Warmth suppresses and desensitizes damage-sensing ion channel TRPA1
© Wang et al; licensee BioMed Central Ltd. 2012
Received: 5 October 2011
Accepted: 29 March 2012
Published: 29 March 2012
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© Wang et al; licensee BioMed Central Ltd. 2012
Received: 5 October 2011
Accepted: 29 March 2012
Published: 29 March 2012
Acute or chronic tissue damage induces an inflammatory response accompanied by pain and alterations in local tissue temperature. Recent studies revealed that the transient receptor potential A1 (TRPA1) channel is activated by a wide variety of substances that are released following tissue damage to evoke nociception and neurogenic inflammation. Although the effects of a noxious range of cold temperatures on TRPA1 have been rigorously studied, it is not known how agonist-induced activation of TRPA1 is regulated by temperature over an innocuous range centred on the normal skin surface temperature. This study investigated the effect of temperature on agonist-induced currents in human embryonic kidney (HEK) 293 cells transfected with rat or human TRPA1 and in rat sensory neurons.
Agonist-induced TRPA1 currents in HEK293 cells were strongly suppressed by warm temperatures, and almost abolished at 39°C. Such inhibition occurred when TRPA1 was activated by either electrophilic or non-electrophilic agonists. Warming not only decreased the apparent affinity of TRPA1 for mustard oil (MO), but also greatly enhanced the desensitization and tachyphylaxis of TRPA1. Warming also attenuated MO-induced ionic currents in sensory neurons. These results suggest that the extent of agonist-induced activity of TRPA1 may depend on surrounding tissue temperature, and local hyperthermia during acute inflammation could be an endogenous negative regulatory mechanism to attenuate persistent pain at the site of injury.
These results indicate that warmth suppresses and desensitizes damage-sensing ion channel TRPA1. Such warmth-induced suppression of TRPA1 may also explain, at least in part, the mechanistic basis of heat therapy that has been widely used as a supplemental anti-nociceptive approach.
Transient receptor potential A1 (TRPA1) is a Ca2+-permeable non-selective cationic channel enriched in a subpopulation of nociceptive sensory neurons [1, 2]. The activation of TRPA1 directly evokes pain and induces vasodilation and neurogenic inflammation. TRPA1 can be activated by a wide range of irritants including mustard oil (MO), cinnamaldehyde, and formaldehyde. Endogenous products generated by tissue damage and oxidative stress, such as H2O2, 4-hydroxynonenal, prostaglandin J2, and reactive oxygen and nitrogen species can also activate TRPA1 . Thus, TRPA1 functions as a sensor of endogenous tissue damage and exogenous harmful compounds, and is implicated in multiple pathological conditions, including chronic pain and respiratory and cardiovascular diseases [3–5]. Recently, a gain-of-function mutation of TRPA1 N855S was found to cause familial episodic pain syndrome , further suggesting a role for TRPA1 in nociception.
Many agonists activate TRPA1 by covalent binding to reactive residues located at an intracellular amino terminal domain [7, 8]. Since the covalent modification of TRPA1 by an electrophilic agonist is not readily reversible after washout, but persists for more than an hour , TRPA1 activation by reactive agonists typically show prolonged residual activity even following washout [7–9]. Thus TRPA1 may be persistently activated by endogenous agonists that are released at the site of injury or inflammation [10–14]. Consistent with this notion, specific antagonists against TRPA1 reverse persistent pain under various pathological conditions in experimental animals [15–18]. Therefore, developing a more effective approach to suppress the activity of TRPA1 may be beneficial in the treatment of chronic pain conditions.
Since mammalian TRPA1 was originally proposed as a noxious cold sensor , the activation of TRPA1 has been rigorously studied over a cold temperature range. Although cold sensitivity of TRPA1 is controversial , a recent study reported a potentiating effect of cold on agonist-induced activation of TRPA1, suggesting it has a role in cold hyperalgesia rather than cold pain . However, it is not known how the agonist-induced activation of TRPA1 is affected by temperature changes at the skin surface. Moreover, acute or chronic inflammatory conditions in human and experimental animals are accompanied by alterations in the local tissue temperature [21–24]. Therefore, investigating the effects of temperature at the skin surface on agonist-induced activation of TRPA1 should provide information about TRPA1 activity under conditions that are more pathophysiologically relevant.
In this study, we assessed the effect of the temperature on agonist-induced activation of TRPA1 in vitro.
To investigate the modulation of agonist-induced activation of TRPA1 over physiologically relevant temperature range, we analysed MO-evoked currents at various temperatures using the whole-cell voltage clamp technique. In order to better quantify the activity of recombinant TRPA1, we generated human embryonic kidney (HEK) 293 cell lines stably expressing either rat or human TRPA1. Unless otherwise indicated, data presented in the Results were obtained from cells stably expressing TRPA1.
