InteractiveFly: GeneBrief

painless: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

In humans and other vertebrates, painful stimuli are sensed by specialized neurons known as nociceptors, which fire in response to noxious heat, mechanical, or chemical stimuli that have the potential to cause tissue damage. The signals are in turn processed by the central nervous system and perceived as pain, which serves an indispensable, protective role. Nociceptors are also involved in pathological pain states caused by inflammation, nerve damage, or cancer. An increased understanding of nociception therefore is of wide interest, and model systems for molecular genetic analysis are desirable (Tracey, 2003).

The painless gene was discovered in a genetic screen for Drosophila mutants defective in noxious heat response, a paradigm for nociception. In response to the touch of a probe heated above 38°C, Drosophila larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. Recordings from wild-type larval nerves identified neurons that initiate strong spiking above 38°C, and this activity is absent in the painless mutant. The painless mRNA encodes a protein of the transient receptor potential ion channel family. Painless is required for both thermal and mechanical nociception, but not for sensing light touch. painless is expressed in peripheral neurons that extend multiple branched dendrites beneath the larval epidermis, similar to vertebrate pain receptors. An antibody to Painless binds to localized dendritic structures that are hypothesized to be involved in nociceptive signaling (Tracey, 2003).

Great progress in understanding the molecular mechanisms underlying pain signaling was made with the cloning and identification of the vanilloid receptor (TRPV1), an ion channel of the transient receptor potential (TRP) ion channel family, which has been proposed to function at the transduction step in nociceptive pathways (Caterina, 1997). In Xenopus oocytes and human embryonic kidney (HEK) cells, TRPV1-dependent currents are gated by stimuli that are also capable of activating nociceptors, including heat in the noxious temperature range, protons, and capsaicin, the spicy ingredient in chili peppers (Tominaga, 1998; Caterina, 1997). In these systems, the TRPV1 channel is gated by heat in the range of 42°C-48°C, and a related channel, TRPV2, is activated at still higher temperatures (Caterina, 1999). More recently, another channel of the TRP family has been shown to be activated by menthol and cool temperatures, leading to the suggestion that the ion channels in the TRP family might play a central role in sensing temperature (McKemy, 2002; Peier, 2002a). Although the vertebrate TRPV1 channel has been shown to be activated by heat and protons in heterologous systems, mice mutant for TRPV1 still show a marked behavioral response to noxious heat and no defect in mechanical nociception, suggesting that other genes are involved in these processes (Caterina, 2000). This may be due to functional redundancy, as several other genes closely related to TRPV1 are expressed in dorsal root ganglia and are activated by heat in heterologous expression systems (Peier, 2002a; Smith, 2002; Xu, 2002; Tracey, 2003 and references therein).

The TRP gene family has been subdivided into major groups based on sequence homology (Montell, 2002). For example, the TRPC class is closely related to the canonical TRPs identified for their role in Drosophila phototransduction, the TRPV class are closely related to the vanilloid receptor, and the TRPN class is closely related to No mechanoreceptor potential-C (Walker, 2000), a Drosophila gene suspected to participate in mechanosensation (Tracey, 2003).

It has been suggested that the role of TRPV channels in sensing noxious stimuli is an ancient one, since a TRPV1-related gene in C. elegans, osm-9, has been shown to be required for avoidance of chemicals and high osmolarity (Roayaie, 1998). A family of genes related to osm-9 and the capsaicin receptor (the ocr gene family) has been identified in C. elegans. These studies have led to the proposal that the transduction mechanisms for TRPs, which sense physical stimuli, depend on combinatorial expression patterns of different TRP genes, allowing a single TRP gene to play a polymodal sensory role (Tobin, 2002). However, osm-9 mutants have also been tested in C. elegans thermal avoidance and show a strong response to noxious heat (Wittenburg, 1999). Invertebrate molecular and cellular mechanisms underlying thermal nociception are unknown (Tracey, 2003).

In models of nociception, noxious heat is often used as the stimulus to elicit a defensive motor output, as in the tail flick response of the rat. Since heat has also been shown to be an effective negatively reinforcing stimulus in adult Drosophila learning (Brembs, 2000; Mariath, 1985), it was conjectured that heat might also be used to study nociception per se. A normal, undisturbed Drosophila larva moves through its environment with a rhythmic motion. In response to light touch with a probe, a larva will pause or make one or more contractile waves, moving away from the stimulus (Kernan, 1994; Tracey, 2003) and references therein).

In contrast, when touched with the same probe heated above a threshold temperature, larvae are seen to vigorously roll sideways in a corkscrew-like motion. The threshold probe temperature for eliciting this behavior is 39°C-41°C (noxious heat), at which temperature several seconds of stimulation are required to induce rolling, but at 42°C or higher, the response occurs in as little as 0.4 s. Importantly, the temperature threshold for firing of nociceptors in vertebrates, including primates, is similar, 39°C-41°C. Since vertebrate nociceptors also respond to noxious mechanical stimuli, the response of Drosophila larvae to strong punctate stimuli or pinching of the cuticle with forceps was examined, and these elicit the same rolling behavior as noxious heat (Tracey, 2003).

In vertebrates, the cell bodies of nociceptors are located in sensory ganglia. These cells have projections to the periphery, where profuse branching of naked dendrites occurs beneath the skin. In contrast to other sensory modalities of the epidermis, which utilize specialized receptor cells to transduce signals, the naked dendrites of nociceptors themselves are thought to contain the transducing machinery for noxious stimuli (Tracey, 2003).

Similar distinctions prevail in the peripheral nervous system of Drosophila. Type I sensory neurons have a single dendrite as part of a specialized sensillum. Type II, or multidendritic sensory neurons, do not appear to be associated with specialized receptor cells but utilize naked dendrites. Although mutations affecting their developmental biology and branching patterns are under active investigation, the function of these cells was previously unknown (Tracey, 2003).

Kernan (1994) isolated Drosophila mutants that were insensitive to a light touch on the nose. Several of the mutants isolated in that screen have now been cloned, and each has been found to be expressed in ciliated Type I sensory organs, but not in the multidendritic neurons. Because the md-da neurons beneath the cuticle project a plexus of naked dendrites similar to the pain-sensing neurons in mammalian skin and because they were of unknown function, it seemed possible that they might function as the nociceptors (Tracey, 2003).

To test that hypothesis, a cell-specific 'driver' strain was used to selectively disrupt the function of these cells. The tetanus toxin light chain (TeTxLC) blocks calcium-dependent evoked synaptic vesicle release through proteolytic cleavage of the v-SNARE synaptobrevin. To create larvae expressing TeTxLC in md neurons, the enhancer trap driver strain GAL4109(2)80 (md-GAL4), in which GAL4 is expressed in all md-da neurons of the larval peripheral nervous system, and in approximately 100 cells of the central nervous system, was used. When crossed to lines bearing UAS binding sites for GAL4 upstream of a gene of interest, md-GAL4 thus causes expression of this gene specifically in the above pattern. md-GAL4 was crossed to a line carrying UAS-TeTxLC and to a negative control line containing UAS-IMPTNT-V, which contains an inactivating point mutation (Tracey, 2003).

To quantify the response, third instar larvae, from vials seeded by adult flies for 7 days at room temperature, were used. The noxious heat probe (a soldering iron sharpened to a chisel tip shape 0.6 mm wide) was set to maintain a temperature of 46°C, and the stimulus was delivered by gently touching the larvae laterally, in abdominal segments four, five, or six. At that temperature, wild-type larvae perform the rolling avoidance behavior within less than 1 s from the initiation of contact (average response time, 0.4 ± 0.06 s) (Tracey, 2003).

In contrast, third instar larvae expressing the tetanus toxin light chain in the expression pattern of md-GAL4 were strikingly unresponsive to noxious heat. Indeed, the majority of md-GAL4/UAS-TeTxLC larvae did not respond at all, even after 10 s of stimulation. These larvae also fail to roll in response to strong mechanical stimuli. However, control larvae with the driver alone or expressing the tetanus toxin with the mutated catalytic domain showed a normal rolling response. Therefore, the neurons targeted by md-GAL4 are essential for nociception. Although one can not exclude a role for the central neurons expressing this driver, this result suggested that the multidendritic neurons, or a subset of them, might be the ones that function as nociceptors (Tracey, 2003).

