Transient receptor potential channels as therapeutic...

Transient receptor potential (TRP) channels (BOX 1; FIG. 1)

are being ardently pursued as targets for drug discovery. There are several factors that make TRP cation channels appealing as drug targets. First, although ion channels have been successful drug targets, achieving subtype-selectivity has always been a major challenge, particu-larly with voltage-gated sodium and calcium channels. As members of the TRP family of channels do not share much homology with one another, the identification of subtype-selective compounds is likely to be more attain-able. Second, TRP channels act as integrators of several well-described signalling systems, including those that are mediated by cell surface receptors (for example, G protein-coupled receptors (GPCRs) and growth fac-tor receptors). Third, mutations in many of the genes that encode TRP channels are sufficient to cause disease in humans.

Pioneering research in the field of pain has established that a subset of TRP channels (those that are activated by temperatures; the so-called thermoTRP channels) are capable of initiating sensory nerve impulses fol-lowing the detection of chemical and thermal stimuli (reviewed in REFS 1,2). Although pain is currently the most advanced TRP channel-related field, an increasing number of gene deletion studies in animals and genetic association studies in humans have demonstrated that the pathophysiological roles of TRP channels extend well beyond the sensory nervous system (reviewed in REF. 3). Indeed, even broadly classifying TRP channels as sensors of environmental cues understates the diversity of their function. In fact, many TRP channels are activated by

second messenger signalling cascades that are initiated by receptor activation, and some TRP channels function on intracellular membranes3.

TRP channels are associated with several pathophysi-ological processes, which include (but are not limited to) pain, respiratory reflex hypersensitivity, cardiac hyper-trophy and ischaemic cell death3. In addition, several gene association studies in humans have indicated that single-nucleotide polymorphisms (SNPs) in the cod-ing regions and/or promoters of genes that encode TRP channels are either associated with an increased risk of multifactorial diseases or they appear to be causative factors in rare heritable conditions4. Interestingly, when these mutated TRP channels are expressed in recombi-nant systems, they generally display enhanced activity, which suggests that blockade of these channels may pro-vide therapeutic benefit.

To date, target validation of TRP channels has largely been generated via genetic studies; by comparison, the identification of chemical modulators of TRP channels is in its infancy. Several natural ligands (for example, capsaicin and menthol) have provided valuable insights into the pharmacology of TRP channels (reviewed in REFS 1,2,5). Although these molecules can be informa-tive when they are used as tools for compound screen-ing, they rarely display the potency, selectivity and/or the physical properties that are desirable in modern drug discovery programmes. However, despite these obstacles several pharmaceutical companies have been able to develop blockers of TRP cation channel subfam-ily V, member 1 (TRPV1; also known as the capsaicin

*Hydra Biosciences, 790 Memorial Drive, Cambridge, Massachusetts 02139, USA.‡Neuronal Targets Discovery Performance Unit, GlaxoSmithKline Pharmaceuticals,King of Prussia, Pennsylvania, USA.§Department of Physiology, Research Center for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary. ||Department of Pathology, Monmouth Medical Center, 300 Second Avenue, Long Branch, New Jersey 07740, USA.¶Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.Correspondence to A.S. e‑mail: [email protected]:10.1038/nrd3456

Transient receptor potential channels as therapeutic targetsMagdalene M. Moran*, Michael Allen McAlexander‡, Tamás Bíró§ and Arpad Szallasi||¶

Abstract | Transient receptor potential (TRP) cation channels have been among the most aggressively pursued drug targets over the past few years. Although the initial focus of research was on TRP channels that are expressed by nociceptors, there has been an upsurge in the amount of research that implicates TRP channels in other areas of physiology and pathophysiology, including the skin, bladder and pulmonary systems. In addition, mutations in genes encoding TRP channels are the cause of several inherited diseases that affect a variety of systems including the renal, skeletal and nervous system. This Review focuses on recent developments in the TRP channel-related field, and highlights potential opportunities for therapeutic intervention.

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PruritusPruritus (also called an itch) is an unpleasant cutaneous sensation that is associated with an urge to scratch. Various categories of pruritus have been suggested, including pruriceptive itch (which arises from skin conditions), neurogenic itch (which is caused by systemic disorders), neuropathic itch (which is due to a primary neurological disorder) and psychogenic itch.

receptor); these blockers have been sufficiently safe and effective in preclinical studies to merit their testing in clinical trials. Many of these trials are still underway (TABLE 1; Supplementary information S1 (box)), but the results of published trials have not been straightforward; rather, they have raised new questions regarding the role of TRPV1 in humans.

Despite these setbacks, we believe that the pace of drug discovery in the field of TRP channels has inten-sified. The diversity in the expression, function and structure of TRP channels provides the opportunity to generate novel, selective chemical entities that can be tested in diverse clinical indications. Molecules that target TRPV3 have entered Phase I trials6 (see the Sanofi website), and blockers of TRP cation channel subfamily A, member 1 (TRPA1)6, TRP cation channel subfamily M, member 8 (TRPM8)7 and TRPV4 (REF. 8) have been efficacious in preclinical disease models and devoid of unexpected acute adverse effects that could limit their tolerability (TABLE 2).

Although results from clinical trials will serve as the final arbiters of the utility of TRP channel modulators as therapeutics, current evidence indicates that these channels contribute to the development and/or progres-sion of the symptoms of many diseases (for example, neuropathic pain, overactive bladder, asthma, anxiety disorders and pruritus) and that they are therapeutic targets that are amenable to blockade by small mole-cules. In this Review, we summarize the state-of-the-art

developments in this rapidly evolving field, highlight crucial advances and look ahead to the next steps in elucidating the roles of TRP channels in neurology, der-matology, pulmonology, cardiology, urology, oncology and heritable diseases.

TRP channels as analgesic targetsThe role of TRP channels is best understood in the pain area (FIG. 2). TRPV1 and TRPV3 antagonists have already advanced to clinical trials (TABLE 1), whereas TRPA1 antagonists (TABLE 2) are still in preclinical development.

TRPV1. As the desensitization of nociceptive neurons to capsaicin has analgesic potential5, the cloning of the cap-saicin receptor, TRPV1 (REF. 9), has spurred considerable efforts in the pharmaceutical community to find TRPV1 antagonists. However, side effects associated with the use of TRPV1 antagonists have so far prevented any com-pounds from progressing beyond testing in Phase II tri-als. Particular concerns have surfaced around the effects of TRPV1 antagonism on the regulation of body tem-perature10 and in the detection of noxious heat (S. Eid, personal communication).

