The archetypal thermoTRP is the vanilloid (capsaicin) receptor TRPV1 [19], was first cloned from rat dorsal root ganglion neurons using an expression-cloning screening strategy [20]. This thermoreceptor is expressed at high levels in a subset of small diameter (C and Aδ) dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia nociceptive neurons [20,21]. TRPV1 is mainly expressed in peptidergic neurons, and to a lesser extent in the non-peptidergic nociceptors. This channel is also present in a wide diversity of brain regions and in several non-neuronal tissues. TRPV1 is up-regulated in animal models of inflammatory and osteoarthritic pain and displays re-distribution and up-regulation in large diameter (Aβ) sensory fibers in neuropathic pain models. TRPV1 expression is also increased in several chronic human pain states [22,23].

TRPV1 has a dynamic threshold of activation. In addition to noxious heat (>42 °C), TRPV1 is also gated by acidic pH (pH < 5.9) and a diverse collection of chemical ligands [9,11,24]. For example, exogenous TRPV1 agonists include naturally occurring substances such as capsaicin, allicin, piperine and camphor (the pungent extracts from chilies, garlic, black pepper and cinnamon, respectively); resiniferatoxin from Euphorbia resinifera; tarantula venom peptide toxins and synthetic agents such as 2-aminoethoxydiphenylborate (2-APB) and olvanil. Some agents in the “inflammatory soup”, including endogenous TRPV1 agonists (so-called “endovanilloids”) like anandamide, act in concert to reduce the heat activation threshold of TRPV1 [25,26].

The activity of TRPV1 is controlled by a multitude of regulatory mechanisms that either causes sensitization or desensitization of the channel. Sensitization of the ion channel depends on several mechanisms among which phosphorylation by protein kinase A (PKA), protein kinase C (PKC) and other kinases is of particular importance [27,28,29,30,31,32,33]. In addition, TRPV1 sensitization involves the rapid recruitment of an intracellular pool of TRPV1 to the cell membrane, a process in which phophoinositide 3-kinase and Src kinase play an important function [34,35]. Since phosphorylation causes sensitization, it follows that TRPV1 dephosphorylation by protein phosphatases promotes channel desensitization which is a inhibitory mechanism of regulation [36]. Furthermore a dynamic balance between phosphorylation and dephosphorylation of TRPV1 by Ca²⁺/calmodulin-dependent kinase II and calcineurin, respectively, appears to control the activation/desensitization state of the channel [36,37]. It has been demonstrated that TRPV1 not only participates in pain evoked by chemicals and noxious heat, but it also contributes to peripheral sensitization [9,20,38], acting as the final substrate for multiple inflammatory mediators that operate via distinct intracellular signaling pathways. Pro-inflammatory agents can induce long-term up-regulation of TRPV1 expression, as well as acute functional modification of the protein and fast mobilization of channels from a subcellular vesicular reservoir located near the plasma membrane [39,40,41]. Indeed, increased expression of the channel has been shown in several chronic inflammatory diseases [42,43,44].

Recent studies have reported that TRPV1 plays a pronociceptive role in some models of acute inflammatory pain. Mice lacking a functional TRPV1 gene (TRPV1^−/−) does not display nocifensive behavior following intraplantar injection of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, suggesting that PMA-induced nociceptive behavior is exclusively dependent on TRPV1 [45]. In a model of mild heat injury, TRPV1^−/− mice have markedly reduced thermal and mechanical hyperalgesia [45], this finding has clinical relevance because cutaneous thermal injury induces heat and mechanical hyperalgesia in human skin [46].