These data provide the first genetic evidence for the sensory function of the multidendritic neurons. The effects of the painless mutations on producing nociception defects are more specific than blocking evoked synaptic vesicle release in the multidendritic neurons with tetanus toxin. In addition to nociception defects, md-GAL4/UAS-TeTLx larvae appear mildly uncoordinated. They do, however, respond to light touch. The more general effect of tetanus toxin driven by md-GAL4 may indicate other roles for multidendritic neurons, or alternatively, it might be due to the cells in the central nervous system that express this driver (Tracey, 2003).

Electrophysiological recordings provided evidence for multiple types of heat sensitive units in larval nerves. Some units show strong activation at temperatures that elicite rolling behavior. Others show markedly increased firing at high temperatures but with a temperature threshold below that for rolling. Not all units identified in nerve recordings are activated by heat, indicating that increased firing is not a nonspecific property of insect neurons. Since temperature-dependent increases are not seen in painless¹ mutant larvae and because Painless is expressed in the chordotonal and multidendritic neurons, the heat-sensitive units must be either chordotonal or multidendritic. Since atonal¹ larvae show a rapid response to noxious heat, chordotonal neurons are not required for this sensory modality. Final demonstration that the heat-sensitive units are multidendritic will require an electrophysiological preparation that allows monitoring of individual identified neurons. A recent study using a genetically encoded calcium sensor observed temperature-dependent calcium increases in a painless-expressing md neuron (dda-B) at high temperatures (Liu, 2003) but relatively small changes in chordotonal neurons (Tracey, 2003).

In addition to the requirement of Painless for sensing high temperature, it was found that painless is required for the rolling response to strong mechanical stimuli. The detection of strong mechanical stimuli may also be mediated by the multidendritic neurons, as it is blocked by expression of tetanus toxin in these cells. Furthermore, in larval Manduca sexta, mechanical stimuli results in spiking from multidendritic neurons that have been proposed to be homologous to those of larval Drosophila (Tracey, 2003).

Anti-Painless staining is highly localized within the dendritic arbor. In contrast, an ion channel important for intrinsic excitability, the Na⁺/K⁺ ATPase (which is detected by anti-HRP, is present throughout the main branches of the dendritic arbor and does not overlap with anti-Painless. Although nonoverlapping with anti-HRP, anti-Painless staining is indeed dendritic, since it directly contacts the anti-HRP stained dendrites. The highly localized nature of Painless immunoreactivity suggests compartmentalization of these dendrites into domains that contain distinct ion channel populations. The lack of overlap with anti-HRP suggests that Painless and the Na⁺/K⁺ ATPase occupy mutually exclusive domains of the multidendritic membrane (Tracey, 2003).

It is hypothesized that the biophysical mechanism underlying the requirement for Painless in both thermal and mechanical nociception might be explained under the combinatorial model for TRP function (Tobin, 2002). The TRPV genes osm-9 and ocr-2 were shown to be mutually required for subcellular localization, suggesting that they might form a complex. It was proposed that combinatorial action of TRPV genes might explain the requirement of a single TRPV in different sensory pathways (Tobin, 2002). In the case of Painless, heteromeric ion channel partners may be the closely related TRPN genes CG10409 and CG17142 and/or the osm-9 related TRPV genes CG5842 and CG4536, all of which are of unknown function. The need for partners in nociceptive transduction mechanisms is suggested by analogy to Drosophila phototransduction, where three closely related TRP genes, TRP, TRPL, and TRPγ, participate (Tracey, 2003).

It will be interesting to determine whether pickpocket, a degenerin-like ion channel expressed in three of the fourteen md neurons per hemisegment, is required for mechanical and/or thermal nociception (Adams, 1998). However, the pickpocket expressing cells may serve a non-nociceptive sensory function. Indeed, it has been proposed that the md neurons be classified into four morphological subtypes, which may relate to as many functional subclasses. The data suggest that the md neurons, or a subset of them, include nociceptors, but the data do not rule out additional sensory functions for these cells (Tracey, 2003).

Isolation of the painless gene provides long-awaited evidence that ion channels in the transient receptor potential family have an ancient heat-sensing function likely present in a common ancestor of vertebrates and insects. Nociception is to pain as phototransduction is to vision. painless is the first of the genes from this screen to be analyzed in detail. Others will lead to a genetic dissection of the pathways involved (Tracey, 2003).

Drosophila model for in vivo pharmacological analgesia research

Only in vivo animal experimentation can lead to successful discovery of new 'pain killers'. Pain (or nociception) research with animal models is bound by ethical concerns. Fruit flies are increasingly being used in neuropharmacology. Since no serious ethical controversies have been raised regarding in vivo experiments in insects, it appears that a Drosophila model for screening putative analgesics would be advantageous in the discovery of new drugs. Adult fruit flies have not as yet been used for pharmacological pain research (Manev, 2004).

Discussing the issue of pain in animals inevitably leads to anthropomorphic references. From a practical point of view, the response of an animal to noxious stimuli, for example, heat, and the capacity of a drug treatment to attenuate this response are the usual components of pain research. Thus, mice and rats are exposed to a hot-plate test or their tails are immersed into hot water and the latency to jump or tail-flick, respectively, is measured. Administration of drugs to these animals, including agonists for GABAB subtype (GABA= gaminobutyric acid) neurotransmitter receptors, can prolong latency to heat response. Drosophila also express GABAB receptors, which can be activated by the agonist 3-APMPA. As a proof of principle for using Drosophila in pharmacological pain studies, whether injecting flies with 3-APMPA would alter their heat response was tested (Manev, 2004).

A test apparatus was devised which consists of a spiral plastic tube that forms a tunnel for flies to negotiate -- hot water (e.g., 40-60°C) is pumped through this tube to produce a heat barrier. Flies are placed inside an empty plastic 'start' tube, which is inserted into the tube holder; the 'end' tube with the heat barrier is inserted into the opposite side. The apparatus is placed horizontally, the tip of the 'end' tube facing a source of light, (flies prefer lightphototaxis). The apparatus is shaken so that all flies fall into the tip of the 'start' tube, and flies are allowed to move for 2 min. The number of flies in the 'end' tube and outside the barrier is counted. Statistical analysis was performed by the Fisher’s exact test (Manev, 2004).

Forty flies (5-7-day-old female Canton S) per group were injected with either vehicle (control) or 3-APMPA. No vehicle-injected flies passed through the tunnel at 60°C, whereas 3-APMPA dose-dependently allows the heat barrier to be crossed. At 42°C, 27.5% of control flies pass through; 2 pmol/fly of 3-APMPA, a dose ineffective at 60°C, allowed 57.5% of the flies to pass the 42°C barrier. Flies under conditions in which phototaxis was exchanged for geotaxis; i.e., the apparatus was placed vertically in the absence of light stimulus (flies prefer to climb), and similar results as in the phototaxis assay were obtained (Manev, 2004).

Thus, these results suggest that adult Drosophila can be used in neuropharmacological nociception research. These findings in flies are consistent with previous results obtained in rats; i.e., a subcutaneous injection of a GABAB receptor agonist to rats produces dose-dependent antinociception in both the tail-flick and hot-plate test. With the Drosophila model describe in this study, it will be possible to further characterize mechanisms involved in the action of GABAB receptor system in heat avoidance (Manev, 2004).

Although anthropomorphizing the behavioral responses of flies observed in the present study as nociception may be controversial, these fly behaviors are remarkably similar to responses of mammals to heat and to the GABAB receptor agonists. In fact, the perception of and behavioral responses to low and high temperatures are well developed in Drosophila. Recent research in Drosophila larvae has identified specific thermosensory neurons and also a gene, painless, encoding a putative heat-sensing receptor/channel. It is proposed that combining the advantages of gene manipulation in Drosophila with the neuropharmacological techniques described in this study may advance the discovery of new analgesic drugs. Moreover, at this time, it appears that the Drosophila model is ethically more acceptable than the in vivo mammalian animal models (Manev, 2004).

