Chapter 12TRPA1 in Auditory and Nociceptive Organs

TRPA1, initially called P120 and ANKTM1, was originally described as a down-regulated protein in mesenchymal tumor cells and was detected in cultured fibroblasts but lost upon oncogenic transformation, although no expression in healthy tissues was described (Schenker and Trueb, 1998; Jaquemar et al., 1999). With in situ hibridization, we found mouse TRPA1 mRNA absent from most major organs (brain, heart, liver, kidneys, skeletal muscles, lungs, spleen, and testes, as well as whisker pad skin and superior cervical ganglia), but present in the inner ear and in certain peripheral sensory ganglia: dorsal root (DRG), trigeminal (TG), and nodose (Nagata et al., 2005). This very restricted pattern of expression suggests specific roles unique to sensory function.

Molecularly, TRPA1 has six membrane-spanning domains and a presumed pore-forming domain characteristic of all TRPs and many other ion channels. Its N- and C-terminal segments are predicted to be cytoplasmic. In addition, a distinguishing feature of TRPA1 is a very long N-terminus with up to 17 predicted ankyrin (ANK) repeats (Figure 12.1). Many similar repeats are present in the protein ankyrin, and 29 of them are present in TRPN1, a mechanosensory channel protein of flies, worms, and fish that has not been found in mammals (Walker et al., 2000; Sidi et al., 2003; Li et al., 2006). ANK repeats may serve at least two functions: (1) to interact with other proteins, particularly those of the cytoskeleton, and (2) to provide elasticity when in tandem, forming a molecular spring (cited as V. Bennet, personal communication, in Corey et al., 2004; Howard and Bechstedt, 2004; Sotomayor et al., 2005; Lee et al., 2006). Because many models of mechanically gated channels postulate linkage to a force-transducing structure like the cytoskeleton or an elastic gating spring (García-Añoveros and Corey, 1997; Gillespie and Walker, 2001), channels like TRPN1 and TRPA1 may be well endowed for the mechanical transduction that characterizes the auditory and vestibular organs as well as the somatosensory and autonomic ganglia that express it.

FIGURE 12.1

Domain structure of TRPA1 with its predicted topology (the lipid membrane is represented by a gray band) and phylogenetic comparison with other TRP channels of mammals (bold) and nematodes (italic). Because there is no mammalian NOMPC (TRPN1), we include (more...)

Based on the hypothesis that the transduction channel of hair cells and somatosensory neurons could be a TRP channel (Duggan et al., 2000), we screened (RT-PCR and in situ hybridization) TRP genes for expression in the inner ear and somatosensory ganglia. We found several TRPs expressed in ganglia and two whose mRNA is expressed in the organ of Corti, the place in the cochlea where the sensory cells reside (unpublished results and Corey et al., 2004). In addition, a third TRP channel protein (MCOLN3, or TRPML3) has also been detected in hair cells (Di Palma et al., 2002). In this chapter we discuss TRPA1, which is expressed both in the inner ear and peripheral ganglia.

DISTRIBUTION OF TRPA1 IN THE INNER EAR

By ISH we found TRPA1 expression in supporting cells of the neonatal organ of Corti, although there was no clearly detectable expression in the mechanosensory hair cells (Corey et al., 2004). However, because very few functional transduction channels are present per hair cell, very low levels of TRPA1 are not inconsistent with this protein contributing to transduction channels. We first considered this exciting, if less straightforward, possibility.

Using an antibody to the N-terminus of TRPA1, we confirmed expression of this protein in all the mechanosensory epithelia of the inner ear: cochlear organ of Corti, saccular and utricular maculae, and crista ampullaris of the semicircular canals. The immunoreactivities were completed by excess antigenic peptide. Further, this antibody recognizes a single band in Western blots from organ of Corti or saccule plus utricle. Therefore our antibody appears to specifically recognize TRPA1. These experiments confirmed strong TRPA1 expression in supporting cells, but also revealed a late onset TRPA1 immunoreactivity to the sensory hair cells at the kinocilia, structures that are distinct from the actin-rich mechanosensory stereocilia and are not thought to be mechanosensory, but also at cuticular plates and, weakly, the stereocilia (Kumar et al., 2005; Nagata et al., 2005). Studies using an antibody raised against the C-terminus, although not as extensively controlled, report similar immunoreactivity (Corey et al., 2004). However, stereocilia have been known for nonspecifically binding antibodies. Further experiments are needed to confirm functional TRPA1 presence in hair cells. In support of this, TRPA1 agonists AITC and icilin were recently reported to activate inward currents in hair cells (Stepanyan et al., 2006). Because mechanosensory transduction takes place at the stereocilia from day 17 of mouse embryogenesis, and the cuticular plate is thought to hold a reserve of transduction components, this localization pattern is consistent with a potential role of TRPA1 in hair cell mechanotransduction, but also suggests that other proteins make transduction channels early in development.