We also examined whether warming suppressed the MO-induced activation of the mutant TRPA1 N855S that underlies familial episodic pain syndrome . Like wild-type TRPA1, the amplitude of currents evoked by 100 μ M MO in HEK293 cells transiently transfected with TRPA1 N855S was significantly reduced by approximately 70% at 33°C compared with the amplitude at 23°C (-262 ± 61 pA/pF and -847 ± 142 pA/pF, respectively, P < 0.05).
The major finding of this study is that agonist-induced activation of TRPA1 is strongly inhibited by innocuous warmth. Such inhibition is mediated by suppression, an attenuated activation, as well as desensitization, an inability of activation following initial activation. Our results suggest that heat reduces the functionality of TRPA1 only after activation by agonists. Pre-emptive heating without co-application of chemical agonists did not affect functionality of recombinant or native TRPA1, indicating that warmth by itself does not non-specifically impair the function of TRPA1. Heat-induced suppression of TRPA1 is apparently not the result of impaired covalent modification. Indeed, the rate constant representing the nucleophilic reaction between cysteine and hydrogen peroxide increases as temperature increases .
One plausible mechanism of heat-induced suppression of TRPA1 is that heat may act as a 'negative' modulator of TRPA1. In this model, agonistic temperatures positively modulate, whereas counter thermal stimuli may negatively modulate thermosensitive TRP channels. Indeed, agonist activation of heat-gated TRPV1 is strongly suppressed by cold, the counter agonistic temperature . Although the time course of activation by MO was accelerated by heating, the EC50 increased at warm temperatures. In contrast, the heat-gated channel TRPV1 shows decreased EC50 upon heating . Since TRPA1 can be gated and potentiated by cold , heat may act as a negative and positive modulator of TRPA1 and TRPV1, respectively. Recently, it was demonstrated that capsaicin- and heat-evoked activation may occur through distinct structural entities, and TRPV1, with simultaneous N628K/N652T/Y653T mutations, showed impaired activation by heat with intact activation by capsaicin . Interestingly, this mutant TRPV1 also showed impairment in heat-induced potentiation and diminished cold-induced suppression of capsaicin-evoked currents , suggesting that the impairment of the heat activation pathway also attenuates cold-induced suppression. Therefore, it is also possible that the decreased EC50 of TRPA1 by warmth may be, at least in part, a result of negative modulation involving the cold activation mechanism of TRPA1.
Another interesting finding was that heat enhanced desensitization and tachyphylaxis of TRPA1 following agonist activation, but not that of TRPM8. The effects of heat on desensitization of TRPA1 cannot be fully explained by the accelerated activation time course. It is not clear how heat induces desensitization of MO-evoked currents, and the mechanisms need to be further investigated in the future. Importantly, heat-induced desensitization of TRPA1 is not the consequence of thermal activation of TRPA1. Although the structural basis of cold-induced activation/potentiation and heat-induced suppression/desensitization of TRPA1 is completely unknown, we speculate that thermal activation and desensitization may represent independent processes. Related to this notion is the finding that the TRPA1 orthologue from insects (drosophila and mosquito) and snake are activated by heat [39–41]. Drosophila TRPA1 is not only activated by heat but is also desensitized when it is expressed in HEK293 cells (M.K. Chung, unpublished observation). This is consistent with heat-induced activation and desensitization of another insect TRPA1 orthologue, a honeybee Hymenoptera-specific TRPA . The heat-desensitizing mechanism of drosophila and mammalian TRPA1 may be evolutionarily conserved, while thermal activating mechanisms are not. Further structure-function studies are required to investigate the distinct region involved in thermal activation and heat desensitization of these channels. Alternatively, it is possible that the agonist-induced desensitization process is highly sensitive to elevated temperature and heat enhances the rate of desensitization induced by agonist. Investigation of the detailed mechanisms of TRPA1 desensitization and their temperature sensitivity may provide further mechanistic insights.
Since desensitization of TRPA1 is related with the internalization of channel proteins , we cannot exclude the possibility that internalization of TRPA1 by warming could contribute the warmth-induced inhibition of TRPA1 activity. However, we think that the warmth-induced inhibition of TRPA1 cannot be mainly due to such mechanism based upon two reasons: 1) Current amplitudes evoked by 1 mM MO were comparable between 23 and 39°C, suggesting the equivalent existence of functional TRPA1 at the surface of plasma membrane, and 2) the decay time constant of inhibition was as small as < 5 sec, which may be too fast for the internalization of the entire channels in the plasma membrane. Further biochemical and cell biological approaches are required to clarify the mechanisms of warmth-induced effects on TRPA1.