Drosophila painless is a Ca2+-requiring channel activated by noxious heat

Thermal changes activate some members of the transient receptor potential (TRP) ion channel super family. They are primary sensors for detecting environmental temperatures. The Drosophila TRP channel Painless is believed responsible for avoidance of noxious heat because painless mutant flies display defects in heat sensing. However, no studies have proven its heat responsiveness. This study shows that Painless expressed in human embryonic kidney-derived 293 (HEK293) cells is a noxious heat-activated, Ca²⁺-permeable channel, and the function is mostly dependent on Ca²⁺. In Ca²⁺-imaging, Painless mediates a robust intracellular Ca²⁺ (Ca²⁺_i) increase during heating, and it showed heat-evoked inward currents in whole-cell patch-clamp mode. Ca²⁺ permeability was much higher than that of other cations. Heat-evoked currents were negligible in the absence of extracellular Ca²⁺ (Ca²⁺_o) and Ca²⁺_i, whereas 200 nM Ca²⁺_i enabled heat activation of Painless. Activation kinetics were significantly accelerated in the presence of Ca²⁺_i. The temperature threshold for Painless activation was 42.6°C in the presence of Ca²⁺_i, whereas the threshold was significantly increased to 44.1°C when only Ca²⁺_o was present. Temperature thresholds were further reduced after repetitive heating in a Ca²⁺-dependent manner. Ca²⁺-dependent heat activation of Painless was observed at the single-channel level in excised membranes. This study found that a Ca²⁺-regulatory site is located in the N-terminal region of Painless. Painless-expressing HEK293 cells were insensitive to various thermosensitive TRP channel activators including allyl isothiocyanate, whereas mammalian TRPA1 inhibitors, ruthenium red, and camphor, reversibly blocked heat activation of Painless. These results demonstrate that Painless is a direct sensor for noxious heat in Drosophila (Sokabe, 2008).

The present study provides direct evidence that the Drosophila TRP channel Painless is a heat sensor. Although the biophysical properties of Painless have yet to be elucidated, members of the Drosophila TRPA subfamily, dTRPA1 and pyrexia, show temperature sensitivity both in vitro and in vivo, from which it has been inferred that Painless might also be a heat-sensitive channel (Montell, 2005). Indeed, a robust Ca²⁺ influx and current activation via Painless was observed during heating in the heterologous expression system. The temperature threshold for Painless activation (~42.6°C) was consistent with the temperature that causes avoidance behavior in vivo. Painless os activated by heat in a membrane-delimited and Ca²⁺-dependent manner, indicating that Painless can detect heat directly by using Ca²⁺_i, but not intracellular signaling pathways. Thus, Painless itself may act as a primary heat detector to facilitate neural activity in vivo (Sokabe, 2008). In vivo and in vitro analyses revealed different temperature thresholds. Previous work reported that there were two types of Painless-expressing neurons with low (~28°C) and high (~39°C) temperature thresholds (Tracey, 2003). However, the temperature thresholds of Painless in the current system were 41° - 44°C, rather than <40°C, in various heating conditions such as Ca²⁺_o and Ca²⁺_i concentrations, membrane potentials, or heat application rates. These differences might be due, in part, to the methods determining the thresholds. Each threshold was defined as a reflex point with an increasing Q₁₀ value >10 in Arrhenius plots. In this case, minuscule current development (Q₁₀ < 10) was not regarded as the onset of Painless activation. However, magnitude of the background current does not affect the thresholds, because there was no correlation between current sizes and thresholds. Single-channel currents of Painless were sometimes observed that were initiated at ~28°, a temperature close to the value at which neural activity differed between wild-type and the painless mutant (Tracey, 2003), during slow heat application (0.2°C/s). This result suggests that Painless has an ability to respond to lower temperature in vitro, which might contribute to neural excitability. Alternatively, there could be biological differences between in vitro and in vivo. The lipid composition of the mammalian plasma membrane may affect Painless activity differently than it would in its native environment. In fact, the functions of TRP channels, including thermosensitive ones, are regulated by a series of lipids (Hardie, 2007). Temperature threshold for activation of mammalian TRPV1, which is activated by noxious heat (>43°C) like Painless, has been reported to be reduced with phosphorylation by PKC (Numazaki, 2002), and it is also known that TRPV1 function is regulated through binding with specific accessory proteins (Kim, 2006; Kim, 2008). Such physiological regulation may also exist to alter Painless property in vivo (Sokabe, 2008).

Ca²⁺ plays four essential roles. (1) Ca²⁺ enables Painless to respond to heat. Whereas higher heat (~50°C) elicited only faint currents in the absence of Ca²⁺_o and Ca²⁺_i, 200 nM Ca²⁺_i was sufficient for heat activation, suggesting that Painless requires Ca²⁺_i for functionality. Thermosensitive TRP channels such as TRPM4, TRPM5, and TRPA1 are activated by Ca²⁺_i (Talavera, 2005; Nilius, 2006; Doerner, 2007; Zurborg, 2007), whereas Painless demands Ca²⁺ as a coagonist for heat. A similar concept has been reported in TRPM8, in that Ca²⁺_i supports robust icilin-evoked responses (Chuang, 2004). [Ca²⁺]_i required for half activation of Painless was ~103 nM, a concentration close to the reported value (Liu, 2003) in the terminal and dorsal organ of the larval head (Sokabe, 2008).

(2) Ca²⁺ accelerates the activation kinetics of Painless. The longer time required for activation in the presence of Ca²⁺_o alone is probably explained by a necessity for Ca²⁺_o to first enter the cytoplasm until [Ca²⁺]_i reaches the threshold for maximal activation, which results in gradual current development, followed by accelerated activation. In the presence of Ca²⁺_i, Painless is activated quickly because sufficient Ca²⁺_i for full activation exists. Indeed, fly larvae moved away from a heated probe within 0.4 s, supporting this idea. Moreover, inactivation rates of Painless seemed to be affected by Ca²⁺ conditions. Ca²⁺_i might delay the inactivation; however, Ca²⁺_i should increase during activation also in the presence of Ca²⁺_o alone, which shows rapid inactivation. Nevertheless, Ca²⁺ is apparently important for regulating the activation kinetics (Sokabe, 2008).

(3) Ca²⁺ reduces temperature thresholds. Painless is 'ready for activation' in the presence of Ca²⁺_i, so that it may respond to heat at its reduced temperature threshold of ~42.6°C. However, the temperature threshold was ~44.1°C in the presence of Ca²⁺_o alone, where heat should initiate the gating of Painless in a Ca²⁺_i-independent manner. Quick avoidance from a heated probe occurred at ~42°C in fly larvae; therefore, the in vivo temperature threshold is close to that obtained in the presence of Ca²⁺_i. Thermosensitive TRP channels such as TRPV1 and TRPM8 have strong voltage dependencies in their temperature thresholds, whereas Painless does not. This is not surprising because dual rectified I-V relationship of Painless is apparently different from outward rectified I-V relationship of TRPV1 and TRPM8 (Sokabe, 2008).

(4) Ca²⁺ contributes to sensitization of Painless during repetitive heating. Significant reduction in the temperature thresholds during repeated heating was observed in the presence of Ca²⁺_o and Ca²⁺_i, but not in the presence of Ca²⁺_i alone, suggesting that Ca²⁺_o and/or Ca²⁺ influx may be involved in the sensitization. This would be physiologically important, because flies are able to escape from a hazardous heat source in less time after second exposure. Sensitization during repetitive heating is a common feature in TRPV1, TRPV2, and TRPV3, although the underlying mechanism is still unknown including the requirement for Ca²⁺ (Sokabe, 2008).