TRPV1 and regulation of body temperature. Trpv1-null9,11 and -knockdown12 mice have an appar-ently normal body temperature, despite the fact that they prefer lower ambient temperatures13. These characteris-tics are also observed in rats in which TRPV1-expressing

Box 1 | Introduction to TRP channels

The transient receptor potential (TRP) cation channel superfamily is a diverse family of 28 cation channels that have varied physiological functions, including thermal sensation, chemosenation, magnesium transport and iron transport (reviewed in REF. 3). The TRP channel superfamily is classified into six related subfamilies: TRP cation channel subfamily C (canonical; TRPC), TRP cation channel subfamily V (vanilloid; TRPV), TRP cation channel subfamily M (melastatin; TRPM), TRP cation channel subfamily A (ankyrin; TRPA), TRP cation channel polycystin subfamily (TRPP) and TRP cation channel mucolipin subfamily (TRPML)3.

The TRPML and TRPP subfamilies were named after the human diseases they are associated with (mucolipidosis and polycystic kidney disease, respectively). The founding member of the TRPM subfamily, TRPM1, was identified via comparative analysis of genes that distinguish benign nevi from malignant melanoma146. The TRPA subfamily has only one known member (TRPA1) and its name refers to the unusually high number of ankyrin repeats at the amino terminus of the channel protein (FIG. 1). Mammalian TRP channels that are most similar to the product of the Drosophila melanogaster Trp gene are referred to as TRPC proteins3. The TRPV subfamily was identified following expression cloning of TRPV1, which is the receptor for the prototypical irritant vanilloid, capsaicin9.

Overall, few generalizations can be made about TRP channels. Most members of the TRP channel superfamily share a low level of structural similarity (FIG. 1), but some channels — such as TRPC3 and TRPC7, as well as TRPV5 and TRPV6 — are highly homologous to each other3. Most of the channels are predicted to have six transmembrane domains and large intracellular amino and carboxyl termini (FIG. 1). Many TRP channels form functional channels as homotetramers, although heteromultimerization is not uncommon3. The latter phenomenon may have important implications in drug discovery as it is crucial for understanding the endogenous subunit composition of the channels so that TRP channels can be appropriately targeted with a pharmacological agent.

Consistent with their diverse structure, TRP channels also serve diverse functions. Although most members of the TRP channel superfamily are cation channels with limited selectivity for calcium, both calcium‑selective (such as TRPV5 and TRPV6) and sodium‑selective (such as TRPM4 and TRPM5) members of the TRP channel subfamilies exist3. In addition, some TRP channels transport non‑canonical cations such as iron (TRPML1) or magnesium (TRPV6). Temperature also exerts profound effects on several TRP channels. Although TRPV1 and TRPM8 have been clearly demonstrated to serve as sensors for changes in environmental temperature, many other TRP channels have temperature coefficients such that a change in temperature of 10°C has profound effects on channel activity3. These include TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5 and TRPA1.

Data from animal models and human genetic studies have shown that TRP channel dysfunction (which is known as TRP channelopathy) can cause various pathological conditions, including an inherited pain syndrome, multiple kidney diseases and skeletal disorders (reviewed in REF. 4).

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http://www.nature.com/nrd/journal/v10/n8/suppinfo/nrd3456.html

http://en.sanofi.com/research_innovation/rd_key_figures/rd_key_figures.asp

neurons have been ablated by high-dose capsaicin treatment, which was administered when the rats were neonates5. Therefore, the discovery that some TRPV1 antagonists cause hyperthermia in preclinical studies and humans was somewhat unexpected10.

Is the hyperthermic action of TRPV1 antagonists separable from their analgesic action? Several studies have noted that treatment with antagonists that block the three primary TRPV1 activators (that is, capsaicin, low pH and heat) in vitro results in transient hyper-thermia in experimental animal models (reviewed in REF. 14). The severity of this effect varies depending on the compound used but it is attenuated after several days of dosing15. In human volunteers, AMG517 (TABLE 1) caused a hyperthermic response that lasted for 1–4 days and raised body temperatures up to 40.2°C10. Other clin-ical studies have also shown that TRPV1 antagonists

(for example, ABT-102 (REF. 16) and AZD1386 (REF. 17);

TABLE 1) are associated with hyperthermia, although the effects of these antagonists were not as pronounced as the effects that were observed with AMG517.

In rats, it was possible to eliminate hyperthermia while preserving analgesic activity by differential block-ade of TRPV1 activation. Compounds (for example, AMG8562; see Supplementary information S2 (table)) that prevented the activation of rat TRPV1 by capsai-cin, but not by low pH or heat, had no effect on body temperatures in the rat models; however, these com-pounds still caused hyperthermia in dogs18. How well this translates into clinical studies remains to be seen. Notably, PHE377 (which is currently in Phase Ib trials) did not cause hyperthermia in rats or dogs, although it did inhibit all three major modalities of TRPV1 activa-tion (see the PharmEste website).

Figure 1 | Diversity in structure among TRP channel families. The six transient receptor potential (TRP) cation families contain very different motifs in their amino and carboxyl termini. The TRP cation channel subfamily V (TRPV), TRP cation channel subfamily A (TRPA) and TRP cation channel subfamily C (TRPC) families have amino terminal ankyrin repeat (AnkR) domains that are not present in other TRP channel subfamilies. The TRP box, which is found in the TRPV, TRP cation channel subfamily M (TRPM) and TRPC families, is thought to be involved in gating. TRP cation channel polycystin subfamily (TRPP) and TRP cation channel mucolipin subfamily (TRPML) proteins both have endoplasmic reticulum (ER) retention domains that may be due to their functional localization on intracellular organelles. aa, amino acids; CIRB, calmodulin/inositol-1,4,5-tris-phosphate (Ins(1,4,5)P

3) receptor binding domain; NUDIX, nucleoside diphosphate-linked moiety X; PDZ, acronym for

postsynaptic density protein 95 (PSD95), Drosophila disc large tumour suppressor (DLGA) and zonula occludens protein 1 (ZO1). Image is reproduced, with permission, from REF. 180 © (2003) Macmillan Publishers Ltd. All rights reserved.