TRPV1 may also play an important role in visceral pain and hypersensitivity states. In irritable bowel syndrome (IBS), abdominal pain is a common and distressing symptom where the administration of TRPV1 inhibitors was effective in reducing pain responses to colorectal distension. Increased expression of TRPV1 in DRGs is evident in this model, as well as in a trinitrobenzenesulfonic acid (TNBS) colitis model [47], suggesting that TRPV1 up-regulation may be a mechanism by which the effect of a TRPV1 antagonist becomes manifest [48]. In a model of pancreatic inflammation this mechanism was confirmed [49]. The up-regulation of function via TRPV1 can be long lasting, and may initiate other events leading to hypersensitivity [47,50]. Alternatively, the translocation of TRPV1 to the cell membrane may have an important influence on its function independent of changes in expression [51].

TRPV1 function has been investigated in models of neuropathic pain as well. For instance, TRPV1 plays an important role in chemical and thermal hyperalgesia in a model of diabetic neuropathy [52,53]; its role may be associated with altered cell-specific expression coupled to an increase in its function. The contribution of TRPV1 has been tested in models of neuropathic pain associated with nerve lesion. After traumatic mononeuropathy caused by ligation of sciatic nerve, the induced cold allodynia can be markedly reduced after treatment with TRPV1 silencer RNA [54]. Additional support for a role of TRPV1 in neuropathic pain is provided by the increase in TRPV1 expression levels observed in uninjured DRG following peripheral nerve injury [55,56].

The activity of TRPV1 has also been implicated in other chronic pain diseases. A recent study showed that activation of TRPV1 is involved in bone cancer pain [57]. It was found that TRPV1 protein level and TRPV1-positive neurons increased in DRG from a murine model of bone cancer, while treatment with a receptor antagonist significantly attenuated painful symptoms [58]. All these studies indicate that TRPV1 contributes to the development and maintenance of chronic pain, participating in both thermal and mechanical hyperalgesia [45]. As many proalgesic pathways converge on TRPV1 and this nocisensor is up-regulated and sensitized by inflammation and injury, TRPV1 is a prime target for the pharmacological control of pain. A great progress has been made both at the bench and bedside to develop therapeutics that selectively targets this TRP channel providing a potential alternative to opioid-based analgesics. Furthermore, all these studies lead to the conclusion that TRPV1 is an important contributor to pain although its role is obviously more complex than firstly anticipated.

Transient Receptor Potential Ankyrin 1 (TRPA1), originally called ANKTM1, is the only member of the ankyrin subfamily found in mammals. TRPA1 was cloned from human lung fibroblast and is characterized by an extended fourteen to seventeen ankyrin repeats per subunit within its very long N-terminal tail [71,72]. TRPA1 is 20% homologous at the amino acid level to TRPV1.

TRPA1 channels are predominantly found in the C-afferent sensory nerve fibers of dorsal root, trigeminal, nodose and vagal ganglions, where they co-localize with TRPV1 in a subset of small diameter, unmyelinated, peptidergic neurons [72,73,74,75]. TRPA1 channels show also high degree of co-expression with trk receptors [76], and their level of expression increases in the presence of neurotrophic factors like nerve growth factor [77,78]. This very restricted pattern of expression suggests an important and specific role for TRPA1 in nociception in conjunction with TRPV1 [79,80]. Consistent with this postulate, new findings have evidenced that variation in the TRPA1 gene can alter pain perception in humans [17]. A point mutation in the TRPA1 gene, N855D amino acid exchange in the TM4, shows altered biophysical properties with five-fold increase in the inward current on activation at normal resting potentials. This mutation is associated with an autosomal dominant familial syndrome of episodes of debilitating upper body pain, triggered by fasting a physical stress [17].

TRPA1 is a polymodal channel gated by exogenous and endogenous chemicals, noxious cold temperatures and mechanical forces. This channel is activated by a diverse assortment of pungent or irritating reactive chemical compounds, including those found in mustard oil (allyl isothiocyanate), cinnamon oil (cinnamaldehyde), gas exhaust (acrolein), raw garlic and onions (allicin) and formalin (formaldehyde); all of these elicit a painful burning or prickling sensation [72,73,81,82,83,84,85]. The number of natural and environmental molecules able to gate TRPA1 is continuously rising, and their chemical structures strongly differ among them [86].

