The Contribution of TRPM8 and TRPA1 Channels to Cold Allodynia and Neuropathic Pain

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.     

Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin

Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.     

Behavioral Testing of the Effects of Thermosensitive TRP Channel Agonists on Touch,Temperature, and Pain Sensations

It was recently found that transient receptor potential (TRP) channels play an important role in the transduction of thermal, mechanical, and chemical stimuli underlying the somatic sensation. Several types of TRP channels exhibit sensitivity to increases or decreases in temperature, as well as to the action of chemical ligands that elicit similar thermal or painful sensations. These agents include menthol, mustard oil, cinnamaldehyde (CA), gingerol, capsaicin, camphor, eugenol, and others. Cinnamaldehyde is a pungent chemical obtained from cinnamon, which acts as an agonist of the TRPA1 channels; these channels were originally reported to be activated by cold temperatures (below 18°C). TRPA1 is also implicated in cold nociception. However, its role in the formation of cold pain is more controversial, with discrepant reports that TRPA1s do or do not respond to intense cooling. Menthol derived from plants of the mint family enhances the feeling of coldness by interacting with the cold-sensitive TRPM8 channels, but its effect on pain is less well understood. Using behavioral methods, we showed that unilateral intraplantar injection of CA (5 to 20%) induced a significant concentration-dependent decrease in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e., heat hyperalgesia. Cinnamaldehyde also significantly reduced mechanical withdrawal thresholds for the injected paw, i.e., evoked mechanical allodynia. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). In contrast to CA, menthol in a dose-dependent manner increased the latency for noxious heat-evoked withdrawal, i.e., exerted an antinociceptive effect. Menthol did not affect mechanosensation except for a weak allodynic effect when applied in the highest concentration used (40 %), indicating that it did not exert a local anesthetic effect. Menthol had a biphasic effect on cold avoidance. High concentrations of menthol reduced cold avoidance, i.e., induced cold hypoalgesia, while low menthol concentrations significantly intensified cold avoidance. The highest menthol concentration provided cold hypoalgesia (cold plate test), while lower concentrations had no effect. Taken together, our data support the idea that TRPA1 and TRPM8 channels represent promising peripheral targets for pain modulation.     

Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice

ABSTRACT: BACKGROUND: Oxaliplatin, a platinum-based chemotherapeutic agent, causes an unusual acute peripheral neuropathy. Oxaliplatin-induced acute peripheral neuropathy appears in almost all patients rapidly after infusion, and is triggered or exacerbated by cold, while its mechanisms are poorly understood. In this study, the involvement of thermosensitive transient receptor potential channels (TRPA1, TRPM8 and TRPV1) in oxaliplatin-induced acute hypersensitivity was investigated in mice. RESULTS: A single intraperitoneal administration of oxaliplatin (5 mg/kg) induced cold but not mechanical hypersensitivity within 2 h. The oxaliplatin-induced acute cold hypersensitivity was abolished by the TRPA1 antagonist HC-030031 (100 mg/kg) and by TRPA1 deficiency. Infusion of another platinum-based chemotherapeutic agent, cisplatin (5 mg/kg), or the non-platinum-containing chemotherapeutic agent, paclitaxel (6 mg/kg) failed to induce mechanical or cold hypersensitivity. The nocifensive behaviors induced by intraplantar injections of allyl-isothiocyanate (AITC; TRPA1 agonist) and menthol (TRPM8/TRPA1 agonist) were significantly enhanced in mice treated for 2 h with oxaliplatin, while capsaicin (TRPV1 agonist)-induced nocifensive behaviors were not affected. By contrast, neither cisplatin nor paclitaxel affected AITC-induced nocifensive behaviors. Pretreatment of cultured mouse dorsal root ganglia (DRG) neurons with oxaliplatin (100 microM) for 1, 2, or 4 h increased the number of AITC-sensitive neurons whereas there was no change in the number of menthol- or capsaicin-sensitive neurons. CONCLUSIONS: Taken together, these results suggest that a brief treatment with oxaliplatin is sufficient to enhance the responsiveness of TRPA1 but not that of TRPM8 and TRPV1 expressed by DRG neurons, which may contribute to the characteristic acute peripheral neuropathy induced by oxaliplatin.     