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http://www.pharmeste.com/home.asp?op=interna&id=2&id_pag=10&tit=Pipeline

TRPV1 antagonists and noxious heat perception in humans. Clinical studies have confirmed the role of TRPV1 as a noxious heat sensor in humans (S. Eid, personal communication). Indeed, the threshold for detecting painful heat was considerably elevated in non-sensitized skin of healthy volunteers following oral administration of 400 mg of SB-705498 per day (Supplementary information S2 (table)), with sub-sequent studies reporting blunted heat perception in healthy human subjects, which was not desensitized after repeated dosing19. This effect could potentially cause scalding injuries during common activities such as taking a hot shower or consuming hot food or bever-ages. Indeed, some subjects taking MK-2295 perceived potentially harmful temperatures as innocuous (S. Eid, personal communication). In randomized clinical trials, similar findings were reported using ABT-102 (which was administered at a dose of up to 4 mg twice a day) and AZD1386 (which was administered at a single daily dose of 95 mg)16,17. Notably, there were no other relevant safety findings in these two trials and the investigators felt that AZD1386 may have clinical potential in relieving pain associated with gastroesophageal reflux disease17.

TRPV1 agonists (capsaicin and resiniferatoxin) in the clinic. Topical TRPV1 agonists (for example, capsaicin creams) have been used clinically for many years to alle-viate chronic painful conditions such as diabetic neurop-athy20. An occlusive high-concentration capsaicin patch

(Qutenza; NeurogesX) (TABLE 1) was recently approved for the treatment of various pain conditions21. Injections of resiniferatoxin, an ultrapotent capsaicin analogue5 (see Supplementary information S2 (table)), are being evaluated as a so-called ‘molecular scalpel’ to achieve long-term analgesia in patients with cancer who have chronic, intractable pain (ClinicalTrials.gov identifier: NCT00854659). A novel approach for minimizing the burning pain reaction at the application site, which is the main adverse effect of capsaicin administration, is the activity-dependent targeting of TRPV1 using perma-nently charged agonists that only permeate the core of the TRPV1 channel when it is open22. Such agonists are expected to target (and subsequently desensitize) hyper-active TRPV1 and spare normal nociception.

TRPA1. TRPA1 is a receptor for a range of environmental irritants and oxidants23–29, and it has an important role in many preclinical models of pain6. TRPA1 is directly acti-vated by structurally diverse chemicals. These include: cinnamaldehyde (which is present in cinnamon), allyl isothiocyanate (which is found in mustard oil), allicin (which is present in raw garlic), formalin (a chemical that is commonly used to induce experimental pain and is also a hazardous respiratory irritant) and icilin, which is a synthetic compound that produces a sensation of extreme cold (Supplementary information S2 (table); reviewed in REF. 6). When they are applied topically, many of these compounds cause pain in humans. Several

Table 1 | Therapeutic targeting of TRPV1

Compound Therapeutic indications Stage of development (status) Published data and press releases

Agonists*

ALGRX-4975 Analgesia after total knee replacement surgery and bunionectomy

Phase III trial (ongoing) See the Drugs.com website for further information on Anasevia’s Phase III trial results

WN-1001 Cluster headache, osteoarthritis Phase III trial (completed) ClinicalTrials.gov identifier: NCT00033839

NGX-4010 (Qutenza; Astellas Pharma/NeurogesX)

Postherpetic neuralgia post-hepatic

Phase III trial (ongoing) See Drugs.com website for further information on FDA approval of Qutenza

Antagonists‡

ABT-102 Pain associated with inflammation, tissue injury and ischaemia

Phase I trial ClinicalTrials.gov identifier: NCT00854659

AMG-517 Pain Phase Ib trial (terminated) REFS 11,21

AZD-1386 Chronic nociceptive pain and GERD Phase II trial (terminated) REF. 19

DWP-05195 Neuropathic pain Phase I trial (ongoing) Press release: 21 January 2009 (Daewoong Pharmaceutical website)

GRC-6211 Pain, migraine, urinary incontinence-associated pain and osteoarthritis

Phase II osteoarthritis trial (suspended)

Press release: 24 October 2008 (Glenmark Pharmaceuticals website)

JTS-653 Pain Phase II trial (ongoing) Clinical Development of Pharmaceuticals; 29 July 2010: Japan Tobacco website

MK-2295 Pain Phase II trial (completed) ClinicalTrials.gov identifier: NCT00387140

PHE377 Neuropathic pain Phase I trial (ongoing) See the PharmEste website for further information

SB-705498 Pain, migraine and rectal pain Phase II migraine and rectal pain trial (terminated) Phase II non-allergic intranasal rhinitis trial (ongoing)

ClinicalTrials.gov identifiers: NCT00269022; NCT00731250

GERD, gastroesophageal reflux disease; TRPV1, transient receptor potential cation channel subfamily V, member 1. *These agonists have been reviewed in REF. 20. ‡See Supplementary information S1 (box) for further information.

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http://www.clinicaltrials.gov/

http://www.drugs.com/clinical_trials/anesiva-phase-3-trial-adlea-meets-primary-endpoint-significantly-reduce-pain-after-total-knee-6536.html


http://www.drugs.com/newdrugs/neurogesx-receives-fda-approval-qutenza-capsaicin-8-patch-postherpetic-neuralgia-phn-1772.html

http://www.daewoong.co.kr/www_pharm/english_new/aboutus/whatsnews_view.asp?idx=60652

http://www.glenmarkpharma.com/GLN_NWS/pdf/GRC_6211.pdf

http://www.jt.com/investors/results/pharmaceuticals/pdf/P.L.20100729_E.pdf


Table 2 | Stage of development of drugs targeting TRP channels

Channel Findings from in vitro studies

Genetic deletion studies

Studies linking mutation to disease

Published pharmacological modulators

Efficacy with pharmacological agents in vivo

Published clinical trial data

TRPV1 Acts as a capsaicin receptor, and is a heat-activated non-selective cation channel2

Decreased sensitivity to noxious heat, and reduced thermal hyperalgesia in numerous pain models2

None reported Discussed in Table 1 Desensitization to agonists; pharmacological blockade by antagonists1,2,5

See Supplementary information S1 (box)

TRPV3 A heat-activated non-selective cation channel that is potentiated by repeated activation6,49