TRPA1 is known to be a sensor of noxious cold stimuli, but this property was initially controversial. Temperature below 18 °C activates recombinant TRPA1 [72,81], and TRPA1^−/− mice have impaired behavioral responses to a cold plate maintained at 0 °C [87]. Although some groups have failed to demonstrate a response of TRPA1 to noxious cold [75,83], subcutaneous administration of a TRPA1 agonist can induce cold hypersensitivity in wild-type but not TRPA1^−/− mice [88]. Besides, TRPA1 is also gated by mechanical forces, and it could play a role in mechanotransduction. Mice with a deletion of the pore domain of TRPA1 exhibit decreased behavioral responses to noxius mechanical forces [89], but these results were not shown in a similar mutant [83]. Although TRPA1 seems not to be essential for mechanotransduction [89], it mediates mechanical plasma membrane currents in sensory neurons [90]. In fact, TRPA1^−/− mice have a reduced rate of action potentials and a limited maxium firing range to noxious mechanical stimuli [89].

TRPA1 can be directly activated but also indirectly sensitized by multiple endogenous inflammatory mediators. For instance, TRPA1 is directly targeted by linoleic acid, arachidonic acid, reactive oxygen and nitrogen species [91,92], hydrogen sulfide [93], hypochlorites, or cyclopentenone prostaglandins, like PGJ₂[92,94]. Key pronociceptive signalling pathways can indirectly potentiate TRPA1 activity by increasing intracellular Ca²⁺, a key regulator of TRPA1 activity, since it activates this channel [95,96] while extracellular Ca²⁺ can both potentiate channel gating and subsequently inactivate TRPA1 activity [97]. Furthermore, similar to TRPV1 channel, proinflammatory mediators can sensitize TRPA1 channel function activity via kinases and/or phospholipase C (PLC) activation [93,98]. For example, TRPA1 sensitization by PLC activation develops through membrane PIP₂ hydrolysis [99]. Endogenous PIP₂ inhibits TRPA1 activity, reduces its sensitivity, and delays its desensitization [99,100,101]. Some studies however have not observed that alteration of PIP₂ levels affect TRPA1 activity [97,102].

Bradykinin, proteinase-activated receptor 2 and nerve growth factor potentiate TRPA1 currents via their respective receptors [82,83] Mice with disrupted TRPA1 function fail to develop pain behaviour and thermal and mechanical hypersensitivity after intraplantar injection of bradykinin [83,87], which provides evidence that this phenomenon is relevant in vivo. TRPA1 nocifensive behavior can be sensitized in vitro and in vivo.

Nociceptive signals can also induce functional TRPA1 trafficking to the plasma membrane by vesicle fusion via PKA/PLC signaling [93], a process already described for TRPV1. Since TRPA1 and TRPV1 are co-expressed in the same subset of nociceptors, it is likely that both channels share similar regulatory pathways. TRPA1 desensitization is regulated by TRPV1-directed internalization [103], TRPV1 regulates TRPA1 activation by Ca²⁺[104], while PIP₂ regulation depends on the co-expression profile of TRPA1 and TRPV1 [102].

TRPA1 has been involved in persistent to chronic painful states such as inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis and emphysema among others [105]. As mentioned before, TRPA1 has a main role in the development and maintenance of cold and mechanical hypersensitivity in chronic inflammatory diseases and neuropathies. In fact, treatment with TRPA1 antisense oligodeoxynucleotides or selective TRPA1 antagonists reduces both cold and mechanical hypersensitivity in several models of inflammatory and neuropathic pain [74,88,106,107,108].

TRPA1 role in visceral pain and gastric nociception has been also recently reported [109,110]. TRPA1 is up-regulated in colonic afferent DRGs after colitis induction [111], and treatment with TRPA1 antisense oligodeoxynucleotides or pharmacological blockage of TRPA1 suppresses colitis-induced hyperalgesia [109,112]. Results obtained in TRPA1^−/− mice support these evidences, as well. Selected classes of visceral afferents show reduced mechanosensitivity [113]; responses to noxius mechanical stimulation are impaired in colon [114]; and mechanical hyperalgesia is decreased in a colitis model [111]. Thus, TRPA1 appears to selectively mediate cold and mechanical hypersensitivity in some pathological conditions.