FUNCTIONAL TESTS OF TRPA1 IN THE INNER EAR

Two general approaches address the function of TRPA1 in hearing: (1) inhibiting its production with acutely applied agents (Corey et al., 2004), and (2) mutating the gene by deleting part of it (Bautista et al., 2006). These two approaches have yielded apparently contradictory results.

TRPA1 knockdown with morpholinos in zebrafish or with viral-mediated RNA interference in cultured mice utricles resulted in a reduction, although not an elimination, in the magnitude of the hair cell transduction currents. Although these agents can have nonspecific effects, an alternative interpretation is that they specifically inhibited TRPA1 production and that this protein participates, directly or indirectly, in generating mechanotransduction currents. On the other hand, a deletion of the pore domain of TRPA1-produced animals with apparently normal auditory function, as assessed by auditory brainstem responses (ABRs) and distortion product otoacustic emissions (DPOAEs), clearly indicates that intact TRPA1 is not necessary for hair cell transduction. However, we should bear in mind that (1) the deletion of TRPA1 left a large portion of the protein intact (the 17 ANK repeats plus the first five membrane-spanning domains), and (2) TRPA1 may act redundantly with other channel subunits to form transduction channels. The fact that there are transduction channels distributed along the length of the cochlea with different conductance levels (Ricci et al., 2003; He et al., 2004) suggests to us that these channels are molecularly heterogeneous, something that could be accomplished by alternative splicing or by heteromultimerization of several distinct channel subunits (like other TRPs that we and others have found in hair cells). In this admittedly hypothetical context, a protein like TRPA1 could contribute to hair cell transducers but be compensated for by other subunits if lacking, perhaps accounting for the lack of phenotype of the mutant and the limited effects of the acute inhibition studies. Although the mutant phenotype proves that TRPA1 is not an essential subunit of the pore, we still need to determine with certainty whether TRPA1 contributes to hair cell transduction or not.

COMPARING CHANNEL PROPERTIES OF TRPA1 AND OF THE HAIR CELL TRANSDUCER

If TRPA1 is indeed a component of the hair cell transducer, we would expect that the pore properties of TRPA1 in heterologous cells and those of the hair cell transducer to be similar, with some distinctions due to the lack of other subunits in heterologous cells. Heterologously expressed TRPA1 can be opened with several agonists such as allyl isothiocyanate (AITC) (Bandell et al., 2004; Jordt et al., 2004; Nagata et al., 2005). Hence, TRPA1 channel properties could be studied and compared with those already known for the endogenous hair cell mechanotransducing channel. Many similarities, and a few differences, have been found (Nagata et al., 2005). The most prominent difference relates to channel gating. Heterologously expressed TRPA1 has not been shown to be mechanically gated. This is not surprising: in hair cells the channel is in a unique subcellular specialization, the stereocilia, with a unique molecular composition of lipids and proteins. Indeed, hair cell stereocilia are rich in certain lipids like PIP₂, and its removal impairs mechanotransduction (Hirono et al., 2004). Most models of hair cell transduction postulate that the channel associates with other proteins, like the extracellular tip link and a linker to the actin cytoskeleton, which transmit gating force to the channel. Without these structures, a channel may not be able to sense displacements.

Despite these differences in gates in distinct cellular settings, if TRPA1 forms part of the hair cell transducer one would expect that, once opened, the properties of their pores would be similar, as it is the pore that TRPA1 would contribute to in the hair cell transduction complex.

