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.

Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-gamma to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family.

Allyl isothiocyanate, the pungent principle of wasabi and other mustard oils, produces pain by activating TRPA1, an excitatory ion channel on sensory nerve endings. Isothiocyanates are membranepermeable electrophiles that form adducts with thiols and primary amines, suggesting that covalent modification, rather than classical lock-and-key binding, accounts for their agonist properties. Indeed, we show that thiol reactive compounds of diverse structure activate TRPA1 in a manner that relies on covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. These findings suggest an unusual paradigm whereby natural products activate a receptor through direct, reversible, and covalent protein modification.chemical modification ͉ irritants ͉ natural products ͉ pain P lants have evolved ingenious defensive strategies to ward off herbivorous predators. Such protective mechanisms often involve the production of chemical irritants that activate sensory nerve fibers of offending creatures to produce discomfort or pain (1, 2). For example, plants of the genus Brassica (mustard), Allium (onion), and Cinnamomum (cinnamon) produce pungent isothiocyanate, thiosulfinate, and ␣,␤-unsaturated aldehyde compounds, respectively, which elicit acute pain and neurogenic inflammation by activating TRPA1, a nonselective cation channel expressed by sensory neurons of the pain pathway (3-7). Each of these compounds is capable of forming covalent adducts with thiols, primary amines, and to a lesser extent, hydroxyl groups (8), raising questions as to how electrophiles with such indiscriminate reactivity function as reversible and specific ionchannel agonists.Here we provide evidence to support a model whereby a variety of structurally distinct environmental irritants activate TRPA1 through a mechanism primarily involving covalent modification of specific cysteine side chains located within the putative cytoplasmic N-terminal domain of the channel. Pharmacological analysis of various sulfhydryl reactive agents provides a biochemical rationale for the reversible properties of mustard oil and other naturally occurring TRPA1 agonists. Importantly, TRPA1 mutants that are insensitive to these electrophilic agonists retain their ability to function as ''receptor operated'' channels, indicating that distinct biochemical pathways can contribute to TRPA1 activation by environmental or endogenous stimuli. These studies provide a mechanistic framework to further elucidate biochemical and structural parameters underlying activation of this member of the TRP ion channel family. ResultsThe diverse chemical nature of TRPA1-activating irritants (Fig. 1a) suggests that reactivity, rather than structure per se, constitutes the critical determinant of TRPA1 agonist activity. To clarify this issue, we first asked whether benzyl thiocyanate (BTC) is able to activate TRPA1. BTC is isosteric with the TRPA1 agonist benzyl isothiocyanate (BITC), and, thus, whereas both compounds possess thiocyanate functional groups of similar size, th...

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.

The capsaicin receptor TRPV1, one of the major transduction channels in the pain pathway, integrates information from extracellular milieu to control excitability of primary nociceptive neurons. Sensitization of TRPV1 heightens pain sensation to moderately noxious or even innocuous stimuli. We report here that oxidative stress markedly sensitizes TRPV1 in multiple species' orthologs. The sensitization can be recapitulated in excised insideout membrane patches, reversed by strong reducing agents, and blocked by pretreatment with maleimide that alkylates cysteines. We identify multiple cysteines required for full modulation of TRPV1 by oxidative challenges. Robust oxidative modulation recovers the agonist sensitivity of receptors desensitized by prolonged exposure to capsaicin. Moreover, oxidative modulation operates synergistically with kinase or proton modulations. Thus, oxidative modulation is a robust mechanism tuning TRPV1 activity via covalent modification of evolutionarily conserved cysteines and may play a role in pain sensing processes during inflammation, infection, or tissue injury.covalent modification ͉ pain