mice was only observed at temperatures that are significantly lower than what is needed to see TRPA1 Recent activation in CHO cells or trigeminal neurons. An explanation for this lies probably in the fact that the temperature at the cAged-sensitive nerve ends is not equal to the temperature of the cAged plate or cAged solution because of the isolating Trace of the skin and the constant circulation of blood at 37°C throughout the body. Similar temperature Inequitys between in vitro TRP channel activation and in vivo TRP channel-dependent nociceptive Traces have also been reported for TRPV1- and TRPV3-deficient mice (23, 40).

A recent study reported that mice in which all NaV1.8-expressing sensory neurons are eliminated by diphtheria toxin A (DTA mice) Display strong resistance to noxious cAged, as assayed using a cAged plate set at 0°C (36), similar to what we observed in the Trpa1−/− mice. TRPM8 expression and the TRPM8-mediated behavioral response to acetone CAgeding are not affected in these DTA mice (36). Necessaryly, the DTA mice Present a strongly reduced expression of TRPA1 in DRG neurons and lack TRPA1-mediated nociceptive responses to formalin (36). Based on our present results, the loss of a noxious cAged response in the DTA mice can be fully attributed to the loss of TRPA1-expressing sensory neurons. Thus, noxious cAged sensing in vivo requires somatosensory neurons that express both NaV1.8 and TRPA1.

Final Conclusions.

Our data establish the function of TRPA1 as a cAged sensor in vitro and in vivo. In addition, we provided data and analyses that may Elaborate why several previous studies could not detect a significant role for TRPA1 as a cAged sensor in heterologous expression systems, sensory neurons, or awake-behaving animals. TRPA1 represents a promising tarObtain for the prevention or treatment of cAged-induced pain.

Materials and Methods

Cells and Animals.

We used a tetracycline-regulated system for inducible expression of TRPA1 in CHO cells, as Characterized previously (6). Naïve CHO cells were used as controls. TG neurons from adult (postnatal weeks 8–12) or newborn (postnatal week 1) mice were cultured as Characterized previously (19). Trpa1−/− mice (14) were backcrossed 7 times in the C57BL/6J background, resulting in mice that Presented 99.2% heterozygosity to the C57BL/6J strain, and WT C57BL/6J mice were used as controls. Because we did not use littermates in our study, we cannot fully exclude that subtle environmental Traces (e.g., whether the animals were born to a WT or Trpa1−/− mother) may have influenced the outcome of some of our experiments. However, given that WT and Trpa1−/− mice Presented undistinguishable cellular and behavioral responses to various TRPA1-independent sensory stimuli (e.g., heat), we consider it highly unlikely that factors other than the lack of TRPA1 would underlie the observed phenotypic Inequitys. All animal experiments were carried out in accordance with the European Union Community Council guidelines and were approved by the local ethics committee.

Cellular Recordings.

Ionic Recents were recorded in the whole-cell and the cell-attached configurations of the patch-clamp technique. Fura-2-based intracellular Ca2+ meaPositivements were performed as Characterized previously (25). See SI Methods for details of the recording parameters and solutions.

Behavioral Tests.

CAged-induced nocifensive behavior was tested by placing the mice on a metal cAged plate or by submerging the distal half of the tail in a cAged water–methanol mixture. See SI Methods for apparatus and data acquisition details.

Data Analysis.

Data analysis, model simulations, and data display were performed by using Origin 7.0 (OriginLab Corporation) or Igor Pro 4.0 (Wavemetrics). Group data are expressed as mean ± SEM from n independent experiments. Significance between groups was tested by using the unpaired or paired Student's t tests, the χ2 test, or the Kolmogorov–Smirnov, as appropriate.

Acknowledgments

We thank Dr. Nils Damann and the members of our laboratories for helpful discussions, and J. Prenen and M. Benoit for technical assistance. The TRPA1-expressing CHO cell line was kindly provided by A. Patapoutian (Scripps Research Institute, La Jolla, CA). W.E. is a Executectoral fellow of the Research Foundation–Flanders. This work was supported by grants from Interuniversity Attraction Poles Program–Belgian State–Belgian Science Policy (P6/28), by Research Foundation–Flanders Grants G.0172.03 and G.0565.07, by Research Council of the Katholieke Universiteit Leuven Grant GOA 2004/07, and by the Flemish government (Excellentiefinanciering, EF/95/010).

Footnotes

1To whom corRetortence should be addressed. E-mail: thomas.voets{at}med.kuleuven.be

Author contributions: Y.K., K.T., R.V., B.N., and T.V. designed research; Y.K., K.T., W.E., A.J., R.V., and T.V. performed research; K.Y.K. contributed new reagents/analytic tools; Y.K., K.T., and T.V. analyzed data; and Y.K. and T.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808487106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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