Heterologous TRPA1 (Nagata et al., 2005) and the hair cell transducer (Jorgensen and Ohmori, 1988; Kroese et al., 1989; Rüsch et al., 1994; Kimitsuki et al., 1996; Ricci, 2002; Farris et al., 2004) are blocked by the same four antagonists (amiloride, gadolinium, gentamicin, and ruthenium red), and all with indistinguishable Hill coefficients, which supports a common mode of action. Of these blockers, the two that act by plugging into the pore, ruthenium red and gentamicin, have IC₅₀s that are indistinguishable between TRPA1 in heterologous cells and the mechanotransducer in hair cells, but the two other blockers act with different affinities. Heterologously expressed TRPA1 is 100 times more sensitive to Gd³⁺ (it is indeed the most gadolinium-sensitive channel known) and 10 times less sensitive to amiloride, revealing some molecular divergence between heterologous TRPA1 and endogenous hair cell transducers.

Another parallel between TRPA1 and the hair cell transducer relates to the effects of calcium on channel gating and conductance. Calcium, a permeant ion of both channels, reduces single channel conductance to the same extent (54 percent of its conductance with no or just micromolar amounts of Ca²⁺) (Ricci et al., 2003; Nagata et al., 2005). In addition, as extracellular Ca²⁺ enters the transduction channel, it binds to a site at or very close to the channel and causes first an increase in open probability (as detected with single channel recordings in hair cells) and then closure, a phenomenon often referred to as fast adaptation (Howard and Hudspeth, 1988; Kennedy et al., 2003). Upon depolarization, the channels open again (Ricci et al., 2000). The same events take place in heterologously expressed TRPA1 chemically activated with AITC (Nagata et al., 2005):

1. Ca²⁺ entering from the outside causes (as mentioned above) a reduction of single channel conductance, but also increases the open probability, resulting in a brief potentiation of the current.
2. Subsequently, Ca²⁺ induces channel closure (Figure 12.2a, b, and c). It should be noted that, while the Ca²⁺-induced closure is thought to mediate fast adaptation in hair cells, the brief potentiation of opening might underlie the “release” that occurs just before adaptation, which fosters a small, additional positive displacement of the hair bundle, a phenomenon that may contribute to amplification of the response of the cochlea, and thus to enhancing hearing sensitivity to soft sounds (Hudspeth, 2005; Lemasurier and Gillespie, 2005) (Figure 12.2d).
3. Through an unknown mechanism, cellular depolarization after calcium-induced closure reopens the channels. Figure 12.2c shows a model of calcium action, modified from one developed to account for the behavior of the hair cell transducer but that also illustrates the phenomena described for heterologously expressed TRPA1.

FIGURE 12.2

Effects of calcium on permeation and gating of chemically activated (with AITC), heterologously expressed TRPA1 shown for (a) single channels (in outside out patches) and (b) whole cells (Nagata et al., 2005). (c) Calcium effects on TRPA1 as well as on (more...)

The primary caveats to this comparison are due to timing, for while the effects of Ca²⁺ on the hair cell transducer take place in milliseconds, in chemically activated, heterologously expressed TRPA1 they take seconds. However, these quantitative differences do not imply a mechanistic difference. What we envision might be happening is that Ca²⁺, as it goes through the channel, either goes across to the cytoplasm or binds to it, in which case it induces first potentiation (by increasing Po, even if single channel conductance diminishes), and, second, closure (Figure 12.2c). Most calcium ions go through, and how long it takes for one to bind would depend on the affinity of the binding site, something that could vary between TRPA1 expressed alone or as part of the transduction complex in hair cells.

Finally, TRPA1 displayed a single channel conductance of ~100 pS (Nagata et al., 2005), similar to that described for the hair cell transducer (Crawford et al., 1991; Denk et al., 1995; Géléoc et al., 1997; Ricci et al., 2003).

Given the many mechanistic similarities between the pores of TRPA1 and the hair cell transducers, but also considering the various quantitative differences, it seems that either TRPA1 or similar channel proteins (perhaps the other TRPs found in stereocilia), or perhaps heteromultimers of TRPA1 with these various proteins, could form the pore of the hair cell mechanotransducer.

