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.

Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.

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.

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.

Visualizing cold spots: TRPM8-expressing sensory neurons and their projections.

Environmental stimuli such as temperature and pressure are sensed by dorsal root ganglion (DRG) neurons. DRG neurons are heterogeneous, but molecular markers that identify unique functional subpopulations are mainly lacking. ThermoTRPs are members of the transient receptor potential family of ion channels and are gated by shifts in temperature. TRPM8 is activated by cooling, and TRPM8-deficient mice have severe deficits in cool thermosensation. The anatomical and functional properties of TRPM8-expressing fibers have not been not comprehensively investigated. We use mice engineered to express the farnesylated enhanced green fluorescent protein (EGFPf) from the TRPM8 locus (TRPM8(EGFPf)) to explore this issue. Virtually all EGFPf-positive cultured DRG neurons from hemizygous mice (TRPM8(EGFPf/+)) responded to cold and menthol. In contrast, EGFPf-positive DRGs from homozygous mice (TRPM8(EGFPf/EGFPf)) had drastically reduced cold responses and no menthol responses. In vivo, EGFPf-positive neurons marked a unique population of DRG neurons, a majority of which do not coexpress nociceptive markers. The fraction of DRG neurons expressing EGFPf was not altered under an inflammatory condition, although an increase in TRPV1-coexpressing neurons was observed. TRPM8(EGFPf) neurons project to the superficial layer I of the spinal cord, making distinct contacts when compared with peptidergic projections. At the periphery, TRPM8(EGFPf) projections mark unique endings in the most superficial layers of epidermis, including bush/cluster endings of the mystacial pads. We show that TRPM8 expression functionally associates with cold sensitivity in cultured DRGs, and provide the first glimpses of the unique anatomical architecture of cold fibers in vivo.

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.

Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin.

Environmental temperature is thought to be directly sensed by neurons through their projections in the skin. A subset of the mammalian transient receptor potential (TRP) family of ion channels has been implicated in this process. These "thermoTRPs" are activated at distinct temperature thresholds and are typically expressed in sensory neurons. TRPV3 is activated by heat (>33 degrees C) and, unlike most thermoTRPs, is expressed in mouse keratinocytes. We found that TRPV3 null mice have strong deficits in responses to innocuous and noxious heat but not in other sensory modalities; hence, TRPV3 has a specific role in thermosensation. The natural compound camphor, which modulates sensations of warmth in humans, proved to be a specific activator of TRPV3. Camphor activated cultured primary keratinocytes but not sensory neurons, and this activity was abolished in TRPV3 null mice. Therefore, heat-activated receptors in keratinocytes are important for mammalian thermosensation.

Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.

Tactile information is perceived by a heterogeneous population of specialized neurons. Neurotrophin receptors (the receptor tyrosine kinases, Trks) mark the major classes of these sensory neurons: TrkA is expressed in neurons that sense temperature and noxious stimuli, and TrkC is expressed in proprioceptive neurons that sense body position. Neurotrophin signaling through these receptors is required for cell survival. To test whether neurotrophins have an instructive role in sensory specification, we expressed rat TrkC from the TrkA (also known as Ntrk1) locus in mice. The surviving presumptive TrkA-expressing neurons adopted a proprioceptive phenotype, indicating that neurotrophin signaling can specify sensory neuron subtypes.

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.

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.

A heat-sensitive TRP channel expressed in keratinocytes.

Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

A TRP channel that senses cold stimuli and menthol.

A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.