A common theme has emerged whereby TRPA1 is found in nociceptors, and the modality(s) of nociception to which it mechanistically contributes appears to be quite diverse. Akin to TRPV1 [20], TRPA1 may be a molecular “switchboard” integrator for a range of diverse noxious stimuli. Due to its role in painful disease states is of considerable interest for drug delivery and design by many pharmaceutical companies.

Transient Receptor Potential melastatin 8 (TRPM8) is one of the eight members of the melastatin subfamily of transient receptor potential (TRP) ion channels. Since initially cloned from the prostate [125], TRPM8 has been identified in a variety of tissues, both neuronal and non-neuronal. Much of the research has focused on its role in sensory neurons. Indeed, TRPM8 is best known for being a sensor of mild cold temperatures being activated at temperatures below 30 °C.

TRPM8 has been cloned from several mammalian and non-mammalian species, including human [125], rat [126], canine [127], chicken [128] and frog [129]. The human, rat, mouse and canine TRPM8 genes each encode a protein product of 1,104 amino acids with a predicted molecular weight of ~128 kDa. The proteins are highly conserved with 93–98% primary sequence identity (only ~75–80% between mammalian and frog/chicken TRPM8). As with other TRPs, TRPM8 contains six putative transmembrane segments (S1–S6) and a TRP domain in the C-terminus [125]. It forms a homotetrameric channel [130].

TRPM8 is a cation-selective channel with high permeability to both monovalent and divalent cations and a current-voltage (I–V) relationship that exhibits strong outward rectification [126,131]. Agonist-activated TRPM8 currents undergo pronounced Ca²⁺-dependent desensitization (during continuous agonist application) and tachyphylaxis (upon repeated agonist applications) [126]. Despite being permeant through the channel, extracellular Ca²⁺ also blocks TRPM8 currents, an effect that is independent of channel desensitization and possibly results from Ca²⁺ inhibition of currents conducted by monovalent ions [127,132]. Measurements of the single channel conductance range from 21 to 83 pS under various experimental conditions [126,132,133].

As with TRPV1, TRPM8 is an ion channel with polymodal gating mechanisms. It can be activated by multiple types of stimuli including innocuous cool to noxious cold temperatures [127,128,128], chemical ligands such as menthol and icilin [126,127,131], and membrane depolarization [133,134]. Effects of these stimuli on TRPM8 activation are additive [134]. It is argued that the temperature sensitivity of TRPM8 gating is a thermodynamic consequence of the difference in activation energy between voltage-dependent channel opening and closing. Cooling and chemical agonists such as menthol act by changing the voltage dependence of activation towards the hyperpolarizing direction (i.e., physiological potentials) [134,135]. Conversely, antagonists exert their inhibitory effects by shifting the voltage dependence of activation towards more positive potentials [135]. As such, thermal activation of TRPM8 can be regarded as a threshold phenomenon only in the context of a given membrane potential (e.g., ~26 °C at the resting membrane potential of a neuron). Other studies, however, indicate that the increase in channel open probability upon cooling is greater than what can be accounted for by a simple left-shift of voltage dependence of activation, suggesting that temperature and voltage interact allosterically to promote channel opening [133].

Cooling, as well as relatively low doses of menthol or icilin, is also reported to attenuate thermal (noxious heat-induced) and mechanical hypersensitivity in rats with chronic constriction injury (CCI) [136]. These effects are reversed by antisense knockdown of TRPM8 expression, supporting a critical role of TRPM8 activation in the analgesic effects. Furthermore, TRPM8 knockout mice lack cooling-induced analgesia normally present in wild type mice following administration of formalin, a stimulus of acute pain followed by inflammation [137]. These results suggest that activation of TRPM8 can also mediate analgesia in certain acute/inflammatory pain states. The molecular mechanism underlying cold pain/analgesia is poorly understood.