At this point, more experimentation is required to determine the function of TRPA1 in hair cells, especially whether it does or does not contribute to mechanotransducing channels. Yet another intriguing question is what the function of TRPA1 might be not in hair cells but in their support cells, where its expression, although not yet thoroughly described, is most prominent.

DISTRIBUTION OF TRPA1 IN SENSORY GANGLIA

Outside the ear, TRPA1 mRNA has only been reproducibly detected in peripheral sensory ganglia that have nociceptive neurons: trigeminal, dorsal root, and nodose. In all these ganglia, the nociceptive neurons are generally the smaller cells in diameter and axon caliber. In situ hybridization in mouse sections demonstrated TRPA1 mRNA expression by a large number of the smaller, nociceptive cells (36.5 percent of all neurons in TG, 56.5 percent of all neurons in cervical DRGs, and 28.4 percent of all neurons in nodose) (Nagata et al., 2005). Similar results were obtained in rats (36.7 percent in TG, 39.5 percent in lumbar, L5 and L6 DRGs) and indicated that these cells do not coexpress neurofilament 200 or the growth factor receptors TrkC and TrkB, but do express the NGF receptor TrkA or no Trk receptor at all; most express other markers of nociceptive neurons like the capsaicin receptor TRPV1, calcitonin gene-related peptide (CGRP), or substance P (SP) (Kobayashi et al., 2005). All these experiments confirm that TRPA1 is expressed by C-fiber nociceptors and not by A-fiber neurons (including the Aδ fibers, the faster-conducting nociceptors, as well as the Aβ and Aα innocuous mechanoreceptors). A previous report indicating TRPA1 expression in only 3.6 percent of the mouse DRG neurons may be an underestimate (Story et al., 2003), whereas another report of expression based on neonatal rat TG (~20%) (Jordt et al., 2004) is similar enough given the potential for developmental differences. However, it is important to realize that experimentally induced inflammation and neuropathic pain increase the number of neurons that express TRPA1 mRNA (Obata et al., 2005), so the precise distribution may depend on the nociceptive history of each animal and ganglion. As a general rule, however, TRPA1 is expressed by half or more of the small, C-fiber nociceptive neurons, a distribution consistent with a prominent role for TRPA1 channels in pain.

Antibodies to TRPA1 confirm expression to small, peripherin-positive nociceptors (Bautista et al., 2005; Nagata et al., 2005). In addition to detecting TRPA1 protein at the cell bodies (the site of protein synthesis), it is also detected in their peripheral nerves, the expected subcellular location for a channel involved in nociceptive transduction.

Unlike many other potential mediators of pain, TRPA1 has a surprisingly restricted expression, having been so far only reproducibly detected in a few cells of the inner ear and in most nociceptors. Therefore, inhibitors of this channel, provided that they are specific (currently known ones are not), could perhaps function as pain-killers with limited side effects. Delivery to the ear may have to be avoided, but this would be easily achieved with local applications, which should be effective because TRPA1 is present at the periphery. A drug with these characteristics could revolutionize the medical treatment of pain. But what kind of pain would a TRPA1 antagonist block?

THE FUNCTION OF TRPA1 IN NOCICEPTORS

Functional evidence that TRPA1 may indeed serve as a pain-receptor channel first came from heterologous expression studies that demonstrate that TRPA1 channels are opened upon exposure to various pain-producing chemicals (the pungent components of many often edible substances): allyl isothiocyanate (horseradish, wasabi, and mustard oil), benzil isothiocyanate (yellow mustard and crushed papaya seeds), phenylethyl isothiocyanate (Brussels sprouts), methyl isothiocyanate (capers and nasturtium seeds), cinnamaldehyde (cinnamon), methyl salicilate (wintergreen oil, often used in mouthwashes like Listerine), eugenol (clove), allicin (freshly crushed garlic), the synthetic compound AG-3–5 (icilin), and acrolein (tear gas and the undesirable by-product of certain chemotherapeutic regimes) (Story et al., 2003; Bandell et al., 2004; Jordt et al., 2004; Bautista et al., 2005; Macpherson et al., 2005; Nagata et al., 2005; Bautista et al., 2006). More recently, mice with a deletion of part of TRPA1 have been generated and found to be insensitive to TRPA1 agonists mustard oil and allicin. Dissociated trigeminal ganglion neurons from mutants did not respond to these agonists, and mutant animals did not react adversely to their acute application and did not develop neurogenic inflammation and hyperalgesia, which wild-type animals do. This suggests that TRPA1 is the primary and perhaps only receptor for these pungent compounds in nociceptive neurons (Bautista et al., 2006).