Effects on skin, heat sensation44,50

None reported Agonists: incensole acetate, 2-APB (nonspecific49) Antagonists: GRC 15300 (no structure available)

CCI, CFA (GRC 15300)49

GRC 15300 (details not reported)6

See the Sanofi website for further information

TRPV4 A non-selective cation channel. Acts as an osmosensor that is activated by metabolites of the arachidonic acid pathway. Involved in osteoclast differentiation and bone resorption114,115

Increased bone density114,115 altered urination

Skeletal dysplasias including SMDK, brachyolmia type 3, hereditary motor and sensory neuropathy type 2C, scapuloperoneal and congenital distal SMA116–120

Agonists: 4aPDD, GSK1016790A

(REF. 69) Antagonist: HC-067047 (REF. 67)

Agonists cause profound circulatory collapse110 and antagonists have efficacy in bladder cystitis (HC-067047; REF. 67)

None

TRPC3 A diacylglycerol-activated non-selective cation channel that is a downstream target of multiple G

q-coupled

GPCRs3

Ataxia and reduced mGluR signalling140

None reported Antagonist: ethyl-1-(4-2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate165

Reduced cardiac hypertrophy in mice

None

TRPC5 A non-selective cation channel that is potentiated by calcium and G

q-coupled

GPCR signalling, and forms heteromultimers with TRPC13

Reduced anxiety behaviours and reduced CCK4-invoked currents in hippocampal slices141

None reported None None None

TRPML1 An intracellular iron-permeable channel131

Deletion in Drosophila melanogaster results in neurodegeneration*166

Mucolipidosis type IV3,4

None None None

TRPM1 Cloned following a screen for genes that mark melanoma cells146; is a marker for melanoma progression146; small current, activated by steroids and blocked by zinc167

Mutation identified as the potential cause of heritable stationary night blindness in horses168

Associated with the complete form of human congenital stationary night blindness169–171

None None None

TRPM2 An oxidant sensor that is potentiated by increases in levels of intracellular calcium, cyclic ADP ribose, hydrogen peroxide and NADP172

Increased blood glucose levels in Trpm2–/– mice172

Downregulation or mutation may be associated with bipolar disorder or hereditary deafness144

Antagonist: N-(p-amylcinnamoyl)anthranilic acid173

None None

TRPM6 An outwardly rectifying channel, involved in magnesium transport4

Genetic deletion is lethal174; heterozygotes show mild hypomagnesaemia174

Inherited hypomagnesaemia4

None None None

TRPM7 An outwardly rectifying channel. siRNAs reduce cell death in hippocampal neurons owing to oxygen and glucose deprivation145

Knockout is lethal; targeted deletion in T cells disrupts thymopoiesis without affecting magnesium homeostasis175

Potential susceptibility locus for Guamanian amyotrophic lateral sclerosis and parkinsonism dementia176

Antagonists: NDGA, AA861 and MK886; Antagonists: nafamostat mesylate‡ activates TRPM7 under physiological conditions177

None None

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endogenous compounds (for example, 4-hydroxynonenal, A- and J-series prostaglandins and hydrogen peroxide) that are released following tissue damage and inflammation also directly activate TRPA1 via covalent binding and induce pain behaviours in mice25,30,31.

Key pronociceptive signalling pathways also indi-rectly potentiate TRPA1 activity by increasing intracel-lular calcium concentration. For example, bradykinin, proteinase-activated receptor 2 and nerve growth factor potentiate TRPA1 currents via their respective receptors (reviewed in REFS 1,6). Furthermore, mice with disrupted TRPA1 function fail to develop pain behaviour and ther-mal and mechanical hypersensitivity after intraplantar injection of bradykinin24,32, which provides evidence that this phenomenon is relevant in vivo.

Collectively, the above results led to efforts that aimed to identify TRPA1 antagonists as therapies for treating pain6. Two moderately potent but highly selective TRPA1 antagonists, HC-030031 (REF. 33) and AP-18 (REF. 34), have subsequently been discovered (see Supplementary information S2 (table)).

In rodents, HC-030031 reduces acute and chronic inflammatory pain, and reduces neuropathic pain with-out having any apparent effect on motor coordination or noxious cold detection33,35. A structurally related compound, Chembridge-5861528 (Supplementary

information S2 (table)), prevents the development of mechanical hyperalgesia in animal models of diabetes-induced pain, which suggests that endogenously pro-duced oxidant and inflammatory mediators may act via TRPA1 to contribute to diabetes-induced pain36. In addi-tion, animals that were treated with Chembridge-5861528 had reduced nerve damage in response to streptozotocin, which indicates that TRPA1 antagonists could be used for disease modification in painful diabetic neuropathy36. In rats, intrathecal TRPA1-targeted antisense oligonucleo-tides37 or TRPA1 antagonists can also dramatically reduce cold hypersensitivity after nerve injury or inflammation35.

Cold temperatures are a considerably weaker activa-tor of the TRPA1 channel38. However, cold temperatures dramatically potentiate TRPA1 activity in the presence of other agonists38. Furthermore, the selective TRPA1 antag-onist HC-030031 reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain without altering normal cold sensation in naive animals33,38,39. Similarly, A-967079 — a compound that is structurally similar to AP-18 (Supplementary information S2 (table)) — reduces cold hypersensitivity after nerve injury without affecting acute responses to environmental cold40. Thus, TRPA1 appears to selectively mediate cold hypersensitiv-ity in pathological conditions in which other activators of the channel are also present.