Studies attempting to address the role that TRPM8 may play in these processes have produced results that are paradoxical. A number of reports describe an increased TRPM8 expression in sensory neurons after nerve injury or inflammation [138,139,140]. Others, however, report no change or a decrease in TRPM8 expression [141,142]. Notably, expression of TRPM8 is markedly augmented in bladder specimens from patients with idiopathic detrusor overactivity and painful bladder syndrome [143]. In addition, there is an increase in the fraction of TRPM8-expressing neurons that also express TRPV1 under inflammatory conditions induced by CFA [144].

TRPM8 appears to play an important part in chronic pain, causing, for instance, cold hypersensitivity or analgesia under conditions of nerve injury and inflammation. However, additionally studies are needed to understand the role of TRPM8 in various pain states.

TRPV2 displays 50% amino acid identity with TRPV1 [143]. It can be activated by noxious heat (>52° C), hypotonicity, membrane stretching and a number of exogenous chemical ligands, however, there appears to be some species-specificity for activation. For example, whilst noxious heat (up to 53 °C), 2-APB and Δ⁹-tetrahydrocannabinol (THC) activate rat and mouse TRPV2, human TRPV2 only responds to Δ⁹ THC [153]. This species-specific pharmacology coupled with problems in developing stable recombinant cell lines due to cytotoxicity may underlie the difficulties experienced by many investigators in the study of this channel and the relative lack of detailed characterization to date [154,155]. Given these species differences, the role of TRPV2 as a thermosensor at least in humans is not fully understood. Whilst no selective exogenous or endogenous activators of TRPV2 have been identified to date, several reports indicate that receptor translocation may be an important regulatory mechanism for this channel. TRPV2 expression and function is up-regulated by growth factors, such as IGF-1, heat, phosphatidylinositol 3-kinase (PI3-kinase) and association with the recombinase gene activator (RGA) chaperone protein [21,156,157].

TRPV2 is expressed in pain pathways including medium to large diameter (Aδ and Aβ) DRG, TG and NG neurons and in the dorsal horn of the spinal cord [158]. TRPV2 immunoreactivity in DRG neurons is augmented 2 days after CFA-induced inflammation, which could suggest a role in peripheral sensitization during inflammation [159]. Sciatic nerve ligation results in a build-up of immunoreactivity proximal to the ligation and increases in TRPV2 expression are also seen in rat DRG after unilateral sciatic nerve chronic constriction injury [138]. An increment in expression is seen in sympathetic, but not sensory neurons after peripheral axotomy suggesting a potential role for TRPV2 in sympathetic mediated neuropathic pain [160].

TRPV2 knock-out mice showed reduced embryonic weight and perinatal viability. As adults, surviving knock-out mice also exhibited a slightly reduced body weight. TRPV2 knock-out mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation [161]. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse.

TRPV3 is activated by innocuous warmth (31 °C–39 °C), with activation maintained at noxious temperatures [164,165]. It is activated by a number of exogenous chemical ligands, including natural irritants (e.g., carvacrol, eugenol and thymol, extracts from oregano, cloves and thyme, respectively) and synthetic ligands (e.g., 2-aminoethoxydiphenyl borate (2-APB), 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA)) [166]. TRPV3 is also sensitized and/or directly activated by endogenous ligands, including downstream elements of the inflammatory cascade, such as unsaturated fatty acids (e.g., arachidonic acid) protein kinases (e.g., PKC), and by nitric oxide [167,168]. TRPV3 would be expected to be basally active at body temperature, given its heat activation threshold. However, TRPV3 activity may be further enhanced under inflammatory conditions since repeated activation by heat or chemical ligands sensitizes the channel [166].

