Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

Transient receptor potential melastatin 8 (TRPM8) is the best molecular candidate for innocuous cold detection by peripheral thermoreceptor terminals. To dissect out the contribution of this cold-and menthol-gated, nonselective cation channel to cold transduction, we identified BCTC [N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide] as a potent and full blocker of recombinant TRPM8 channels. In cold-sensitive trigeminal ganglion neurons of mice and guinea pig, responses to menthol were abolished by BCTC. In contrast, the effect of BCTC on cold-evoked responses was variable but showed a good correlation with the presence or lack of menthol sensitivity in the same neuron, suggesting a specific blocking action of BCTC on TRPM8 channels. The biophysical properties of native cold-gated currents (I cold ), and the currents blocked by BCTC were nearly identical, consistent with a role of this channel in cold sensing at the soma. The temperature activation threshold of native TRPM8 channels was significantly warmer than those reported in previous expression studies. The effect of BCTC on native I cold was characterized by a dose-dependent shift in the temperature threshold of activation.The role of TRPM8 in transduction was further investigated in the guinea pig cornea, a peripheral territory densely innervated with cold thermoreceptors. All cold-sensitive terminals were activated by menthol, suggesting the functional expression of TRPM8 channels in their membrane. However, the spontaneous activity and firing pattern characteristic of cold thermoreceptors was totally immune to TRPM8 channel blockade with BCTC or SKF96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1 H-imidazole hydrochloride). Cold-evoked responses in corneal terminals were also essentially unaffected by these drugs, whereas responses to menthol were completely abolished. The minor impairment in the ability to transduce cold stimuli by peripheral corneal thermoreceptors during TRPM8 blockade unveils an overlapping functional role for various thermosensitive mechanisms in these nerve terminals.

Discomfort in dry eye is possibly caused by an augmented ongoing activity in corneal cold neurons secondary to dryness-induced alterations in sodium and potassium channel expression.

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Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterolrich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn 934 residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol-and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.Ambient temperature detection is a critical biological process carried out by terminals of primary afferent sensory neurons of the dorsal root (DRG) 2 and trigeminal ganglia in the mammalian sensory system (1). Thermosensitive nerves express a subset of proteins of the transient receptor potential (TRP) ion channel family that are activated at different temperatures, making these cationic ion channels central elements in the temperature sensing machinery of peripheral nerve endings (2). Among thermoTRPs, TRPM8 (transient receptor potential melastatine 8) is characterized by its enhanced activity at low temperatures (threshold, ϳ25°C) and by application of cooling compounds such as menthol and icilin (3, 4). These properties, and its selective expression in a subset of small sensory neurons, make TRPM8 an excellent candidate for transducing cold temperatures at nerve endings, a view supported by behavioral findings in TRPM8 knock-out mice (5-7). In addition to mild cold temperature detection, several recent studies (8, 9) have attributed a role to TRPM8 in noxious cold transduction and nociception.Functional studies in chimeric channels suggest that the C terminus of TRPM8 contains structural elements important in temperature-dependent gating (10). However, the molecular mechanisms underlying temperature sensitivity of TRPM8 are still unknown. It has been hypothesized that TRP channels might sense temperature-mediated changes in lipid bilayer tension (11,12). Whereas at physiological temperatures most of the plasma membrane remains in a l...

Summary Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods and cones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs where they associate with voltage-gated ion channels, enabling light to control action potential firing. All charged photoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches and membrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently-labeled RGC dendritic projections together indicate that photosensitization is highly selective for OFF-RGCs. Hence P2X receptors are a natural conduit allowing cell type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease.

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