Recently, mammalian TRPA1 was reported to be activated by Ca²⁺ (Doerner, 2007; Zurborg, 2007). Ca²⁺_i-dependent activation significantly deteriorates when the EF-hand-like motif in the N-terminal region is mutated. Amino acid sequences between Painless and TRPA1 were compared and the candidate region for Ca²⁺_i regulation was determined in Painless. It was found to be located in the ankyrin repeat domain. The Painless N363A mutant displayed small heat-evoked currents, increased temperature thresholds, and higher [Ca²⁺]_i requirement for half activation with a reduced Hill coefficient. These features could be explained if the 'Ca²⁺-regulatory region' in Painless included N363, and its affinity to Ca²⁺_i is reduced by mutation. Thus, the mutant channel requires increasing [Ca²⁺]_i to be fully activated, which results in a higher temperature threshold and small size in currents. These results suggest that N363 is a key residue in Ca²⁺ sensitivity of Painless, although the function is different from the EF-hand-like motif in mammalian TRPA1. Painless is not activated by high [Ca²⁺]_i alone and requires much less [Ca²⁺]_i for its regulation, whereas TRPA1 requires Ca²⁺ at a micromolar level for activation. Furthermore, mutation of N356, S357, or D366 does not affect the heat responsiveness of Painless, whereas the corresponding amino acids in the EF-hand-like motif are necessary for Ca²⁺_i-dependent TRPA1 activation (Sokabe, 2008).

Thermosensitive TRP channels can be activated by various stimuli and one stimulant sometimes activates multiple thermosensitive TRP channels (Dhaka, 2006; Ramsey, 2006; Tominaga, 2007). Accordingly, Painless could mediate several stimuli such as allyl isothiocyanate (AITC) or a mechanical stimulus (Tracey, 2003; Al-Anzi, 2006). However, Painless-expressing HEK293 cells did not respond to a range of possible activators. Recently, AITC was reported to activate mammalian TRPA1 through covalent modification (Hinman, 2006; Macpherson, 2007). However, several cysteines important for covalent modification were not present in Painless, which might explain its insensitivity to AITC, cinnamaldehyde, allicin, acrolein, and formalin. Thus, Painless is not likely to be a direct receptor for these cysteine modifiers. Alternatively, interaction with accessory protein(s), formation of heteromeric channel, and/or splice variants might alter properties of Painless in vivo. These possibilities remain to be addressed. Painless is expressed in the CNS as well as peripheral sensory neurons (Tracey, 2003; Al-Anzi, 2006), where Painless could be activated by endogenous ligands. Heat activation of Painless is inhibited by ruthenium red and camphor, indicating that Painless shares some properties with mammalian TRPA1. Camphor, a wood derivative from camphor laurel, has been used as a repellent for pests and proved to be effective for mosquitoes (Gillij, 2007). The repellent may inhibit the noxious heat sensor, perhaps interfering with the normal sensing ability of flies. It would therefore be intriguing to test the effects of camphor at the behavioral level (Sokabe, 2008).

Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain

Acute avoidance of dangerous temperatures is critical for animals to prevent or minimize injury. Therefore, surface receptors have evolved to endow neurons with the capacity to detect noxious heat so that animals can initiate escape behaviors. Animals including humans have evolved intrinsic pain-suppressing systems to attenuate nociception under some circumstances. Using Drosophila melanogaster, this study uncovered a new mechanism through which thermal nociception is suppressed. A single descending neuron was identified in each brain hemisphere, which is the center for suppression of thermal nociception. These Epi neurons, for Epione-the goddess of soothing of pain-express a nociception-suppressing neuropeptide Allatostatin C (AstC), which is related to a mammalian anti-nociceptive peptide, somatostatin. Epi neurons are direct sensors for noxious heat, and when activated they release AstC, which diminishes nociception. Epi neurons also express the heat-activated TRP channel, Painless (Pain), and thermal activation of Epi neurons and the subsequent suppression of thermal nociception depend on Pain. Thus, while TRP channels are well known to sense noxious temperatures to promote avoidance behavior, this work reveals the first role for a TRP channel for detecting noxious temperatures for the purpose of suppressing rather than enhancing nociception behavior in response to hot thermal stimuli (Liu, 2023).

Endogenous pain inhibitory systems can temporarily provide relief. Millions of people suffer from chronic and debilitating pain, some of which might be induced by abnormalities in the descending pain modulatory system. In mammals, neurotransmitters and neuromodulators, including endogenous opioids (β-endorphin, encephalin, and dynorphin) and endogenous cannabinoids, play important roles in nociception inhibition. Brain imaging and electrophysiological studies indicate that the pain-suppressing descending modulatory circuit receives input from multiple brain regions including the rostral anterior cingulate cortex, the periaqueductal gray region, and the rostral ventromedial medulla. However, the key neurons that are activated in the inhibitory pathway, and the target neurons that are silenced, have not been clearly delineated (Liu, 2023).

A pain inhibitory system has also been documented in worms. In C. elegans avoidance responses that are mediated through the polymodal ASH neurons are suppressed by complex signaling pathways initiated by octopamine and neuropeptides. Drosophila has also been employed to study the inhibition of nociception in addition to the far more extensive studies focusing on the mechanisms for detecting noxious stimuli, such as excessive heat, has been shown to to initiate escape responses. A Drosophila channel, Painless (Pain), which is related to the TRP channel in the fly's compound eye, is critical for sensing noxious heat. This work, which followed the seminal discovery of TRPV1 as a heat sensor in mammals and the finding that a related TRPV channel (Osm-9) contributes to several other sensory modalities in C. elegans, contributed significantly to the notion that TRP channels are evolutionarily conserved polymodal sensors (Liu, 2023).

In addition to Pain, two other Drosophila TRP channels also function in sensing high temperatures to promote escape behavior: Pyrexia (Pyx) and TRPA1. However, it is unclear whether any TRP channel serves to detect noxious heat for the purpose of alleviating thermally induced nociception (Liu, 2023).

This work used the fruit fly, Drosophila melanogaster, to investigate an intrinsic system for suppression of thermal nociception. A pair of bilaterally symmetrical neurons in the brain was identified that is required for decreasing the nociceptive response to hot temperatures. These Epi neurons respond directly to heat and release a neuropeptide, Allatostatin C (AstC), which is required for suppression of nociception. The ability of Epi neurons to sense noxious heat depends on Pain, demonstrating a role for a thermo-TRP in suppressing rather than enhancing the nociceptive response to high temperatures (Liu, 2023).

This study found that a single pair of bilaterally symmetrical Epi neurons in the fly brain is critical for suppressing thermal nociception. The importance of Epi neurons is underscored by the observation that artificial activation of these neurons is sufficient to suppress the aversive jump response to hot temperatures and that inhibition of signaling from these neurons increases the jump responses to moderate heat. The profound effect of a single pair of neurons in reducing thermal nociception is surprising given that multiple brain regions appear to function in pain suppression in mammals (Liu, 2023).

The dendrites of Epi neurons arborize to multiple regions of the brain, such as the optic lobes (OLs), the lateral horn (LH), and a region near the mushroom bodies, indicating that Epi neurons receive multiple signal inputs. The LH is a higher-order processing center that receives input from the antennal (olfactory) lobes and then sends relays to other brain regions such as the mushroom bodies. Therefore, it is intriguing to speculate that the Epi neurons may be activated by noxious odorants and aversive visual cues, which attenuate the avoidance behavioral responses to these stimuli. Epi neurons might also receive input from attractive olfactory and visual cues, which in turn diminish the escape responses to noxious stimuli such as high temperatures. In addition, the axons of Epi neurons project to the VNC, consistent with a role in descending control of motor output (Liu, 2023).

A key question is the mechanism through which Epi neurons respond to hot temperatures and alleviate thermal nociception. Epi neurons were found to be directly activated by hot temperatures and do so through activation of the thermo-TRP channel, Pain, which is expressed in Epi neurons. The Pain channel is critical for suppressing nociception since mutation of the pain gene causes an increase in thermal pain sensitivity (hyperalgesia). While Epi neurons respond directly to heat and are anti-nociceptors, other neurons in the fly brain, the so-called anterior cell neurons, respond directly to suboptimal warm temperatures. In contrast to the anti-nociceptive Epi neurons, the AC neurons function in thermal avoidance, which is mediated through thermal activation of TRPA1 (Liu, 2023).