How all these different compounds activate TRPA1 is not clear. Their low hydrophilicity and extremely slow activation (tens of seconds to minutes), plus the fact that they can activate a cell-attached patch by exposure to the outside of the rest of the cell membrane, imply a mechanism of action that might require prior partition of these chemicals into the lipid membrane to then, directly or indirectly, activate TRPA1. Because these pungent chemicals are not endogenous to animals and no evidence indicates that they would appear in damaged tissues, they would not, under normal physiological conditions, activate TRPA1. What does?

Another mechanism that opens TRPA1 in heterologous cells is through metabotropic receptors that activate phospholipase C (PLC) second-messenger pathways (Jordt et al., 2004). This is interesting because many endogenous pro-algesics and pro-inflammatory agents (bradykinin, histamine, serotonin, ATP, and neurotrophins) act this way. Indeed, bradykinin has been shown to activate TRPA1 in cells that also expressed the bradykinin receptor (Bandell et al., 2004). Accordingly, TRPA1 mutant mice did not develop hyperalgesia in response to bradykinin. Interestingly, for responding to bradykinin, trigeminal neurons require both TRPA1 and TRPV1 (Bautista et al., 2006). Hence TRPV1, which is an ionotropic sensor for noxious heat and acidosis, can also act as a mediator of second messenger–induced sensitization. We wonder if there may also be an ionotropic noxious stimulus, besides the above-mentioned exogenous agonists, that activates TRPA1.

An interesting hypothesis is that TRPA1 is a sensor for painfully cold temperatures. This idea was suggested by heterologous expression studies of TRPA1 that showed channel activation by temperatures in the painfully cold range (below 17°C) and by icilin (Story et al., 2003; Bandell et al., 2004), a synthetic compound that produces a cool sensation. A major drawback to this hypothesis is that, in attempts by others, heterologously expressed TRPA1 has been activated by icilin, but not by cold (Jordt et al., 2004; Nagata et al., 2005). In addition, TRPA1 mutant mice respond normally to cold, and their sensory ganglia have a normal distribution of menthol-sensitive (i.e., TRPM8 expressing) and insensitive neurons (Bautista et al., 2006). Because icilin also activates the cold receptor channel TRPM8, and it produces both a cold and a prickling sensation, it may be that the cold sensation is mediated by TRPM8 activation and the prickling by TRPA1 activation. Perhaps in certain cellular settings cold triggers a cellular response that can activate TRPA1, which is not intrinsically sensitive to cold itself, thus explaining the occasional currents observed upon cooling in cells expressing TRPA1. But whether TRPA1 plays a physiological role in sensing painful cold seems at present unlikely (Reid, 2005). In addition, all other chemical agonists of TRPA1 produce subjective sensations of pain, but not of cold, arguing against a role of TRPA1 in sensing cold. It is certainly possible that painful cold sensation results from the combined activation of TRPA1 and TRPM8, whereas pungent chemicals that activate TRPA1 do not act on TRPM8 and could thus produce a different pain. But even this would imply that TRPA1 mediates something else than painful cold.

One hypothesis is force. Regardless of the function of TRPA1 in the ear, its pharmacology indicates that it is blocked by a set of four chemicals (amiloride, Gd³⁺, ruthenium red, and gentamycin) that are characteristic blockers of mechanosensory channels found in several cell types besides hair cells (Hamill and McBride, 1996; Nagata et al., 2005). The wide distribution of TRPA1 among nociceptors is consistent with the large number of c-fibers that are or can become mechanosensitive. A caveat to this hypothesis is that in heterologous cells, TRPA1 has not thus far been shown to be mechanically gated, although this may be explained by the lack of accessory molecules, both proteins and lipids, that would confer mechanosensitivity to a channel. Perhaps more important is that TRPA1 mutant mice have the same paw withdrawal thresholds to mechanical stimulation as wild-type mice (Bautista et al., 2006). However, this is not necessarily unexpected if we consider the lack of naturally occurring mutants, either in mice or in humans, with congenital insensitivity to touch or mechanically induced nociception. This rarity, striking in comparison with the hundreds of genes that, when mutated, produce other sensory defects like deafness or blindness, suggests that either (1) very few genes participate in somatosensory mechanoreception, which seems unlikely, or (2) many genes participate, and do so redundantly. Therefore determination of TRPA1’s potential role in mechanonociception may require detailed phenotypic analysis or the generation of animals with mutations in other genes in addition to TRPA1.