Mild heating not only suppressed recombinant TRPA1, but also attenuated MO-evoked ionic currents in TG neurons, suggesting that warmth suppresses the activation of endogenous TRPA1. We cannot exclude a possibility that MO-induced responses partly involve other molecules, e.g., TRPV1 . Indeed, we also observed a weak activation of TRPV1 by a high concentration of MO and superimposition of MO on warmth potentiated currents (M.K. Chung, unpublished observation). Therefore, MO-evoked currents at warm temperature in sensory neurons could be partly the result of activation of TRPV1. However, such agonistic effects of MO on TRPV1 cannot explain heat-induced suppression of MO-evoked currents in TG, which strongly suggest substantial suppression of agonist-induced activation of TRPA1 in nociceptors.
Persistent activation of TRPA1 may contribute to the maintenance of persistent pain at the site of injury or inflammation [14, 16]. We demonstrated that innocuous heating suppressed MO-induced nociceptor activation. Tissue temperature has been found to increase by approximately 3 - 4°C at the site of acute inflammation in experimental animals [22, 23], which may substantially attenuate activation of TRPA1 by putative endogenous agonists. Therefore, local hyperthermia during acute inflammation may be an endogenous anti-nociceptive mechanism preventing excessive pain at the site of injury. Also heating of local tissues or the whole body is a remedy that has been widely used for attenuating chronic pain conditions, such as low back pain and osteoarthritis [45–47]. Our results suggest that heat-induced suppression of TRPA1 may provide, in part, a peripheral molecular mechanism of heat therapy. It is worthwhile to note that warmth desensitizes TRPA1 only when the channel is activated, but not in the resting state (Figures 2C and 7D). Thus innocuous heating may preferentially desensitize TRPA1 in an active but not a resting state, thus preserving the role of TRPA1 as a sensor of tissue damage.
In conclusion, we demonstrated that TRPA1 is modestly activated and potentiated by cold, but is strongly suppressed by innocuous heat. Warmth-induced suppression is a result, not only of reduced activation, but also of enhanced desensitization. These results may provide a novel insight for thermosensitivity of TRPA1 and a mechanistic rationale for the application of mild heating to attenuate nociception.
Rat TRPA1, human TRPA1, rat TRPM8 cDNAs were generously gifted by Dr. David Julius (University of California, San Francisco, CA). Mouse TRPA1 cDNA was kindly gifted by Dr. Ardem Patapoutian (Scripps Research Institute, San Diego, CA). To generate the human TRPA1 N855S mutant, site directed mutagenesis was performed by overlap extension polymerase chain reaction methods using a pair of specific primers.
HEK293 cells were maintained as previously described . Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and penicillin/streptomycin/glutamine (PSG) in 5% CO2 at 37°C. Lipofectamine-2000 (Invitrogen) was used for the transient transfection of cDNA. To identify transfected cells, cDNA encoding mCherry was co-transfected. HEK293 cells stably transfected with rat or human TRPA1 were generated by selection of transfected cells with G418. Resistant clones were screened for functional expression of TRPA1 by electrophysiology and constitutive expression of mRNA of TRPA1. Transfected HEK293 cells were plated onto polyornithine-coated round coverslips (8-mm in diameter) and used for experiments after 16 to 24 hours.
Rat TG neurons were dissected as described previously . All procedures were conducted using twelve male Sprague-Dawley rats (body weight 200-300 g, Harlan) in accordance with the NIH Guide for the Care and Use of Laboratory Animals and under a University of Maryland-approved Institutional Animal Care and Use Committee protocol. Rats were euthanized using a lethal dose of sodium pentobarbital. The ganglia were dissected out and minced in cold DMEM/F12 containing 10% horse serum and PSG. TG were incubated in 1 mg/ml collagenase (type XI, Sigma) for 30 min at 37°C and triturated with flame-polished Pasteur pipettes. The ganglia were incubated in phosphate-buffered saline (PBS) containing 0.05% trypsin and 0.1% EDTA for 2 min at 37°C. After washing with serum-containing culture medium, dissociated cells were added to a 25% Percoll gradient, centrifuged for 12 min at 900 g, and plated on to polyornithine- and laminin-coated glass coverslips. The neurons were cultured in DMEM containing 10% horse serum, 1% PSG, and 100 ng/ml nerve growth factor, in 5% CO2 at 37°C. The neurons were assayed 16-48 h later.