The next question is the mechanism through which activation of Epi neurons suppresses thermal pain. Epi neurons express a neuropeptide, AstC, which binds to receptors that have sequence homology (39.0% identity for AstC-R1; 38.5% identity for AstC-R2) to human opioid receptors, which function in the suppression of nociception in mammals. Moreover, mutation of AstC or knockdown of AstC in Epi neurons causes thermal hyperalgesia, and mutation of AstC-R1 elicits a similar phenotype. Heat stimulation diminishes the level of AstC in Epi neurons, indicating that activation of these neurons promotes release of AstC. It is concluded that Epi neurons alleviate thermal nociception through a mechanism that depends on heat sensing by the Pain channel, leading to release of AstC (Liu, 2023).

Surprisingly, mutation of pain also reduced expression of AstC in Epi neurons below the level of detection. This effect was not due to elimination of Epi neurons since pain mutant brains express UAS-GCaMP6f under control of the Epi-Gal4. Expression of neuropeptides has been linked to neuronal activity. Moreover, there is an example in which a thermosensory TRPV channel affects expression of a neuropeptide receptor. Pain is activated by thermal heat, with the most pronounced activation in the noxious heat range. However, even at temperatures significantly below the flex point in which a given temperature rapidly opens the gate of a thermosensory TRP, such as Pain, there is some channel activity. It is suggested that low levels of Pain and Epi neuron activities are necessary for expression of AstC, while high levels of activities that are induced by noxious heat are required for release of the AstC (Liu, 2023).

A feature of activation of Epi neurons is that the pain suppression due to an acute 30-second activation of Epi neurons is sustained for several minutes. It is suggested that the slow termination of the pain suppression following stimulation of these neurons is mediated by release of the neuromodulator AstC, which persists for several minutes. Epi neurons appear to be non-adapting, as chronic activation of these neurons with the NaChBac channel leads to similar levels of pain suppression as acute stimulation with channelrhodopsin. This non-adapting feature of Epi neurons may be beneficial because it allows for pain suppression under conditions in which the aversive response to heat needs to be suppressed sufficiently long enough to allow activities that promote survival. Given that fruit flies are poikilothermic, and their body temperature equilibrates with the environment, direct activation of Epi neurons would allow the flies to suppress nociception and enter excessively warm environments to feed or avoid predators (Liu, 2023).

In conclusion, this study unveils a molecular and cellular basis for pain suppression in Drosophila. The observation that Pain is essential for suppressing nociception is surprising given that all other thermal-TRP channels function in avoidance of suboptimal or noxious temperatures. Mutation of pain in fly larvae eliminates the sensitivity to hot temperatures (hypoalgesia). Thus, it is remarkable that the same TRP channel has opposite functions in nociception and anti-nociception in larvae and adults (Liu, 2023).

REGULATION

Embryonic

To determine the pattern of painless mRNA expression, whole-mount in situ hybridization on embryos was performed with anti-sense RNA probes. Beginning at stage 13 of embryonic development, painless mRNA is detected in a small number of cells in the central nervous system and in a subset of neurons of the peripheral nervous system. The cell bodies of these latter neurons are in positions suggesting that they might include sensory precursors of multidendritic (md) neurons; this was confirmed in double labeling experiments. At embryonic stage 16, prior to when dendritic process are elaborated, the painless mRNA appears to be distributed in the cytoplasm of the multidendritic neurons in a polarized manner. At stage 17 of development, when the md neurons first initiate dendritogenesis, the painless RNA becomes localized to branched projections initiating from clusters of multidendritic neurons. The structures projecting from the dorsal cluster of multidendritic neurons project dorsally. In still older embryos with a more developed dendritic arbor, the pattern of expression evolves into a more elaborate subepidermal plexus of staining. Combined, these data suggest that the painless mRNA is present in md neuron precursors at early stages and becomes localized to their dendrites at later stages. However, further experimentation will be needed to formally demonstrate the latter. In addition, strong expression is also observed in sensory neurons of the antennal maxillary complex. Weak expression of painless mRNA was also observed in chordotonal neurons. Outside of the nervous system, painless is expressed in the embryonic gonad and in the dorsal vessel, the insect heart equivalent (Tracey, 2003).

A GAL4 enhancer trap allele of painless (pain-GAL4) was generated by P element replacement. The pain-GAL4 insertion is within the painless transcription unit at an identical site to the pain¹ EP insertion. Consistent with in situ hybridization results, pain-GAL4 driven expression of UAS-GFP shows fluorescence in multidendritic neurons, a subset of cells in the central nervous system, and a subset of sensory neurons in the antennal-maxillary complex. Unlike for painless mRNA, no expression of UAS-GFP driven by pain-GAL4 was detected in the dorsal vessel or in the gonad. Although chordotonal neurons express painless, these neurons are not required for nociception since atonal¹ mutant larvae, which lack most chordotonal organs, show rapid, though uncoordinated, responses in both the thermal and mechanical nociception paradigms. Thus, it is hypothesized that the md-da neurons include the primary nociceptors in the larval abdominal segments (Tracey, 2003).

Immunostaining of embryos using the anti-Painless antibody shows strong staining in chordotonal organs. Consistent with the results of Western blots, which show the presence of novel bands rather than a reduction in protein levels in pain¹, pain¹ mutant embryos show apparently normal staining. Staining in pain² mutants, however, is greatly reduced, again consistent with the Western analysis. In addition to the staining seen in embryonic chordotonal organs, punctate staining was seen beneath the embryonic epidermis. Given the expression pattern of pain-GAL4, it was reasoned that this staining might correspond to structures associated with the fine dendrites of the multidendritic neurons. Staining of the md neuron arbors was therefore examined in filleted preparations of third instar larvae (Tracey, 2003).

Anti-painless immunoreactivity was tightly associated with the dendritic arbors. The most intense anti-Painless staining was present in bright puncta that were juxtaposed with the dendritic arbor, but Painless was not strongly detected throughout the main branches of the arbor. The anti-Painless staining was highly localized and often clearly seen to be attached to the dendrite. The highly localized nature of Painless immunoreactivity may indicate a specialized region of the dendrite used for nociceptive signaling (Tracey, 2003).

DEVELOPMENTAL BIOLOGY

Embryonic

To determine the pattern of painless mRNA expression, whole-mount in situ hybridization on embryos was performed with anti-sense RNA probes. Beginning at stage 13 of embryonic development, painless mRNA is detected in a small number of cells in the central nervous system and in a subset of neurons of the peripheral nervous system. The cell bodies of these latter neurons are in positions suggesting that they might include sensory precursors of multidendritic (md) neurons; this was confirmed in double labeling experiments. At embryonic stage 16, prior to when dendritic process are elaborated, the painless mRNA appears to be distributed in the cytoplasm of the multidendritic neurons in a polarized manner. At stage 17 of development, when the md neurons first initiate dendritogenesis, the painless RNA becomes localized to branched projections initiating from clusters of multidendritic neurons. The structures projecting from the dorsal cluster of multidendritic neurons project dorsally. In still older embryos with a more developed dendritic arbor, the pattern of expression evolves into a more elaborate subepidermal plexus of staining. Combined, these data suggest that the painless mRNA is present in md neuron precursors at early stages and becomes localized to their dendrites at later stages. However, further experimentation will be needed to formally demonstrate the latter. In addition, strong expression is also observed in sensory neurons of the antennal maxillary complex. Weak expression of painless mRNA was also observed in chordotonal neurons. Outside of the nervous system, painless is expressed in the embryonic gonad and in the dorsal vessel, the insect heart equivalent (Tracey, 2003).

A GAL4 enhancer trap allele of painless (pain-GAL4) was generated by P element replacement. The pain-GAL4 insertion is within the painless transcription unit at an identical site to the pain¹ EP insertion. Consistent with in situ hybridization results, pain-GAL4 driven expression of UAS-GFP shows fluorescence in multidendritic neurons, a subset of cells in the central nervous system, and a subset of sensory neurons in the antennal-maxillary complex. Unlike for painless mRNA, no expression of UAS-GFP driven by pain-GAL4 was detected in the dorsal vessel or in the gonad. Although chordotonal neurons express painless, these neurons are not required for nociception since atonal¹ mutant larvae, which lack most chordotonal organs, show rapid, though uncoordinated, responses in both the thermal and mechanical nociception paradigms. Thus, it is hypothesized that the md-da neurons include the primary nociceptors in the larval abdominal segments (Tracey, 2003).

