Adrienne E. Dubin

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

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.

GPR92 as a New G12/13- and Gq-coupled Lysophosphatidic Acid Receptor That Increases cAMP, LPA5

The signaling effects of lysophospholipids such as lysophosphatidic acid (LPA) are mediated by G protein-coupled receptors (GPCRs). There are currently four LPA receptors known as LPA1–4. Genetic deletion studies have identified essential biological functions for LPA receptors in mice. However, these studies have also revealed phenotypes consistent with the existence of as yet unidentified receptors. Toward identifying new LPA receptors, we have screened collections of GPCR cDNAs using reverse transfection and cell-based assays. Here we report an interim result of one screen to identify receptors that produced LPA-dependent changes in cell shape: the orphan receptor GPR92 has properties of a new LPA receptor. Sequence analyses of human GPR92 and its mouse homolog have ∼35% amino acid identity with LPA4/GPR23. The same cell-based approaches that were used to identify and/or characterize LPA1–4, particularly heterologous expression in B103 cells or RH7777 cells, were utilized and compared with known LPA receptors. Retroviral-mediated expression of epitope-tagged receptors was further combined with G protein minigenes and pharmacological intervention, along with calcium imaging and whole-cell patch clamp electrophysiology. LPA-dependent receptor internalization following exposure to LPA but not related lysophospholipids was observed. Furthermore, LPA induced concentration-dependent activation of G12/13 and Gq and increased cAMP levels. Specific [3H]LPA binding was detected in cell membranes heterologously expressing GPR92 but not control membranes. Northern blot and reverse transcriptase-PCR studies indicated a broad low level of expression in many tissues including embryonic brain and enrichment in small intestine and sensory dorsal root ganglia, as well as embryonic stem cells. These results support GPR92 as a fifth LPA receptor, LPA5, which likely has distinct physiological functions in view of its expression pattern.

Piezo proteins are pore-forming subunits of mechanically activated channels

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

Selective Blockade of the Capsaicin Receptor TRPV1 Attenuates Bone Cancer Pain

Cancer colonization of bone leads to the activation of osteoclasts, thereby producing local tissue acidosis and bone resorption. This process may contribute to the generation of both ongoing and movement-evoked pain, resulting from the activation of sensory neurons that detect noxious stimuli (nociceptors). The capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1) is a cation channel expressed by nociceptors that detects multiple pain-producing stimuli, including noxious heat and extracellular protons, raising the possibility that it is an important mediator of bone cancer pain via its capacity to detect osteoclast- and tumor-mediated tissue acidosis. Here, we show that TRPV1 is present on sensory neuron fibers that innervate the mouse femur and that, in an in vivo model of bone cancer pain, acute or chronic administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in a significant attenuation of both ongoing and movement-evoked nocifensive behaviors. Administration of the antagonist had similar efficacy in reducing early, moderate, and severe pain-related responses, suggesting that TRPV1 may be a novel target for pharmacological treatment of chronic pain states associated with bone cancer metastasis.

Nociceptors: the sensors of the pain pathway

Specialized peripheral sensory neurons known as nociceptors alert us to potentially damaging stimuli at the skin by detecting extremes in temperature and pressure and injury-related chemicals, and transducing these stimuli into long-ranging electrical signals that are relayed to higher brain centers. The activation of functionally distinct cutaneous nociceptor populations and the processing of information they convey provide a rich diversity of pain qualities. Current work in this field is providing researchers with a more thorough understanding of nociceptor cell biology at molecular and systems levels and insight that will allow the targeted design of novel pain therapeutics.

The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit

While homomers containing 5-HT3A subunits form functional ligand-gated serotonin (5-HT) receptors in heterologous expression systems (Jackson, M. B., and Yakel, J. L. (1995) Annu. Rev. Physiol. 57, 447–468; Lambert, J. J., Peters, J. A., and Hope, A. G. (1995) in Ligand-Voltage-Gated Ion Channels (North, R., ed) pp. 177–211, CRC Press, Inc., Boca Raton, FL), it has been proposed that native receptors may exist as heteromers (Fletcher, S., and Barnes, N. M. (1998) Trends Pharmacol. Sci. 19, 212–215). We report the cloning of a subunit 5-HT3B with ∼44% amino acid identity to 5-HT3A that specifically modified 5-HT3Areceptor kinetics, voltage dependence, and pharmacology. Co-expression of 5-HT3B with 5-HT3A modified the duration of 5-HT3 receptor agonist-induced responses, linearized the current-voltage relationship, increased agonist and antagonist affinity, and reduced cooperativity between subunits. Reverse transcriptase-polymerase chain reaction in situhybridization revealed co-localization of both 5-HT3B and 5-HT3A in a population of neurons in the amygdala, telencephalon, and entorhinal cortex. Furthermore, 5-HT3Aand 5-HT3B mRNAs were expressed in spleen and intestine. Our data suggest that 5-HT3B might contribute to tissue-specific functional changes in 5-HT3-mediated signaling and/or modulation.

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.