Gene name - painless
Cytological map position - 60E5
Function - calcium channel
Symbol - pain
FlyBase ID: FBgn0060296
Genetic map position -
Classification - TRPN family
Cellular location - surface
|Recent literature||Tokusumi, Y., Tokusumi, T. and Schulz, R. A. (2017). The nociception genes painless and Piezo are required for the cellular immune response of Drosophila larvae to wasp parasitization. Biochem Biophys Res Commun 486(4):893-897. PubMed ID: 28342875
In vertebrates, interaction between the nervous system and immune system is important to protect a challenged host from stress inputs from external sources. This study demonstrates that sensory neurons are involved in the cellular immune response elicited by wasp infestation of Drosophila larvae. Multidendritic class IV neurons sense contacts from external stimuli and induce avoidance behaviors for host defense. The findings show that inactivation of these sensory neurons impairs the cellular response against wasp parasitization. It was also demonstrated that the nociception genes encoding the mechanosensory receptors Painless and Piezo, both expressed in class IV neurons, are essential for the normal cellular immune response to parasite challenge.
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).
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 Fishers 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).
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).
The EP(2)2451 insertion is located 3.6 kb upstream of the predicted gene CG15860. Alterations to CG15860 indeed result from painless mutations. The closest protein relatives of CG15860 are ion channels of the transient receptor potential family. Consistent with this, the predicted protein contains eight ankyrin repeats at its N terminus and a TRP-like ion channel domain near its C terminus. While Painless (CG15860) is distantly related to the TRPV genes previously implicated in nociception, it represents a distinct member of the TRP ion channel family. The protein of known function most closely related to Painless is NOMP-C. Painless thus belongs in the TRPN class of ion channels (Montell, 2002). When the predicted channel region was used for the purpose of comparison, the closest vertebrate homolog found was the human gene ANKTM1 (Jaquemar, 1999; Tracey, 2003 and references therein).
painless does not encode a Drosophila ortholog of TRPV1 but instead represents a second member of the TRPN gene family whose founding member, no mechanoreceptor potential-C (NOMP-C), is essential for Drosophila mechanosensation. Indeed, the closest vertebrate homolog of painless, ANKTM1 (Story, 2003), has been shown to be expressed in a subset of pain sensing neurons of the mouse (Tracey, 2003 and references therein).
Using the reverse transcriptase polymerase chain reaction (RT-PCR) with primers designed across the region, the structure of a cDNA from the painless locus was determined; the cDNA encodes a 105 kDa protein identical to that predicted by BDGP for CG15860 (Painless). Several features of this cDNA were not predicted by BDGP, namely a small, 54 bp noncoding exon 3.6 kb upstream of CG15860, and a 260 bp 3' untranslated region. All four of the painless P element insertions disrupt the 5' noncoding exon of this transcript. A Northern blot showed that the 3.0 kb transcript of painless1 mutant larvae migrates at approximately 3.5 kb. The Northern blot also indicated smaller, approximately 2.0 kb and 1.0 kb transcripts that were unaffected by the mutation (Tracey, 2003).
Using RT-PCR, it was found that the 3.0 kb painless transcript encoding Painless/CG15860 is indeed altered as a result of splicing the P element derived sequences into the wild-type message. In one mutant transcript, 441 base pairs of EP element sequence are spliced into the second exon of the wild-type transcript. This mutant stretch of 441 nucleotides contains four start codons (ATGs), each shortly followed by an in-frame stop codon. Since upstream small open reading frames greatly reduce the efficiency of translational initiation from those downstream, this transcript is unlikely to produce a Painless protein. Another mutant transcript utilizes the 3' splice donor site of the noncoding painless 5'UTR but contains an upstream in-frame mutant ATG encoded by sequences from within the 3' end of the P element. This transcript is thus predicted to encode a mutant Painless protein that has 65 additional amino acids at its N terminus. From pain3, a similar mutant transcript was isolated, which also contains an upstream, in-frame ATG that is predicted to add 24 amino acids to the N terminus of Painless (Tracey, 2003).
date revised: 10 November 2003
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