Immunostaining of embryos using the anti-Painless antibody shows strong staining in chordotonal organs. Consistent with the results of Western blots, which show the presence of novel bands rather than a reduction in protein levels in pain¹, pain¹ mutant embryos show apparently normal staining. Staining in pain² mutants, however, is greatly reduced, again consistent with the Western analysis. In addition to the staining seen in embryonic chordotonal organs, punctate staining was seen beneath the embryonic epidermis. Given the expression pattern of pain-GAL4, it was reasoned that this staining might correspond to structures associated with the fine dendrites of the multidendritic neurons. Staining of the md neuron arbors was therefore examined in filleted preparations of third instar larvae (Tracey, 2003).

Anti-painless immunoreactivity was tightly associated with the dendritic arbors. The most intense anti-Painless staining was present in bright puncta that were juxtaposed with the dendritic arbor, but Painless was not strongly detected throughout the main branches of the arbor. The anti-Painless staining was highly localized and often clearly seen to be attached to the dendrite. The highly localized nature of Painless immunoreactivity may indicate a specialized region of the dendrite used for nociceptive signaling (Tracey, 2003).

EFFECTS OF MUTATION

To identify genes important for nociception, a genetic screen was perfomred for mutations that cause insensitivity to noxious heat. A collection of fly lines, carrying randomly inserted EP transposable elements, was screened. A line was considered to have impaired sensitivity to noxious heat if stimulation longer than 3 s was required to produce the rolling response (Tracey, 2003).

Among the 1500 EP lines screened, 49 were identified with reproducibly decreased sensitivity to noxious heat. EP(2)2451, among the most insensitive and carrying an insertion on the second chromosome, was chosen for further study. Larvae homozygous for EP(2)2451 had a defective response to noxious heat, some failing to roll even after 10 s. To reflect this phenotype, the mutant was named painless¹ (pain¹). Although painless¹ larvae show a defect in the writhing response, they still showed a normal response to a light touch on the nose. Also, they did not display the highly uncoordinated movement and poor adult viability typical of the touch-insensitive mutants (Kernan, 1994) having defects in mechanosensory external sensillae (Tracey, 2003).

Nociceptors in vertebrates have been found to be divisible into several classes. Low threshold, polymodal nociceptors respond to noxious heat in the range of 42°C-48°C and to noxious mechanical stimuli, while high-threshold nociceptors respond at even higher temperatures. To test whether the painless¹ mutation blocks all nociception, the response of the mutant larvae was examined over a range of temperatures. The larvae also showed a delayed response to a 48°C stimulus, but 52°C or higher elicited a rapid response, similar to that of normal larvae. Since the response to high temperature is seen even in putative null alleles of painless, this result may indicate that moderate and intense levels of noxious heat are processed via separate pathways. In addition, these data imply that the motor system needed for a rapid response is not abolished by mutations in painless; the defect is at the sensory level (Tracey, 2003).

painless mutants were also examined for the rolling response exhibited by wild-type flies given strong mechanical stimuli. To do this, larvae were stimulated with calibrated Von Frey filaments (0.2 mm diameter), which are calibrated to deliver a controlled stimulus. In the wild-type Canton S strain, rolling was not observed when larvae were stimulated with a filament delivering 10 mN of force (n = 19); instead touch responses (larva will pause or make one or more contractile waves, moving away from the stimulus) occurred. When stimulated with a 45 mN fiber, vigorous rolling was observed in 92% of the larvae (n = 36). In contrast, only 13% of painless¹ mutant larvae (n = 31) rolled in response to the 45 mN fiber. Nevertheless, with an even stronger mechanical stimulus (100 mN), a high proportion (81%) of painless¹ mutant larvae (n = 43) responded by rolling. These data demonstrate that the painless¹ mutation results in an increased threshold for both thermal and mechanical nociception (Tracey, 2003).

The painless¹ larvae that did not roll with the 45 mN fiber were not completely insensitive to the stimulus; instead, they responded by pausing their feeding movement, as in the wild-type response to light touch. Although thus defective in responding to strong mechanical stimuli, the response to light touch on the nose was unaffected in painless¹. The painless¹ mutation therefore genetically separates mechanical nociception from mechanosensation (Tracey, 2003).

Next, tests were performed to see whether the behavioral defect exhibited by painless¹ mutants could be correlated with a specific defect in the response of abdominal primary afferents. Suction-electrode recordings were performed from sectioned abdominal nerves in third instar larvae containing axons of peripheral sensory neurons. All recordings were done blind to the genotype. At room temperature, the mean bulk spiking frequencies of nerves from wild-type and painless¹ larvae were not significantly different (wild-type 8 ± 2 Hz, pain¹ 11 ± 4 Hz). Data were continuously recorded from the nerve as the temperature of the saline bathing the larvae was gradually increased. The bulk spiking rate of wild-type nerves increased more than 2-fold at the temperatures that elicited rolling behavior. The temperature threshold for the increase in firing rate was near 38°C; frequency at 38°C-42°C was 2.6 ± 0.8 times greater than at room temperature. By contrast, the firing rate of painless¹ nerves did not increase in the noxious temperature range (Tracey, 2003).

Some of these recordings contained a variety of sufficiently distinctive spike waveforms to permit separation of spikes originating from different individual neurons. In wild-type nerves, many neurons showed a marked firing rate increase near 38°C-40°C but little spontaneous spiking activity below that temperature, as expected for thermal nociceptors. Other wild-type neurons had a lower temperature threshold. Neurons relatively insensitive to temperature were recorded simultaneously in most wild-type nerves, implying that the thermoresponses of neurons like E1 and E2 are not a nonspecific general property of insect sensory neurons. Importantly, the spiking rate of individual painless¹ neurons almost never increased at elevated temperatures. Thus, Painless is required for the excitatory response of abdominal sensory neurons to noxious heat (Tracey, 2003).

To determine whether the insensitivity of painless¹ was due to the presence of the EP element insertion, painless¹ to a transposase line to mobilize the EP element. Of 80 excision alleles obtained, 73 were homozygous viable, and 29 of those were tested as third instar larvae for the painless phenotype. Among them, 22 alleles showed reversion of the larval response to noxious heat. Therefore, the painless phenotype can be reverted by excision of the P element (Tracey, 2003).

In addition to EP(2)2451, three other P element insertions have been identified within 16 bp of EP(2)2451 by the Berkeley Drosophila Genome Project (BDGP). Larvae homozygous for EP(2)2621 (painless²) and EP(2)2251 (painless³) were found to be strongly insensitive to noxious heat in the nociception paradigm. The fourth insertion, EP(2)2462 (painless⁴), showed reduced viability, but those larvae that did survive to third instar displayed the same insensitive phenotype as did the other alleles. All of the alleles failed to complement one another for the nociception defect when tested in trans, indicating that the behavioral defects of the lines were due to mutations in the same gene. pain³, like pain¹, is recessive. pain² and pain⁴ are semidominant and show mild nociception defects when heterozygous (Tracey, 2003).

To examine effects of the mutations on the Painless protein, rabbit antisera were raised against peptides from the Painless sequence. The affinity purified anti-serum GN6620, raised against an intracellular loop in the six-transmembrane region, stained sense organs of the peripheral nervous system, which also expressed the painless mRNA. The antibody also detects alterations on Western blots of extracts from painless¹ and painless ² (Tracey, 2003).

In wild-type extracts, the serum detects a band of 105 kd, consistent with the predicted molecular weight of Painless. That band is absent in the painless¹ and painless² strains. However, an abnormal, higher molecular weight species is detected in both mutants. The presence of this band is consistent with the finding that an upstream in-frame ATG from the painless¹ mutant transcript is predicted to add 65 amino acids (18 kd) to the Painless N terminus. The data also suggest that a mutant Painless protein is produced in the painless² strain (Tracey, 2003).