TRPA1 may very well be polymodal, activated by more than one noxious form of stimulation, as other nociceptive receptor channels have been found to be (for example, the capsaicin-, heat-, and acid-sensitive TRPV1, which also mediates bradykinin-induced sensitization). In this regard, it is worth noting that in Drosophila the painless gene, which encodes a TRPA homologue (not the TRPA1 orthologue, but a close relative with no mammalian orthologue), mediates the aversive response to touch with a heated probe and is required for both thermal and mechanical nociception (Tracey et al., 2003).

A CHANNEL PROPERTY OF TRPA1 FOR NOCICEPTION

Whatever the stimulus that gates TRPA1 in nociceptors may be, one property of this channel renders it well suited for nociception (Nagata et al., 2005). As described above, after the channel opens in response to a pungent agonist, extracellular calcium enters, and within tens of seconds it causes channel closure. But this phenomenon is voltage sensitive, so that inactivation occurs if the cell is at a potential of −80 mV but not (or very slowly) at −20 mV, which is the potential reached by a stimulated nociceptive DRG neuron (Blair and Bean, 2003). Therefore, if TRPA1 is weakly stimulated and the resulting currents fail to depolarize the neuronal terminal sufficiently, the channels would close. But if a stronger stimulation triggers enough cation influx to sufficiently depolarize the neuron, the channel would remain open as long as it is stimulated by the noxious agonist. In this way TRPA1 could distinguish between subthreshold (i.e., innocuous) stimulation, to which it would inactivate, and suprathreshold (i.e., noxious) stimulation, to which it would remain open as long as it lasted. This channel property might account for the lack of habituation that is so characteristic of pain sensation. Furthermore, it is easy to envision that depolarization due to activation of another nociceptive channel (i.e., heat activation of TRPV1, which is expressed by the TRPA1-expressing neurons) would lower the threshold stimulation required to keep TRPA1 open. This scenario could account for other characteristic pain phenomena like coincidence detection and perhaps also allodinia (if a sensitizing stimulus were to leave the neuron or its terminal depolarized).

TRPA1 IN INVERTEBRATES

Another aspect in which TRPA1 is remarkable is its evolutionary conservation. A phylogenetic comparison of TRP channels from mammalian and nematode genomes (Figure 12.1) reveals that both contain a TRPA1 orthologue, closer to each other than to any other TRP channel produced by their respective genomes, and these orthologues have the same overall protein structure (17 ANK repeats followed by the TRP-like channel domains). We have found expression of C. elegans TRPA1 in presumed nociceptive and mechanosensory neurons, as well as in neuronal support cells, implying a conservation of function from nematodes to mammals (Mancillas et al., 2005, and our unpublished results). This suggests that TRPA1 was used by organisms 600 million years ago in ancestral sensory systems.

On the other hand, a distinction of TRPA1 in nematodes is that it is also expressed, rather prominently, in various nonneural cells, some of which are essential for survival. Curiously, most of the pungent agonists of TRPA1 kill worms (our unpublished results), and worm parasites are commonly ingested, especially with uncooked foods. This distinction in TRPA1 expression, in sensory cells in humans but also in vital organs in nematodes, might provide a rationale for why so many cuisines from all over the world mix pungent TRPA1 agonists with nutrients, as humans can tolerate and even acquire a “taste” for the same chemicals that kill their parasites.

NOTE

Another targeted mutation of TRPA1 that also deletes the pore domain was recently published (Kwan et al., 2006). A detailed analysis showed that the mutant mice have raised thresholds and decreased withdrawal responses to mechanical stimulation with Von Frey hairs to the paw. These results add support to our hypothesis that, at least in nociceptors, TRPA1 may form mechanosensory channels.

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