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ～25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.     

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR‐32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009. © 2009 Wiley‐Liss, Inc     

Effects of the neurotrophic factor artemin on sensory afferent development and sensitivity

Artemin is a neuronal survival and differentiation factor in the glial cell line-derived neurotrophic factor family.Its receptor GFRα3 is expressed by a subpopulation of nociceptor type sensory neurons in the dorsal root and trigeminal ganglia(DRG and TG).These neurons co-express the heat,capsaicin and proton-sensitive channel TRPV1 and the cold and chemical-sensitive channel TRPA1.To further investigate the effects of artemin on sensory neurons,we isolated transgenic mice(ARTN-OE mice) that overexpress art...     

Novel menthol-derived cooling compounds activate primary and second-order trigeminal sensory neurons and modulate lingual thermosensitivity

We presently investigated 2 novel menthol derivatives GIV1 and GIV2, which exhibit strong cooling effects. In previous human psychophysical studies, GIV1 delivered in a toothpaste medium elicited a cooling sensation that was longer lasting compared with GIV2 and menthol carboxamide (WS-3). In the current study, we investigated the molecular and cellular effects of these cooling agents. In calcium flux studies of TRPM8 expressed in HEK cells, both GIV1 and GIV2 were approximately 40- to 200-fold more potent than menthol and WS-3. GIV1 and GIV2 also activated TRPA1 but at levels that were 400 times greater than those required for TRPM8 activation. In calcium imaging studies, subpopulations of cultured rat trigeminal ganglion and dorsal root ganglion cells responded to GIV1 and/or GIV2; the majority of these were also activated by menthol and some were additionally activated by the TRPA1 agonist cinnamaldehyde and/or the TRPV1 agonist capsaicin. We also made in vivo single-unit recordings from cold-sensitive neurons in rat trigeminal subnucleus caudalis (Vc). GIV 1 and GIV2 directly excited some Vc neurons, GIV1 significantly enhanced their responses to cooling, and both GIV1 and GIV2 reduced responses to noxious heat. These novel cooling compounds provide additional molecular tools to investigate the neural processes of cold sensation.     

Effects of Antagonists and Heat on TRPM8 Channel Currents in Dorsal Root Ganglion Neuron Activated by Nociceptive Cold Stress and Menthol

Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperature and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of environmental cold stress such as cold allodynia in dorsal root ganglion (DRG) neuron; however, the underlying mechanisms of action are unclear. We tested the effects of physiological heat (37°C), anthralic acid (ACA and 0.025 mM), 2-aminoethyl diphenylborinate (2-APB and 0.05) on noxious cold (10°C) and menthol (0.1 mM)-induced TRPM8 cation channel currents in the DRG neurons of rats. DRG neurons were freshly isolated from rats. In whole-cell patch clamp experiments, TRPM8 currents were consistently induced by noxious cold or menthol. TRPM8 channels current densities of the neurons were higher in cold and menthol groups than in control. When the physiological heat is introduced by chamber TRPM8 channel currents were inhibited by the heat. Noxious cold-induced Ca²⁺ gates were blocked by the ACA although menthol-induced TRPM8 currents were not blocked by ACA and 2-APB. In conclusion, the results suggested that activation of TRPM8 either by menthol or nociceptive cold can activate TRPM8 channels although we observed the protective role of heat, ACA and 2-APB through a TRPM8 channel in nociceptive cold-activated DRG neurons. Since cold allodynia is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.     

How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation

Recognition of temperature is a critical element of sensory perception and allows us to evaluate both our external and internal environments. In vertebrates, the somatosensory system can discriminate discrete changes in ambient temperature, which activate nerve endings of primary afferent fibers. These thermosensitive nerves can be further segregated into those that detect either innocuous or noxious (painful) temperatures; the latter neurons being nociceptors. We now know that thermosensitive afferents express ion channels of the transient receptor potential (TRP) family that respond at distinct temperature thresholds, thus establishing the molecular basis for thermosensation. Much is known of those channels mediating the perception of noxious heat; however, those proposed to be involved in cool to noxious cold sensation, TRPM8 and TRPA1, have only recently been described. The former channel is a receptor for menthol, and links the sensations provided by this and other cooling compounds to temperature perception. While TRPM8 almost certainly performs a critical role in cold signaling, its part in nociception is still at issue. The latter channel, TRPA1, is activated by the pungent ingredients in mustard and cinnamon, but has also been postulated to mediate our perception of noxious cold temperatures. However, a number of conflicting reports have suggested that the role of this channel in cold sensation needs to be confirmed. Thus, the molecular logic for the perception of cold-evoked pain remains enigmatic. This review is intended to summarize our current understanding of these cold thermoreceptors, as well as address the current controversy regarding TRPA1 and cold signaling.     