Channel Findings from in vitro studies

Genetic deletion studies

Studies linking mutation to disease

Published pharmacological modulators

Efficacy with pharmacological agents in vivo

Published clinical trial data

TRPM8 A cold- and menthol-activated non-selective cation channel (reviewed in REFS 1,2)

Affects the sensation of environmental cold; mild pain phenotype52–54

None reported Agonists: menthol, icilin, WS-12 and WS-5 (REF. 1) Antagonists: benzimidazoles, AMTB (REF. 70)

Agonists: wet dog shakes; analgesia in CCI model of chronic pain55 Antagonists: AMTB decreased volume-induced voiding events70

Agonists: cutaneous menthol (migraine); menthol patch (mild to moderate muscle strain)78 Antagonists: D-3263

(ClinicalTrials.gov identifier: NCT00839631)

TRPP2 A non-selective cation channel that forms heteromultimers with several other TRP channels. Involved in ciliary movement121,123

Pkd2–/– mice usually die mid-gestation as a result of kidney cysts, oedema and placental abnormalities179

Mutation in gene encoding polycystic kidney disease 2 protein122

None None None

TRPA1 Channel activated by reactive chemicals, potentiated by cold

Reduced sensitivity to environmental irritants; reduced pain behaviours in numerous models; reduced responses in an allergic asthma model

Mutations cause familial episodic pain syndrome43

Agonists: multiple reactive chemicals (endogenous and exogenous) that lead to sulfhydryl modification of cysteine reidues Antagonists: HC-030031 (REF. 33), AP-18 (REF. 34), A-967079 (REFS 40,41)

Formalin, AITC, cinnamaldehyde-flinch33–35; CFA-cold38, mechanical35; SNL-mechanical; SNI-cold; MIA-induced OA40

None reported

2-APB, 2-aminoethoxydiphenyl borate; 4aPDD, a-phorbol 12,13-didecanoate; AITC, allyl isothiocyanate; AMTB, N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride; CCI, chronic constriction injury; CCK4, cholecystokinin 4; CFA, complete Freund’s adjuvant; DAG, diacylglycerol; GPCR, G protein-coupled receptor; IC

50, half-maximal inhibitory concentration; mGluR, metabotropic glutamate receptor; MIA, monoiodoacetate; OA,

osteoarthritis; SMA, spinal muscular atrophy; SMDK, spondylometaphyseal dysplasia, Koslowski type; SNI, spared nerve injury; SNL, spinal nerve ligation; TRPA1, transient receptor potential cation channel subfamily A, member 1; TRPC1, TRP cation channel subfamily C, member 1; TRPM1, TRP cation channel subfamily M, member 1; TRPML1, TRP cation channel mucolipin subfamily 1; TRPP2, TRP cation channel polycystin subfamily 2 (also known as PKD2); TRPV1, TRP cation channel subfamily V, member 1. *This is possibly owing to reduced clearance of macromolecules and apoptotic cells. ‡IC

50 varies with concentration of divalent cations.

Table 2 cont. | Stage of development of drugs targeting TRP channels

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Although TRPA1 may have an analogous role in nociceptor mechanotransduction, whether TRPA1 is mechanically activated per se remains to be determined. In rats, A-967079 reduced the frequency of firing of wide-range dynamic neurons in lamina V of the spinal cord in response to noxious mechanical stimuli41. These data are consistent with data taken from an isolated rat skin-nerve preparation using HC-030031 and from mice lacking TRPA1, which suggests that TRPA1 is required for pro-longed neuronal firing in response to certain high-inten-sity mechanical stimuli42.

In conclusion, preclinical data (and data from a recent human genetic study43) highlight TRPA1 antago-nists as a promising new approach for the treatment of acute and chronic pain. The current status of several drug development programmes is presented in TABLE 2.

TRPV3. Changes in the levels of expression of TRPV3, which is usually highly expressed in the skin44,45, can occur in human disease states. For example, TRPV3 expres-sion is increased in painful breast tissue46 and decreased in basal keratinocytes that are recovered from patients

with diabetic neuropathy47. TRPV3 appears to be unique among TRP channels in that repeated stimulation of the channel leads to sensitization that depends on the pres-ence of calcium48. Activation of Gq-coupled GPCRs — including the histamine and bradykinin receptors — also potentiates TRPV3 activity45, thus allowing TRPV3 to serve as a convergence point for multiple pain pathways (reviewed in REF. 49). TRPV3 is activated in response to temperatures in the range of 31–39 °C and to the chemicals camphor and 2-aminoethoxydiphenyl borate (reviewed in REFS 1,49). Trpv3-null mice had defects in thermal selec-tion behaviour in response to innocuous heat, and defects in withdrawal behaviour in response to noxious heat50. Taken together, these findings suggest an important role for TRPV3 in pain transduction. TRPV3 antagonists have shown efficacy in preclinical neuropathic and inflamma-tory pain models (TABLE 2), and one molecule has entered Phase I clinical trials (see the Sanofi website).

TRPM8. In sensory ganglia, TRPM8 expression dis-tinguishes a specific subpopulation of primary sensory neurons that are cold-sensitive (reviewed in REFS 1,2).

Figure 2 | TRP channels as nociceptors. Sensory neurons express multiple transient receptor potential (TRP) channels. TRP cation channel subfamily V, member 1 (TRPV1), TRPV3 and TRPV4 all respond to warming temperatures. Noxious heat activates TRPV2, but the physiological relevance of this is unclear. Acids are robust activators of TRPV1 and bases have emerged as activators of TRP cation channel subfamily A, member 1 (TRPA1). TRPA1 is a key chemoreceptor that responds to scores of reactive chemicals. At higher concentrations, some of these chemicals also activate TRPV1. TRP cation channel subfamily M, member 8 (TRPM8) serves as the key receptor for environmental cold, although TRPA1 also has a role in cold hyperalgesia. Activation of any of these TRP cation channels can trigger action potentials in the sensory neuron. Some of these channels, such as TRPV1, are also expressed in the spinal cord, where they seem to have an important role in the central nervous system as well. DRG, dorsal root ganglion. Image is reproduced, with permission, from REF. 181 © (2002) Macmillan Publishers Ltd. All rights reserved.

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The finding that the heat-sensitive TRPV1 and the cold-sensitive TRPM8 are co-expressed in some neurons51 underlines the hypothesis that individual neurons may be able to sense ranges of both hot and cold temperatures.

In vitro, TRPM8 is activated by cool temperatures in the range of 10–23 °C, as well as the cooling agents men-thol (Supplementary information S2 (table)) and icilin (reviewed in REFS 1,2). Furthermore, TRPM8-deficient mice showed no preference for warm temperatures over cool temperatures, and they exhibited impaired cold avoidance behaviour, which highlights the essential role of TRPM8 in the sensation of environmental cold52–54.

Controversy surrounds the utility of TRPM8 as an analgesic target. The TRPM8 agonist menthol decreases nociceptive responses in animal models of inflammatory and neuropathic pain55. However, TRPM8 antagonism

may also provide relief from some forms of pain. For example, Trpm8–/– mice failed to develop cold allodynia after chronic constriction injury or injection of complete Freund’s adjuvant (CFA)54. Importantly, a TRPM8 antag-onist reduced cold allodynia in a chronic constriction injury model of chronic neuropathic pain, thus recapitu-lating the phenotype of genetic deletion56.