In humans, TRPV3 has been reported to be present in pain pathways including DRG and TG neurons, spinal cord, skin and brain [165]. Functional TRPV3 has been found in corneal epithelial cells where may play a role not only in thermosensation, but also in the regulation of cell proliferation [169]. In mouse and rat the distribution of TRPV3 is more controversial. Peier and colleagues reported confinement of TRPV3 to keratinocytes [165], however Zimmermann et al. suggested that TRPV3 may be present, although not functional, in adult mouse DRG neurons [170]. Acute noxious heat thermosensation is diminished in TRPV3^−/− mice, but there is no evidence to date for a role of TRPV3 in thermal or mechanical hyperalgesia after inflammatory insult (CFA, bradykinin) or in behavioural responses to formalin [171].

In certain human disease states there are changes in the level of TRPV3 expression. For example, TRPV3 expression is increased in painful breast tissue [120] and decreased in basal keratinocytes that are recovered from patients with diabetic neuropathy [172]. Activation of Gq-coupled GPCRs, including the histamine and bradykinin receptors, also potentiates TRPV3 activity [166], thus allowing TRPV3 to serve as a convergence point for multiple pain pathways. Taken together, these findings suggest an important role for TRPV3 in pain transduction.

TRPV4, is activated by innocuous warmth (27 °C–35 °C), hypotonicity and shear stress, as well as by chemical ligands [174]. Endogenous chemical ligands (e.g., endocannabinoids and arachidonic acid metabolites and nitric oxide) and exogenous natural plant extracts (e.g., bisandrographolide A) and synthetic ligands (e.g., phorbol ester 4-α-phorbol 12,13-didecanoate, (4α-PDD)) have been identified [175]. Given the thermal threshold for TRPV4 activation, it is expected to be active at body temperature. However it seems that TRPV4 activity is enhanced under inflammatory pain conditions due to the fact that the injection of inflammatory mediators (prostaglandin E2 and serotonin) into the mechanical receptive fields of C-fibres significantly increases the percentage of C-fibres responding to hypotonic stimulus and the magnitude of the response in wild-type, but not TRPV4 knockout mice [159]. Coupled with a widespread tissue distribution, this activation profile has resulted in a large number of distinct physiological functions for TRPV4. These range from temperature monitoring in skin keratinocytes to osmolarity sensing in kidneys, sheer stress detection in blood vessels and osteoclast differentiation control in bone. As knowledge of its physiological roles has expanded, interest in targeting TRPV4 modulation for therapeutic purposes has arisen and is now focused on several areas [176,177].

TRPV4 is expressed in pain pathways in DRG, TG and NG neurons, with an increase in TRPV4 mRNA and protein reported after chronic compression of DRGs [160]. TRPV4 mRNA is highly enriched in colonic sensory neurons and TRPV4 protein is found in colonic nerve fibres from patients with inflammatory bowel disease. In patients with active colitis or inflammation, serosal blood vessels are more densely innervated with TRPV4-expressing fibres compared with non-inflamed tissue [178]. TRPV4 protein is also increased in breast skin in patients with breast pain [179].

Trpv4^−/− mice showed impaired sensitivity to acid, an increase in mechanical nociceptive threshold and altered thermal selection behavior [180,181,182]. In contrast, these mice have normal response to noxious heat and low-threshold mechanical stimuli. Finally, it was demonstrated that agonists of TRPV4 promote the release of the neuropeptides substance P and CGRP from the central projections of primary afferents in the spinal cord [183]. These studies suggest a role of TRPV4 in nociception.

