Matt J. Petrus

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.

Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels

Mechanical Responders Identified Although many cells appear to respond to mechanical stimulation through increased conductance of ion channels in the plasma membrane, the actual channels that mediate these effects—which are important in diverse processes from hearing and touch to control of blood pressure—have remained elusive. Coste et al. (p. 55, published online 2 September) used RNA interference to decrease expression of candidate genes systematically in a mouse neuroblastoma cell line and identified two genes that encode proteins, Piezo1 and Piezo2, which are required for mechanically stimulated cation conductance in these cells and in cultured dorsal root ganglion neurons. Similar proteins are expressed in a range of species from protozoa to vertebrates. The proteins are not similar to known pore-forming proteins and thus could be unusual channels or regulatory components of a channel complex. Cation channel genes encode for a transducer molecule that converts mechanical stimuli into cell signaling. Mechanical stimuli drive many physiological processes, including touch and pain sensation, hearing, and blood pressure regulation. Mechanically activated (MA) cation channel activities have been recorded in many cells, but the responsible molecules have not been identified. We characterized a rapidly adapting MA current in a mouse neuroblastoma cell line. Expression profiling and RNA interference knockdown of candidate genes identified Piezo1 (Fam38A) to be required for MA currents in these cells. Piezo1 and related Piezo2 (Fam38B) are vertebrate multipass transmembrane proteins with homologs in invertebrates, plants, and protozoa. Overexpression of mouse Piezo1 or Piezo2 induced two kinetically distinct MA currents. Piezos are expressed in several tissues, and knockdown of Piezo2 in dorsal root ganglia neurons specifically reduced rapidly adapting MA currents. We propose that Piezos are components of MA cation channels.

TRPM8 Is Required for Cold Sensation in Mice

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling; however, it is unclear whether either ion channel is required for thermosensation in vivo. We show that mice lacking TRPM8 have severe behavioral deficits in response to cold stimuli. In thermotaxis assays of temperature gradient and two-temperature choice assays, TRPM8-deficient mice exhibit strikingly reduced avoidance of cold temperatures. TRPM8-deficient mice also lack behavioral response to cold-inducing icilin application and display an attenuated response to acetone, an unpleasant cold stimulus. However, TRPM8-deficient mice have normal nociceptive-like responses to subzero centigrade temperatures, suggesting the presence of at least one additional noxious cold receptor. Finally, we show that TRPM8 mediates the analgesic effect of moderate cooling after administration of formalin, a painful stimulus. Therefore, depending on context, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia.

A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition

Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18) that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.

An Ion Channel Essential for Sensing Chemical Damage

Tissue damage and its downstream consequences are experimentally assayed by formaldehyde application, which indiscriminately modifies proteins and is presumed to cause pain through broadly acting mechanisms. Here we show that formaldehyde activates the ion channel TRPA1 and that TRPA1-deficient mice exhibit dramatically reduced formaldehyde-induced pain responses. 4-Hydroxynonenal, a reactive chemical produced endogenously during oxidative stress, and other related aldehydes also activate TRPA1 in vitro. Furthermore, painful responses to iodoacetamide, a nonspecific cysteine-alkylating compound, are abolished in TRPA1-deficient mice. Therefore, although these reactive chemicals modify many proteins, the associated pain appears mainly dependent on a single ion channel.

Piezo2 is required for Merkel-cell mechanotransduction

How we sense touch remains fundamentally unknown. The Merkel cell–neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aβ sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell–neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell–neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

TRPA1 Contributes to Cold Hypersensitivity

TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1−/− mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.

High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of ∼14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.Note: The AOP version of this article was corrected on 19 March 2006. Please see the PDF for details.

Nociceptive Signals Induce Trafficking of TRPA1 to the Plasma Membrane

Transient receptor potential A1 (TRPA1) ion channel senses a variety of noxious stimuli and is involved in nociception. Many TRPA1 agonists covalently modify the channel, which can lead to desensitization. The fate of modified TRPA1 and the mechanism of preserving its response to subsequent stimuli are not understood. Moreover, inflammatory signals sensitize TRPA1 by involving protein kinase A (PKA) and phospholipase C (PLC) through unknown means. We show that TRPA1-mediated nocifensive behavior can be sensitized in vivo via PKA/PLC signaling and by activating TRPA1 with the ligand mustard oil (MO). Interestingly, both stimuli increased TRPA1 membrane levels in vitro. Tetanus toxin attenuated the response to the second of two pulses of MO in neurons, suggesting that vesicle fusion increases functional surface TRPA1. Capacitance recordings suggest that MO can induce exocytosis. We propose that TRPA1 translocation to the membrane might represent one of the mechanisms controlling TRPA1 functionality upon acute activation or inflammatory signals.