Involvement of Rabring7 in EGF receptor degradation as an E3 ligase

Thermosensitive TRP channels display unique thermal responses, suggesting distinct roles mediating sensory transmission of temperature. However, whether relative expression of these channels in dorsal root ganglia (DRG) is altered in nerve injury is unknown. We developed a multiplex ribonuclease protection assay (RPA) to quantify rat TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8 RNA levels in DRG. We used the multiplex RPA to measure thermosensitive TRP channel RNA levels in DRG from RTX-treated rats (300 microg/kg) or rats with unilateral sciatic nerve chronic constriction injury (CCI). TRPV1 and TRPA1 RNA were significantly decreased in DRG from RTX-treated rats, indicating functional colocalization of TRPA1 and TRPV1 in sensory nociceptors. In DRG from CCI rats, TRPA1, TRPV2, and TRPM8 RNA showed slight but significant increases ipsilateral to peripheral nerve injury. Our findings support the hypothesis that increased TRP channel expression in sensory neurons may contribute to mechanical and cold hypersensitivity.     

A fluorescence-immunohistochemical study on phosphorylation of ERK1/2, p38, and STAT3 in rat dorsal root ganglia following noxious stimulation of hind paw sensory neurons

A fluorescence-immunohistochemical investigation was performed in lumbar dorsal root ganglia (DRGs) neurons of the rat with regard to ERK1/2-, p38- and STAT3-phosphorylation in response to nociceptor activation in the rat. The stimuli applied were perineural capsaicin treatment of the sciatic nerve, mustard oil application to the hind paw and heat or cold stimulation of the hind paw. The time points of investigations were 15 min/30 min after perineural capsaicin, 30 min/2 h/4 h for mustard oil, 10 min/4 h for cold and 30 min/2 h/8 h for the heat stimulus. All four stimuli lead to a time-dependent, significant 2-3 fold increase in the number of small and medium size DRG cells displaying cytoplasmic staining for p-ERK1/2, but to no activation of satellite cells. Phosphorylated p38 immunoreactivity was increased in the cytoplasma of DRG cells at 2 h after the mustard oil treatment of the hind paw and 30 min after the perineural capsaicin application to the sciatic nerve axons, but not following heat or cold stimuli to the hind paws. Phospho-STAT3 staining was characteristically observed as nuclear and cytoplasmic staining. It was found increased after the perineural capsaicin application to the sciatic nerve axons, however, no marked increase was found with the other 3 noxious stimuli. The present results show that sensory neurons respond with a selective long-lasting increase in p-ERK1/2 in small and medium-size DRG cells, when their axons or axon terminals are stimulated by capsaicin, mustard oil, noxious heat or noxious cold.     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

Relationship of single-nucleotide polymorphism rs11562975 in thermo-sensitive ion channel TRPM8 gene with human sensitivity to cold and menthol

The examination of people belonging to the Russian ethnic group revealed that 20.3% of subjects had heterozygous genotype, containing the C-allele in single nucleotide polymorphism rs11562975, located in exon 7 of the gene encoding the temperature-sensitive ion channel TRPM8. Functional differences, associated with sensitivity to cold and menthol were identified between subjects with different genotypes of the polymorphism rs11562975 (GG and GC). Subjects with heterozygous genotype GC were characterized by increased sensitivity to cold and reduced sensitivity to menthol, agonist of the ion channel TRPM8, compared with subjects with homozygous genotype GG.     

ANKTM1, a TRP-like channel expressed in nociceptive neurons,is activated by cold temperatures

Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.     

Human TRPM8 and TRPA1 pain channels,including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca²⁺ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)₁₀ retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.