The observation that both TRPM8 agonists and TRPM8 antagonists may be useful for the treatment of pain highlights the importance of selecting appropriate models to examine TRP channel modulators.

TRP channels in bladder disordersSeveral TRP channels are expressed in the bladder — in the urothelium, nerve endings and detrusor muscle (FIG. 3) — where they are thought to function as sensors of

Figure 3 | Roles of TRP channels in bladder functions. The micturition reflex is mediated by transient receptor potential cation channel subfamily V, member 1 (TRPV1)-positive nerves and probably — albeit to a lesser degree — also by TRP cation channel subfamily M, member 8 (TRPM8)-positive nerves. These same neurons convey nociceptive information (for example, bladder pain that is secondary to cystitis) to the central nervous system. TRPV4 is a key player in bladder function because it is present in both the urothelium and the detrusor muscle, and it is activated by stretch (bladder distension) and hypo-osmolar urine. The activator of TRPM8 in the bladder remains to be determined. The micturition reflex is under the control of a descending central nervous system pathway; when this pathway is disrupted (for example, as a result of spinal cord injury or multiple sclerosis), it becomes autonomous and partly driven by TRPV1. The existence of functional TRPV1 in urothelial cells remains controversial, as evidence for (REFS 64, 66) and against (REF. 65) the presence of a funtional channel has been presented. Collectively, these findings imply a therapeutic potential of TRPV1 antagonists (or TRPV1 desensitization) and/or TRPM8 agonists as therapies in painful bladder disorders (and in pain induced by benign prostatic hyperplasia) and in an overactive bladder. These findings also suggest that TRPV4 blockers could be useful in the management of an overactive bladder. DRG, dorsal root ganglion.

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Prurigo nodularisA skin condition that is characterized by itchy nodules (circumscribed, solid elevations on the skin), which usually appear on the arms or legs.

PruritogensAgents that induce itch by stimulating pruritoceptive sensory afferent neurons. In the skin, they are synthesized by and released from multiple non-neuronal cell types and include histamine, acids, ATP, prostaglandins and pro-inflammatory interleukins.

stretch and chemical irritation (reviewed in REFS 57,58). Intravesical administration of TRPV1 agonists has been used in the management of the overactive bladder for many years, largely on an empirical basis (reviewed in REF. 58). The recent recognition of disease state-related changes in the expression of TRP channels has provided a new impetus to investigate the roles of these channels in normal bladder function and dysfunction.

TRPV1. There is a dense network of nerve fibres that express TRPV1 in the suburothelium and muscular layer of the human renal pelvis, ureter, bladder and urethra57,58, but the existence of functional TRPV1 in the urothelium and detrusor smooth muscle remains controversial59. The involvement of neuronal TRPV1 in the micturition reflex is well established57,58. Indeed, rats in which TRPV1-expressing nerves have been ablated by neonatal capsaicin treatment develop a dis-tended bladder5. Trpv1–/– mice, however, show only spotty incontinence60. It is possible that another protein compensates for the lack of TRPV1 or that TRPV1-containing, neuron-specific gene products other than TRPV1 contribute to the phenotype that is observed in these animals.

In humans, desensitization therapy using intravesical TRPV1 agonists (capsaicin or resiniferatoxin) is based on the concept that the C fibre-driven micturition reflex — which is inactive in adult life — reassumes control of micturition in the overactive bladder, in both neu-rogenic and non-neurogenic cases57,58. In patients with neurogenic detrusor overactivity disorders, intravesical administration of resiniferatoxin provides symptomatic relief by increasing bladder capacity and decreasing the frequency of daily episodes of incontinence61. Moreover, intrathecal administration of resiniferatoxin blocks detrusor overactivity in rats that have complete spinal cord transection62, and intravesical administration of resiniferatoxin reduces the frequency of incontinence episodes in patients with spinal cord injury (reviewed in REFS 61,63).

The therapeutic value of TRPV1 antagonists in manag-ing detrusor muscle overactivity is unclear, as no endoge-nous agonist has been identified in the bladder of patients who suffer from incontinence. By contrast, the pain and bladder hyperactivity that accompany interstitial cystitis are thought to be amenable to therapy with TRPV1 antag-onists63. In a feline model of interstitial cystitis, abnor-mally enhanced responses to capsaicin were detected after TRPV1 phosphorylation by protein kinase C64. In mice, genetic manipulation of the Trpv1 gene prevents bladder reflex hyperactivity and spinal FOS overexpres-sion in experimental models of cystitis60. The TRPV1 antagonist GRC-6211 (Supplementary information S2 (table)) ameliorates micturition reflex activity in the chronically inflamed bladder65. Collectively, these find-ings imply a therapeutic value for TRPV1 antagonists in the symptomatic treatment of interstitial cystitis.

TRPV4. In the bladder, TRPV4 is predominantly expressed in the urothelium, but it is also present in the detrusor muscle57,59. Trpv4–/– mice show an altered

micturition pattern that is characterized by increased intermicturation intervals and spotting behaviour due to spontaneous (that is, not reflex-driven) contractions of the detrusor muscle66. In addition, Trpv4–/– mice show reduced urinary frequency and increased void volume after bladder damage caused by intravesical administra-tion of cyclophosphamide67. Based on these findings, it has been postulated that TRPV4 plays a crucial part in the mechanosensory pathway in the bladder by detect-ing changes in intravesical pressure. Indeed, mechani-cal stretch has been shown to activate urothelial TRPV4 in vitro68.

Activation of TRPV4 that is located in the detrusor muscle can lead to muscle contractions. Accordingly, the TRPV4 agonist GSK1016790A (Supplementary information S2 (table)) causes direct contraction of the bladder, even in the absence of the urothelium69. As predicted by data generated from Trpv4–/– mice, the TRPV4 antagonist HC-067047 (Supplementary infor-mation S2 (table)) improved bladder function in mice with cyclophosphamide-induced cystitis67. Acute dosing of HC-067047 did not alter water intake, core body tem-perature, thermal selection behaviour, heart rate, loco-motion or motor coordination in vivo. Taken together, these observations imply that TRPV4 antagonists could be valuable in the management of an overactive bladder.