Following previously described methods [32, 38], conventional whole-cell patch clamp recordings were performed using the Axopatch 200B with Digidata 1440 interface (Molecular Devices). Unless otherwise indicated, the bath solution contained 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES (in mM, pH 7.4 adjusted with NaOH, 300-310 mOsm). The pipette solution contained 140 KCl, 5 NaCl, 10 EGTA, 1 CaCl2, 1 MgCl2, 2.5 Mg-ATP (in mM, pH 7.3 adjusted with KOH, 290-300 mOsm). As indicated in the Results section, external Ca2+ was replaced with Ba2+ in some experiments. To reduce tachyphylaxis of TRPA1 activation in Figure 5, we used divalent-free internal and external solutions containing 150 X-OH, 10 HEPES, 10 EDTA (X = Na in the internal and N-methyl-d-glucamine (NMDG) in the external solution; pH 7.4 adjusted with HCl, 290-300 mOsm). Osmolarity of every solution was measured using a vapour pressure osmometer (Wescor) and adjusted using mannitol as necessary. Borosilicate glass electrodes with tip resistances of 2-4 MΩ were fabricated using a pipette puller (Sutter). Series resistance was compensated > 75%. A 3 M KCl agar salt bridge was used throughout the experiments. To evaluate current amplitudes and current-voltage relationships at the same time in HEK293 cells, we applied repetitive 200 ms voltage ramp pulses (-100 to +100 mV, 1 mV/ms at 0.5 Hz) from a holding potential of 0 mV. Voltage protocols were delivered and currents were acquired using Clampex (Molecular devices).
In sensory neurons, membrane potential was continuously clamped at -70 mV. Following the application of MO, neurons showing currents greater than -4 pA/pF, which was twice the mean rms noise, were regarded as responders. In groups exposed to 39°C heat, the threshold was determined as -8 pA/pF, which was twice the mean heat-induced change.
Bath temperature was controlled using an in-line heater (Warner instruments) connected to a valve-controlled perfusion system driven by gravity. Actual bath temperature was monitored continuously with a thermocouple (IT-18, Physitemp) placed within 4 mm of the patch-clamped cell and recorded in Clampex throughout the experiment. The bath (< 200 μ l) was continuously perfused at a rate of ~3 ml/min. Temperature values referred to in the results section represent the mean of the actual temperatures measured for each group. In this study, we used the term 'warmth' to indicate an innocuous range of heat above that of the skin surface temperature, i.e., approximately 32°C to 40°C.
Ratiometric Ca2+ imaging experiments and analysis were performed as described previously  with slight modifications. Sensory neurons were loaded with 1 μ M fura-2 acetoxymethyl ester (Anaspec, Inc) with 0.01% pluronic acid (Anaspec, Inc) for 40 min at 37°C in Ca2+ imaging buffer containing 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 10 sucrose, 1.2 NaHCO3 (in mM, pH 7.45, 310 mOsm after adjustment with mannitol). Measurement of fluorescence was performed using an inverted fluorescence microscope (Nikon) equipped with a filter changer (Sutter Instruments) and a CCD camera (Nikon). Paired images (340 nm and 380 nm excitation, 510 nm emission) were collected every 2 s and the fura ratios (emission at 340 nm excitation/emission at 380 nm excitation) were calculated. Data acquisition and analysis were performed using NIS Elements (Nikon).
MO (allylisothiocyanate, Sigma) was diluted in DMSO for cellular electrophysiological experiments. Stocks of HC030031, menthol and flufenamic acid (Sigma) were dissolved in DMSO and diluted in the external solution. Formaldehyde (37%, Fisher) was diluted directly in the external solution.
To minimize variability due to the differences in individual cell size, we determined current densities (current amplitude/membrane capacitance). Statistical analysis was performed using Prism or SigmaStat. Curve fitting of current traces was performed using pClamp (Molecular Devices). Concentration-dependent relationships were fitted by a logistics function: Y = Min + (Max-Min)/(1 + 10^(LogEC 50 - X) × H) where Y = current density, Min and Max = Minimum and maximum plateaus, EC 50 = half-maximal concentration, X = log of concentration, H = Hill slope. The curves were fitted without constraining the four parameters. Data in all figures represent mean ± s.e.m. Unless otherwise indicated, the Student's t test was used to compare two groups and P < 0.05 was considered to be statistically significant.
Dulbecco's Modified Eagle Medium
Human embryonic kidney
Transient receptor potential A1
Transient receptor potential v1
We would like to thank to Drs. David Julius and Ardem Patatpoutian for generously providing cDNA constructs and to Drs. Matthias Ringkamp and Michael Caterina for critical reading of manuscript. This work was supported by University of Maryland Dental School start-up fund (to MKC) and NIDCR (DE019694 to MKC and DE016062 to JYR).
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