As mentioned, a large number of TRPV1 antagonists, mostly competitive antagonist, have been identified with high efficacy and potency. However, despite the claimed therapeutic potential of these compounds, a rather disappointing number of candidates have progressed through clinical trials because of the initially unpredicted secondary effects such as hyperthermia. In addition, it seems that complete blockade of TRPV1 in some models of chronic pain models results in enhanced hypersensitivity [195]. These observations are consistent with its widespread distribution in neuronal and non-neuronal tissues that suggests an involvement in body functions other than nociception and pain; for instance body temperature regulation [196]. Indiscriminate pharmacological blockade of the receptor by using high affinity, quasi-irreversible, competitive vanilloid antagonists may be responsible for the observed side effects [195]. In this regard, high affinity antagonists that bind to the receptor in an activity-independent manner should show limited therapeutic index, since these compounds will interact with both resting and active channels. Taken together, these data support the need of a different class of antagonists, either by acting on a specific mode of activation or that function on an activity-dependent fashion primarily targeting overactivated sensitized receptors.

Uncompetitive antagonists can be adequate candidates because they are a class of activity-dependent inhibitors that specifically bind to the agonist-receptor complex or to the open state of the channel [197] This property is considered as a solid argument for their preferential blockade of highly activated receptors and minimal interaction with physiologically working or silent channels. Because of their interaction with active receptors, these compounds have attracted a notable interest as potent and safe drugs. A case in point of the therapeutic benefit is memantine, an uncompetitive L-glutamate antagonist of the NMDA receptor approved for the treatment of Alzheimer’s dementia [198]. Accordingly, the identification and validation of uncompetitive antagonists acting as open channel blockers of TRPV1 receptors warrant exploration as a strategy to develop selective analgesic drugs with higher therapeutic index than currently discovered channel antagonists.

Recently, a compound able to block TRPV1 channel (triazine 8aA) by an activity-dependent mechanism was reported [194]. This compound did not alter the capsaicin IC₅₀ consistent with an uncompetitive antagonist. Furthermore, this compound showed a strong voltage-dependent TRPV1 blockage by inhibiting channel activity at negative membrane potential, a hallmark of open-channel blockers. In addition, the observed fast kinetics of channel blockade suggests that it acts from the extracellular side of the channel. At variance with first generation of channel blockers, triazine 8aA displayed a true blockage of TRPV1 activity, since it did not activated the channel at low nanomolar concentrations. This compound holds promise for therapeutic development, although no in vivo activity in pain models has been yet reported. Nonetheless, an important outcome of this study is the unveiling of a pharmacophoric group that acts as an open channel blocker for TRPV1, and that most likely could be modified to develop similar activity-dependent compound for TRPA1 and TRPM8.

The search for new drugs that modulate thermoTRPs activity has focused on finding either natural ligands or functional analogs that can act as ligands. The ability to modulate signaling through a location distinct from the orthosteric ligand-binding site, a property known as allosterism, may also provide novel drug target opportunities [199].

The search for allosteric sites that can modulate protein function implies a detailed knowledge of structure-function relationships and channel activity. In this regard, structure-function analysis of TRPV1 channels has demonstrated that the intracellular TRP domain, a region in the C-terminus adjacent to the receptor internal gate that is highly conserved among many TRP channels [13], is essential for subunit tetramerization and channel gating [15,16,54]. These studies suggested that protein-protein interactions in the TRP region are involved in the functional coupling of stimulus sensing and gate opening [14]. Thus, this protein interface could be used as allosteric site to modulate channel function.

A well-established strategy to interfere with protein-protein interactions is the use of peptides mimicking their sequence [39,200,201]. This strategy has been used to modulate G protein-coupled receptors (GPCR) activity by pepducins, lipid-conjugates peptides that mimic the sequence of CGRP cytoplasmic loops. Pepducins potently and selectively modulate the activity of PAR2 receptor by targeting the intracellular receptor-G protein assembly and have been proposed as potential therapeutic leads [202].

This strategy used in metabotropic receptor signaling has been extended to modulate the protein-protein interactions involved in channel gating in TRPV1 [18]. In this case, several peptides patterned after the C-terminal domain of TRPV1 were tested to modulate TRPV1 activity. These peptides were palmitoylated at the N-terminus to favor membrane anchoring and intracellular delivery. The study identified peptide TRP-p5 as the most potent inhibitory peptide, whose activity is sequence specific and receptor selective. Peptide TRP-p5 has also been used as a pharmacological tool for in vitro and in vivo modulation of the receptor activity in intact cells and in the peripheral nervous system. Because these peptides have been patterned after the TRP domain of TRPV1 and behave as moderate and selective channel inhibitors, they were coined with the term TRPducins [18] (Figure 4).