By P element transformation, transgenic flies were created containing a genomic DNA rescue fragment (P-pain-rescue). This fragment consists of the painless transcription unit, as well as 2.0 kb of upstream genomic DNA, which is hypothesized to contain critical cis-regulatory sequences. The results indicate that sequences sufficient to restore the response to noxious heat are present in the 8.5 kb of rescue DNA (Tracey, 2003).

Drosophila nociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. This study shows that the class IV multiple-dendritic (mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1 (PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses (Johnson, 2012).

EVOLUTIONARY HOMOLOGS

TRPN gene family founding member no mechanoreceptor potential-C is essential for Drosophila mechanosensation

Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, Drosophila mechanoreceptive mutants were analyzed for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolishes mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs (Walker, 2000).

OSM-9, a C. elegans TRP family protein involved in mechanosensory response

Although cyclic nucleotide-gated channels mediate sensory transduction in olfaction and vision, other forms of sensory transduction are independent of these channels. Caenorhabditis elegans cyclic nucleotide-gated channel mutants respond normally to some olfactory stimuli and to osmotic stimuli, suggesting that these chemosensory responses use an alternative sensory transduction pathway. One gene that may act in this pathway is osm-9, which is required for each of these responses as well as a mechanosensory response to nose touch. osm-9 encodes a protein with ankyrin repeats and multiple predicted transmembrane domains that has limited similarity to the Drosophila phototransduction channels transient receptor potential (TRP) and TRP-like (TRPL). The sequence of OSM-9 and other TRP-like genes reveals a previously unsuspected diversity of mammalian and invertebrate genes in this family. osm-9 is required for the activity of the predicted G-protein-coupled odorant receptor ODR-10, which acts in the AWA olfactory neurons; its similarity to other G-protein-regulated transduction channels suggests that OSM-9 is involved in AWA signaling. osm-9:: GFP fusion genes are expressed in a subset of chemosensory, mechanosensory, and osmosensory neurons. osm-9 also affects olfactory adaptation within neurons that require the cyclic nucleotide-gated channel for olfaction; in these neurons, the gene has a regulatory function and not a primary role in sensory transduction (Colbert, 1997).

C. elegans OSM-9 is a TRPV channel protein involved in sensory transduction and adaptation. Distinct sensory functions arise from different combinations of OSM-9 and related OCR TRPV proteins. Both OSM-9 and OCR-2 are essential for several forms of sensory transduction, including olfaction, osmosensation, mechanosensation, and chemosensation. In neurons that express both OSM-9 and OCR-2, tagged OCR-2 and OSM-9 proteins reside in sensory cilia and promote each other's localization to cilia. In neurons that express only OSM-9, tagged OSM-9 protein resides in the cell body and acts in sensory adaptation rather than sensory transduction. Thus, alternative combinations of TRPV proteins may direct different functions in distinct subcellular locations. Animals expressing the mammalian TRPV1 (VR1) channel in ASH nociceptor neurons avoid the TRPV1 ligand capsaicin, allowing selective, drug-inducible activation of a specific behavior (Tobin, 2002).

Natural Caenorhabditis elegans isolates exhibit either social or solitary feeding on bacteria. Social feeding is induced by nociceptive neurons that detect adverse or stressful conditions. Ablation of the nociceptive neurons ASH and ADL transforms social animals into solitary feeders. Social feeding is probably due to the sensation of noxious chemicals by ASH and ADL neurons; it requires the genes ocr-2 and osm-9, which encode TRP-related transduction channels, and odr-4 and odr-8, which are required to localize sensory chemoreceptors to cilia. Other sensory neurons may suppress social feeding, since social feeding in ocr-2 and odr-4 mutants is restored by mutations in osm-3, a gene required for the development of 26 ciliated sensory neurons. These data suggest a model for regulation of social feeding by opposing sensory inputs: aversive inputs to nociceptive neurons promote social feeding, whereas antagonistic inputs from neurons that express osm-3 inhibit aggregation (de Bono, 2002).

All animals detect osmotic and mechanical stimuli, but the molecular basis for these responses is incompletely understood. The vertebrate transient receptor potential channel vanilloid subfamily 4 (TRPV4) (VR-OAC) cation channel has been suggested to be an osmo/mechanosensory channel. To assess its function in vivo, TRPV4 was expressed in Caenorhabditis elegans sensory neurons and its ability to generate behavioral responses to sensory stimuli was examined. C. elegans ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors require the TRPV channel OSM-9 because osm-9 mutants do not avoid nose touch, high osmolarity, or noxious odors. Expression of mammalian TRPV4 in ASH neurons of osm-9 worms restores avoidance responses to hypertonicity and nose touch, but not the response to odorant repellents. Mutations known to reduce TRPV4 channel activity also reduces its ability to direct nematode avoidance behavior. TRPV4 function in ASH requires the endogenous C. elegans osmotic and nose touch avoidance genes ocr-2, odr-3, osm-10, and glr-1, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoidance responses of TRPV4- expressing animals are different in their sensitivity and temperature dependence from the responses of wild-type animals, suggesting that the TRPV4 channel confers its characteristic properties on the transgenic animals' behavior. These results provide evidence that TRPV4 can function as a component of an osmotic/mechanical sensor in vivo (Liedtke, 2003).

ANKTM1, a member of the TRPN gene family, is expressed in cold-sensitive neurons

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. ANKTM1 is 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. ANKTM1 is the closest vertebrate homolog of painless. 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, noxious cold-sensitive sensory neurons have been identified that also respond to capsaicin but not to menthol (Story, 2003).

Identification and characterization of Capsaicin receptors, TRP family protein involved in pain and heat responses

Capsaicin, the main pungent ingredient in 'hot' chilli peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. An expression cloning strategy based on calcium influx was used to isolate a functional cDNA encoding a capsaicin receptor from sensory neurons. This receptor is a non-selective cation channel that is structurally related to members of the TRP family of ion channels. The cloned capsaicin receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo (Caterina, 1997).

Capsaicin, the main pungent ingredient in 'hot' chili peppers, elicits buming pain by activating specific (vanilloid) receptors on sensory nerve endings. The cloned vanilloid receptor (VR1) is a cation channel that is also activated by noxious heat. Analysis of heat-evoked single channel currents in excised membrane patches suggest that heat gates VR1 directly. Protons decrease the temperature threshold for VR1 activation such that even moderately acidic conditions (pH < or = 5.9) activate VR1 at room temperature. VR1 can therefore be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. Immunocytochemical analysis indicates that the receptor is located in a neurochemically heterogeneous population of small diameter primary afferent fibers. A role for VR1 in injury-induced hypersensitivity at the level of the sensory neuron is presented (Tominaga, 1998).

Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds. The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43°C) thermal stimuli. VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. A structurally related receptor, VRL-1, has been identified that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of approximately 52 degrees C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. It is proposed that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities (Caterina, 1999).

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the 'pain' pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43°C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Sensory neurons from mice lacking VR1 have been demonstrated to be severely deficient in their responses to each of these noxious stimuli. VR1-/- mice show normal responses to noxious mechanical stimuli but exhibit no vanilloid-evoked pain behavior. They are impaired in the detection of painful heat, and show little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia (Caterina, 2000).

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chili peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviors (that is, responses to painful stimuli in animals) have led to VR1 being considered important for pain sensation. The mouse VR1 gene has been disrupted using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lack many of the capsaicin-, acid- and heat-gated responses that have been well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appear normal in a wide range of behavioral tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. It is concluded that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat (Davis, 2000).

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. Described here is 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. The 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 (Peier, 2002a).

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. 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 (Peier, 2002b).