TRPM8. In the human bladder, TRPM8 is present in both urothelial and suburothelial myelinated nerve fibres, with increased expression in patients who have bladder pain or idiopathic detrusor overactivity70. The TRPM8 agonist menthol evokes the micturition reflex in humans70. Conversely, the TRPM8 antagonist AMTB (Supplementary information S2 (table)) decreases the frequency of volume-induced bladder contractions in a rat model of painful bladder syndrome71. These findings imply that TRPM8 antagonists have therapeutic potential in the management of bladder disorders that are charac-terized by pain and/or overactivity of the detrusor muscle.

TRP channels in the skinPopulations of non-neuronal cells within the skin express many different types of TRP channels (FIG. 4), which are thought to be involved in various key cutaneous func-tions including skin-derived pruritus, proliferation, dif-ferentiation, cancer and inflammatory processes (BOX 2).

TRPV1 as a key molecule in itch. TRPV1 is involved in the development of skin-derived pruritus, which is thought to occur through itch-specific subpopulations of TRPV1-expressing sensory afferent neurons (also known as pruritoceptive neurons; reviewed in REFS 72,73). TRPV1 is also expressed in non-neuronal cell types of human skin74, and its expression is elevated in epidermal keratinocytes of patients with prurigo nodularis75.

Certain endogenous signalling molecules that poten-tiate TRPV1 activity (including acids, ATP, lipoxyge-nase products, prostaglandins and histamine) are also potent pruritogens (reviewed in REFS 72,73). It is probable that on sensory neurons, histamine indirectly activates TRPV1 through histamine H1 receptor-dependent

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AlopeciaA type of pathological hair loss that mostly affects the scalp. The most common forms of alopecia are alopecia universalis, alopecia areata and alopecia androgenetica. Telogen Effluvium, which is characterized by diffuse hair shedding, is a form of alopecia.

HirsutismExcessive and increased hair growth (especially in women) on regions of the body where the occurrence of hair normally is minimal or absent.

DermatitisA universal term describing inflammation of the skin. It can be induced by various factors such as allergens (allergic dermatitis), infections, eczema (atopic dermatitis) or external compounds (contact dermatitis).

ApnoeaProlonged periods of time without respiratory flow. Although humans can perform this manoeuvre voluntarily (by holding their breath), reflex apnoeas can be induced in human volunteers and animals by irritant stimulation of the respiratory tract.

synthesis of 12-hydroperoxyeicosatetraenoic acid, which is an endogenous activator of TRPV1 (reviewed in REF. 2). Consistent with this finding, genetic deletion of Trpv1 in mice substantially suppressed histamine-induced scratching behaviour76. In humans, TRPV1 mediates histamine-induced experimental itch77, as well as pruritus in patients with seasonal allergic rhinitis78. By contrast, histamine-independent itch — caused by chlo-roquine or the endogenous pruritogen peptide BAM8-22 (originally isolated from bovine adrenal medulla) — is predominantly mediated by TRPA1 (REF. 79).

Paradoxically, the loss of TRPV1-expressing neu-rons is also associated with excessive itch. Rats that have had their capsaicin-sensitive neurons chemically ablated may develop skin ulcers as a result of extensive scratching80. Mice lacking the vesicular glutamate trans-porter 2 — thereby ostensibly disabling glutamatergic neurotransmission in the spinal dorsal horn, which is the termination site of TRPV1-positive primary afferent neurons — demonstrated enhanced scratching behav-iour and reduced sensitivity to noxious heat81,82. Clearly, further neurophysiological studies in humans are needed to dissect the multifaceted relationship between TRPV1-positive primary afferent neurons and pruritus.

Role of TRPV1 in the control of skin growth, skin cell survival and cutaneous inflammation. It has been sug-gested that TRPV1 participates in the regulation of cutaneous growth and differentiation. TRPV1-mediated calcium influx in cultured human keratinocytes sup-presses proliferation and promotes apoptosis83,84. In addition, activation of TRPV1 by either capsaicin or heat alters the formation of the epidermal permeability barrier in human skin in vivo85.

TRPV1 has also been suggested to regulate cutaneous inflammation. Capsaicin-induced activation of TRPV1 on human epidermal and hair follicle-derived keratino-cytes in vitro results in the release of several pro-inflam-matory cytokines83. In addition, as ultraviolet irradiation upregulates TRPV1 expression in human skin86, TRPV1 that is expressed on keratinocytes is a specific media-tor of heat shock-induced and ultraviolet irradiation-induced expression of matrix metalloproteinase 1 (REF. 87), an enzyme that is implicated in skin inflam-mation and remodelling. Taken together, these findings imply that topical TRPV1 modulators may be used in the treatment of sunburn, acne vulgaris and alopecia or hirsutism.

TRPV3. TRPV3, which is expressed at high levels by keratinocytes88, is crucial in promoting epidermal bar-rier formation and hair morphogenesis in mouse skin. This probably occurs through the formation of a signal-ling complex between TRPV3 and the epidermal growth factor receptor44. Consistent with this concept, genetic deletion of Trpv3 in mice caused hair abnormalities (that is, wavy hair coat and curly whiskers)44,50, whereas the constitutively active gain-of-function mutation Trpv3Gly573Ser resulted in a spontaneous hairless pheno-type in DS-Nh mice (a mouse model of dermatitis)89,90. As the activation of TRPV3 in organ cultures of human

hair follicles also inhibited hair growth91, these findings imply a therapeutic potential for topical TRPV3 agonists and antagonists in the treatment of hirsutism and alo-pecia, respectively.

TRPV3 is also involved in the development of skin inflammation. Stimulation of TRPV3 in cultured keratinocytes induced the release of pro-inflammatory mediators45. Importantly, DS-Nh mice develop skin alterations that are similar to those seen in human atopic dermatitis89. Moreover, Trpv3Gly573Ser transgenic mice that express high levels of the mutated, constitutively active TRPV3 protein in epidermal keratinocytes also sponta-neously develop an inflammatory condition that is simi-lar to human atopic dermatitis90. As the skin-targeted Trpv3Gly573Ser transgenic mice also exhibit scratching behaviour, these data suggest that TRPV3 channels may function as important transducers of pro-inflammatory signals in the pathogenesis of various forms of dermatitis.

