The cellular and molecular mechanisms that enable us to sense cold are not well understood. Insights into this process have come from the use of pharmacological agents, such as menthol, that elicit a cooling sensation. Here we have characterized and cloned a menthol receptor from trigeminal sensory neurons that is also activated by thermal stimuli in the cool to cold range. This cold- and menthol-sensitive receptor, CMR1, is a member of the TRP family of excitatory ion channels, and we propose that it functions as a transducer of cold stimuli in the somatosensory system. These findings, together with our previous identification of the heat-sensitive channels VR1 and VRL-1, demonstrate that TRP channels detect temperatures over a wide range and are the principal sensors of thermal stimuli in the mammalian peripheral nervous system.

Wasabi, horseradish and mustard owe their pungency to isothiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust hypersensitivity to thermal and mechanical stimuli. Despite their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Here we show that mustard oil depolarizes a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the TRP ion channel family recently implicated in the detection of noxious cold. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors.

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca 2؉ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca 2؉ on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca 2؉ permeability was greatly reduced, extracellular Ca 2؉ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca 2؉ buffering, which allowed intracellular Ca 2؉ levels to become elevated upon entry through D918A channels. Application of Ca 2؉ to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca 2؉ by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg 2؉ , Ba 2؉ , and Ca 2؉ but potentiated only by Ba 2؉ and Ca 2؉ . Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca 2؉ -binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca 2؉ influx through TRPA1 to nociceptive signaling. Members of the transient receptor potential (TRP)2 family of ion channels that are expressed by sensory neurons in dorsal root and trigeminal ganglia serve as sensors for temperature and noxious stimuli (1, 2). Of these, TRPA1 is one of the key sensors for pungent chemicals and environmental irritants and is essential for behavioral responses of mice to conditions that evoke inflammatory pain (3-7). Inflammatory mediators, such as bradykinin, bind to G protein-coupled receptors on nociceptors, initiating a second messenger signaling cascade that leads to Ca 2ϩ influx mediated in part by the opening of Ca 2ϩ -permeable TRPA1 channels (5,8,9). TRPA1 is also activated directly by a wide range of chemicals that cause painful sensations, including food additives, such as mustard oil (MO), cinnamaldehyde (Cin), onion, raw garlic, and thyme; environmental irritants, such as formaldehyde and acrolein (a component of automobile exhaust); and products of oxidative stress (4, 8, 10 -16). Many of these chemicals activate TRPA1 by binding covalently to reactive cysteine residues in the amino terminus of the protein (17, 18), producing a modification of the channel that can last for more than 1 h and which leads to persistent activation of TRPA1 currents (18,19). Ca 2ϩ plays at...

Many primary sensory neurons are polymodal, responding to multiple stimulus modalities (chemical, thermal, or mechanical), yet each modality is recognized differently. While polymodality implies that stimulus encoding occurs in higher centers such as the spinal cord or brain, recent sensory neuron ablation studies find that behavioral responses to different modalities require distinct subpopulations, suggesting the existence of modality-specific labeled-lines at the level of the sensory afferent. Here we provide evidence that neurons expressing TRPM8, a cold- and menthol-gated channel required for normal cold responses in mammals, represents a labeled-line solely for cold sensation. We examined the behavioral significance of conditionally ablating TRPM8+ neurons in adult mice, finding that, like animals lacking TRPM8 channels (Trpm8−/−), animals depleted of TRPM8 neurons (ablated) are insensitive to cool to painfully cold temperatures. Ablated animals showed little aversion to noxious cold and did not distinguish between cold and a preferred warm temperature, a phenotype more profound than that of Trpm8−/− mice which exhibit only partial cold avoidance and preference behaviors. In addition to acute responses, cold pain associated with inflammation and nerve injury was significantly attenuated in ablated and Trpm8−/− mice. Moreover, cooling-induced analgesia after nerve injury was abolished in both genotypes. Lastly, heat, mechanical, and proprioceptive behaviors were normal in ablated mice, demonstrating that TRPM8 neurons are dispensable for other somatosensory modalities. Together these data show that while some limited cold sensitivity remains in Trpm8−/− mice, TRPM8 neurons are required for the breadth of behavioral responses evoked by cold temperatures.

Somatosensory neurons detect environmental stimuli, converting external cues into neural activity that is relayed first to second-order neurons in the spinal cord. The detection of cold is proposed to be mediated by the ion channels TRPM8 and TRPA1. However, there is significant debate regarding the role of each channel in cold-evoked pain, complicating their potential as drug targets for conditions such as cold allodynia and hyperalgesia. To address this debate, we generated mice lacking functional copies of both channels and examined behaviors and neural activity in response to painful cold and noxious cooling compounds. Whereas normal mice display a robust preference for warmth over cold, both TRPM8-null (TRPM8−/−) and TRPM8/TRPA1 double knockout mice (DKO) display no preference until temperatures reach the extreme noxious range. Additionally, in contrast to wildtype mice that avoid touching cold surfaces, mice lacking TRPM8 channels display no such avoidance and explore noxious cold surfaces, even at 5°C. Furthermore, nocifensive behaviors to the cold mimetic icilin are absent in TRPM8−/− and DKO mice, but are retained in TRPA1-nulls (TRPA1−/−). Lastly, neural activity, measured by expression of the immediate early gene c-fos, evoked by hindpaw stimulation with noxious cold, menthol, or icilin is reduced in TRPM8−/− and DKO mice, but not in TRPA1−/− animals. Thus our results show that noxious cold signaling is exclusive to TRPM8, mediating neural and behavioral responses to cold and cold mimetics, and that TRPA1 is not required for acute cold pain in mammals.