The cellular and molecular mechanisms that enable sensing of cold are not well understood. Insights into this process have come from the use of pharmacological agents, such as menthol, that elicit a cooling sensation. A menthol receptor from trigeminal sensory neurons that is also activated by thermal stimuli in the cool to cold range, has been cloned and characterized. This cold- and menthol-sensitive receptor, CMR1, is a member of the TRP family of excitatory ion channels, and it is proposed that this receptor functions as a transducer of cold stimuli in the somatosensory system. These findings, together with the identification of the heat-sensitive channels VR1 and VRL-1, demonstrate that TRP channels detect temperatures over a wide range and are the principal sensors of thermal stimuli in the mammalian peripheral nervous system (McKemy, 2002).

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments, such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons, suggest the existence of thermosensitive receptors distinct from VR1. A member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3) is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39°C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels (Smith, 2002).

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin, noxious heat, and cooling (TRPM8) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22°C and 40°C. A member of the vanilloid channel family, human TRPV3 (hTRPV3), has been identified that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22°C to 40°C in mammalian cells transfected with hTRPV3 elevates intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current is steeply dependent on temperature, sensitized with repeated heating, and displays a marked hysteresis on heating and cooling. On the basis of these properties, it is proposed that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1 (Xu, 2002).

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. Ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells respond to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiates the response of VR1 to capsaicin, protons and heat and lowers the threshold for heat activation of VR1 from approximately 42°C to approximately 34°C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds (Trevisani, 2002).

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using VRL1 (its capsaicin-insensitive homolog), key amino acids that determine ligand binding were localized (Arg-114 and Glu-761, in the N- and C-cytosolic tails, respectively). Point mutations of the two key residues result in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [(3)H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids block capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket (Jung, 2002).

The capsaicin receptor transient receptor potential V1 (TRPV1; also known as vanilloid receptor 1) is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that extracellular ATP potentiates the TRPV1 currents evoked by capsaicin or protons and reduces the temperature threshold for its activation through metabotropic P2Y receptors in a PKC-dependent pathway, suggesting that TRPV1 activation could trigger the sensation of pain at normal body temperature in the presence of ATP. ATP-induced thermal hyperalgesia is abolished in mice lacking TRPV1, suggesting the functional interaction between ATP and TRPV1 at a behavioral level. However, thermal hyperalgesia is preserved in P2Y1 receptor-deficient mice. Patch-clamp analyses using mouse dorsal root ganglion neurons indicate the involvement of P2Y2 rather than P2Y1 receptors. Coexpression of TRPV1 mRNA with P2Y2 mRNA, but not P2Y1 mRNA, occurs in the rat lumbar DRG using in situ hybridization histochemistry. These data indicate the importance of metabotropic P2Y2 receptors in nociception through TRPV1 (Moriyama, 2003).

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. The Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain (Numazaki, 2003).

Phosphorylation of vanilloid receptors

The capsaicin receptor, VR1 (also known as TRPV1), is a ligand-gated ion channel expressed on nociceptive sensory neurons; the receptor responds to noxious thermal and chemical stimuli. Capsaicin responses in sensory neurons exhibit robust potentiation by cAMP-dependent protein kinase (PKA). PKA reduces VR1 desensitization and directly phosphorylates VR1. In vitro phosphorylation, phosphopeptide mapping, and protein sequencing of VR1 cytoplasmic domains delineate several candidate PKA phosphorylation sites. Electrophysiological analysis of phosphorylation site mutants clearly pinpoints Ser116 as the residue responsible for PKA-dependent modulation of VR1. Given the significant roles of VR1 and PKA in inflammatory pain hypersensitivity, VR1 phosphorylation at Ser116 by PKA may represent an important molecular mechanism involved in the regulation of VR1 function after tissue injury (Bhave, 2002).

The responses of vanilloid receptor (VR) channels to changing membrane potential were studied in Xenopus oocytes and rat dorsal root ganglion (DRG) neurons. In oocytes, capsaicin-evoked VR currents increase instantaneously upon a step depolarization and thereafter rise biexponentially with time constants of approximately 20 and 1000 ms. Similarly, upon repolarization the current abruptly decreases, followed by a biexponential decay with time constants of approximately 4 and 200 ms. Qualitatively similar effects are observed in single channel recordings of native VR channels from DRG neurons and with endogenous VR activators, including heat (43 degrees C), H(+), anandamide and protein kinase C (PKC). The magnitude of the time-dependent current rise increases with membrane depolarization. This effect is accompanied by an increase in the relative proportion of the fast kinetic component, A(1). In contrast, the time constants of the activation and deactivation processes are not strongly voltage dependent. Increasing the agonist concentration both reduces the magnitude of the current rise and increases its overall rate, without significantly altering the deactivation rate. In contrast, PKC both speeds the current rise and slows its decay. These results suggest that voltage interacts with agonists in a synergistic manner to augment VR current and this mechanism will be enhanced under conditions of inflammation when VRs are likely to be phosphorylated (Ahern, 2002).

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activates Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu is antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist. This indicates that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulates PKC(alpha) in either the dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlates with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (1) direct ligand binding (anandamide, vanilloids) and (2) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes show that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia (Olah, 2002).

Inflammatory mediators not only activate 'pain-sensing' neurons (the nociceptors), to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. At the cellular level heat-activated ionic currents are potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation is prevented in the presence of a selective PKA inhibitor, suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits translocated to the cell periphery when activated. The translocation and the current potentiation are abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation is greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia (Rathee, 2002).

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, direct sequencing was used to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. The ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1 was tested. Mutation of S800 to alanine significantly reduces the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduces PMA enhancement of capsaicin-evoked currents, but has no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine has no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduces direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation (Bhave, 2003).

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. The influence of PKA on Ca(2+)-dependent desensitization of capsaicin-activated currents was investigated. By using site-directed mutagenesis, point mutations at PKA consensus sites were created and wild-type (WT) and mutant channels transiently expressed in HEK293t cells were studied under whole-cell voltage clamp. Forskolin, a stimulator of adenylate cyclase, decreases desensitization of TRPV1. The selective PKA inhibitor H89 inhibits this effect. Mimicking phosphorylation at PKA consensus sites by replacing S6, S116, T144, T370, S502, S774, or S820 with aspartate (D) results in five mutations (S116D, T144D, T370D, S774D, S820D) that exhibit decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine (A) results in four mutations (S6A, T144A, T370A, S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca(2+) over Na(+) or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D pre-treatment of cells with forskolin do not reduce desensitization as compared to WT and other mutant channels. It is concluded that S116 and possibly T370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents (Mohapatra, 2003).

Endogenous ligands for vanilloid receptors

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. The vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibits the vasodilator effect of anandamide. Other endogenous and synthetic CB1 and CB2 receptor agonists do not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibits anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induces a capsazepine-sensitive current in whole cells and isolated membrane patches. These results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems (Zygmunt, 1999).

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, it has been shown that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possess the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids fail to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction (Hwang, 2000).

The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitate transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibit and enhance, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhances anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following: (1) anandamide activates VR1 receptors by acting at an intracellular site; (2) degradation by fatty acid amide hydrolase limits anandamide activity on VR1 and (3) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A also inhibits the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism (De Petrocellis, 2001a).

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. The potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect is decreased five-fold in the presence of forskolin or the cAMP analog, 8-Br-cAMP. The effects of 8-Br-cAMP and FRSK are blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhances the sensory neuron- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect is abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induces release of substance P (an effect mediated by VR1 activation) is enhanced three-fold by FRSK. Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions (De Petrocellis, 2001b).

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and capsaicin, the pungent ingredient of 'hot' chili peppers. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. The study examines the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous 'capsaicin-like' substance in mammalian nervous tissues. NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. Evidence was gained for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency [EC(50) approximately 50 nM] and efficacy similar to that of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia. These data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors (Huang, 2002).

N-Arachidonoyldopamine (NADA) is an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicates the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Mass spectrometric analysis of bovine striatal extracts reveals the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA fail to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there is no evidence of spontaneous pain behavior. By contrast, OLDA induces calcium influx, reduces the latency of paw withdrawal from a radiant heat source in a dose-dependent manner, and produces nocifensive behavior. These effects are blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin. These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA is only a weak ligand for rat CB1 receptors; but like NADA, it is recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamines indicates the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors (Chu, 2003).

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