Given that TRP domain is widely present in the TRP family of channels, the use of TRPducins provides a strategy that could be exploited for the identification and generation of novel TRPducins targeting virtually all TRP channels. Furthermore, TRPducins are based on the known sequences of thermoTRPs, so they obviate the need to know the molecular identity of the natural ligand, presenting a new range of possible thermoTRP modulators, especially those for which there are not yet available antagonists. Therefore, the TRPducin strategy may open a molecular novel avenue to develop selective pharmacological tools for the TRP superfamily of ion channels providing novel therapeutical strategies based in allosteric modulation.

Constitutive and regulated vesicular trafficking mechanisms have a critical role in controlling the surface expression of TRP channels as well as their activation in response to external stimuli. A number of cellular components such as cytoskeletal and scaffolding proteins also contribute to TRP channel trafficking. Thus, mechanisms involved in the assembly and trafficking of TRP channels control their plasma membrane expression and critically impact their function and regulation [204]. For instance, Recombination Gene Activator (RGA), a protein localized in the Golgi apparatus and endoplasmic reticulum, increases cell surface levels of TRPV2. Furthermore, a role in the early biosynthesis of the TRPV2 channel and its trafficking between the endoplasmic reticulum and the different compartments of the Golgi apparatus has been suggested [205,206]. In addition, TRPV4 binding to PACSIN3, a protein associated with synaptic vesicular trafficking and endocytosis, enhances receptor surface expression probably through endocytosis inhibition [207]. PACSIN3 modulates TRPV4 function in a stimulus-specific manner [208] Similarly, TRPV4 interacts with the microtubule-associated protein 7 (MAP7) enhancing receptor surface expression of TRPV4 [209].

Similar to other TRP channels, TRPV1 is arranged in major molecular complexes establishing high-order signaling networks that notably determine the response to external stimuli. A yeast-two hybrid screen of a rat brain library using the N-terminus of TRPV1 as bait identified two synaptic vesicle proteins that were interacting partners of TRPV1: Snapin and Synaptotagmin IX [40]. These proteins bind to the SNARE proteins and participate in neuronal exocytosis, suggesting that surface delivery of TRPV1 channels is a highly regulated, Ca²⁺-dependent exocytotic process. Indeed, the interaction of both vesicular proteins with TRPV1 appears temporal and seems uninvolved in the formation of the molecular complexes at the cell surface [210]. It has been shown that these interactions are pivotal for the trafficking and surface expression of TRPV1 channels in response to activation of intracellular pathways by inflammatory mediators [39,40,41]. The observation that botulinum neurotoxin A attenuates heat hyperalgesia substantiates this hypothesis [211]. Furthermore, the participation of regulated exocytosis was inferred from the potent, almost complete, inhibition of TRPV1 inflammatory potentiation by peptide DD04107, a small peptide patterned after the N-terminal domain of SNAP-25 that efficiently and specifically blocks neuronal exocytosis [200]. Because peptides that mimic the sequence of an SNARE protein have been proven to block vesicular fusion by interfering with the formation of the neuronal SNARE complex [200], these data suggest that rapid membrane recruitment of TRPV1 channels evoked by acute exposure to some inflammatory mediators occurs via SNARE-dependent, Ca²⁺-mediated exocytosis. Compounds such as peptide DD04107 or its derivatives that abrogate SNARE-dependent exocytosis of activated nociceptors could represent a new family of anti-inflammatory and analgesic molecules.

Therefore, regulation of TRPV1 surface density in nociceptor peripheral terminals could be a therapeutic paradigm to treat pain conditions avoiding the abrogation of the TRPV1 subpopulation involved in anti-inflammatory and protective roles.