TRP channels in the pulmonary systemThe mammalian respiratory tract is lined with a dense plexus of sensory fibres, including those that express TRPA1 and TRPV1 (FIG. 5). Activation of this subset of nerve fibres by irritant and/or inflammatory stimuli triggers multiple reflexes — such as sneezing, cough-ing, mucus secretion, bronchospasms and apnoea — that limit ventilation and dilute and/or expel foreign materials. Analogous to pain, in which inflammatory mediators produce a hypersensitive response to vari-ous stimuli, reflexes such as coughs are proposed to be sensitized by mediators of inflammation and oxidant stress, such that they become triggered by innocuous stimuli. Thus, although a distinct and unusual subpopu-lation of afferent fibres may be responsible for coughing reflexes92, airway nociceptors appear to exert a consider-able amount of control over the sensitivity of this reflex.

TRPV1. Capsaicin is a prototypical respiratory irritant that causes noxious sensations as well as reflexes such as coughing, sneezing and fluid secretion when it is applied to the human respiratory mucosa (reviewed in REF. 5). Increased sensitivity to capsaicin aerosols occurs in several respiratory disorders of varying severity and aetiology (reviewed in REF. 93). Alterations in capsaicin-induced coughing may be due to enhanced TRPV1 expression or activity in airway sensory neurons, altera-tions in the permeability of the epithelial barrier that allows capsaicin to more readily access airway nocicep-tor terminals and/or heightened responsiveness of the central nervous system to afferent inputs. Intriguingly, treatment with the non-selective cyclooxygenase inhibi-tor indomethacin increases capsaicin-induced cough thresholds in patients with asthma or chronic bronchitis but not in healthy subjects94. These results offer com-pelling evidence that inflammatory mediators that are produced in diseased airways enhance the sensitivity of airway reflexes in a manner that can be at least partially reversed by pharmacological intervention.

Therapy using intranasal capsaicin to desensitize TRPV1-containing sensory neurons provides symp-tomatic relief in patients with rhinitis95; however, this

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Hair cycleA life-long regeneration programme of the hair follicles that can be divided into three phases: anagen (growth), catagen (apoptosis-driven regression or involution) and telogen (resting or quiescence, preparation for the next anagen phase). This cycle is controlled by promoters (for example, insulin-like growth factor 1 and hepatocyte growth factor) and inhibitors (for example, interleukin-1β, and transforming growth factor-β2)

Figure 4 | TRP channels in human skin. The role of transient receptor potential (TRP) channels in the skin is supported by strong evidence (shown using continuous arrows) indicating that TRP cation channel subfamily V, member 1 (TRPV1) is expressed by various cell types (sebocytes, keratinocytes, sensory neurons and cells of the hair follicles). TRPV1 activation is shown to induce heat sensation and the development of skin-derived pruritus, and suppress sebaceous lipid synthesis. Activation of TRPV1 and TRPV3 shifts the proliferation–differentiation balance of epidermal keratinocytes towards differentiation and, along with TRPV4 and TRPV6, TRPV1 and TRPV3 are involved in the regulation of epidermal barrier formation. Moreover, activation of TRPV1 and TRPV3 either on epidermal or hair follicle-derived keratinocytes results in increased pro-inflammatory cytokine release, which suggests that these channels are key players in the in situ immunoregulation of human skin. In addition, the hair cycle is regulated both directly (via stimulation of TRPV1 and TRPV3) and indirectly (via TRPV1 activation that results in follicular growth factor production) by TRP channels. Preliminary findings (indicated using dashed arrows) suggest that TRP cation channel subfamily C, member 1 (TRPC1) and TRPC4 are likely to have antitumour effects, whereas TRP cation channel subfamily M, member 7 (TRPM7) regulates melanogenesis of melanocytes. In addition, TRP cation channel subfamily A, member 1 (TRPA1) and TRPM8 may have a synergistic effect with other TRP channels in the regulation of epidermal barrier formation and maturation (that is, differentiation) of keratinocytes.

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Respiratory irritationA stereotypical reflex reduction in respiratory rate exhibited by small laboratory animals following irritant aerosol provocation. This behaviour generally predicts the irritation threshold for molecules in humans.

Pilosebaceous unitConsists of the hair shaft, the hair follicle, the sebaceous gland and the erector pili muscle; causes the hair to stand up when it contracts.

SebumA lipid-enriched, oily exocrine product of the sebaceous glands that has various functions including waterproof barrier formation, antimicrobial activity, transport and thermoregulation.

Basal cell carcinomaThe most common type of malignant skin tumour, which develops from the basal cell layer of the epidermis. It rarely metastasizes, but without treatment it may cause substantial destruction by invading the deeper skin tissues.

Darier’s diseaseA congenital skin condition that is characterized by dyskeratosis (abnormal keratinization of the epidermis) and the appearance of pruritic, greasy and scaly skin papules (circumscribed, solid elevations on the skin) and plaques (confluences of papules).

VitiligoA skin disorder that is characterized by depigmentation of patches of skin. It develops as a result of impaired functions or death of skin melanocytes, which can be induced by various factors, such as autoimmune conditions, genetic factors, oxidative stress and infections.

Penh (enhanced pause)A derived value that is supposed to characterize the ventilatory activity of freely moving rodents in plethysmography chambers where airflow is measured. Interpretation of this measurement is debated within the respiratory field.

procedure is poorly tolerated and has therefore not been widely adopted. The explanation for the efficacy of capsaicin desensitization therapy remains elusive. The beneficial effects of capsaicin may paradoxically extend beyond its actions on TRPV1, in that it may functionally inactivate the entire TRPV1-expressing neuron5. Such neurons express many other gene products in addition to TRPV1, including the related polymodal nocisensor TRPA1 (see below).

TRPA1. Many of the irritants that activate TRPA1 are air pollutants that are produced by the combustion of materials (including tobacco products) that cause pro-nounced cutaneous, ocular and respiratory irritation in humans. Several classes of anaesthetic molecules — including lidocaine, propofol, etomidate and volatile gaseous anaesthetics — also act as TRPA1 agonists96. Although these data raise the possibility that anaesthesia may paradoxically increase postoperative pain, the more immediate impact of these data is the identification of TRPA1 as a possible mediator of the respiratory com-plications of gaseous anaesthetics, which can include coughing and laryngospasms. In support of this hypoth-esis, the TRPA1 blocker HC-030031 has been shown to prevent desflurane-induced increases in airway resist-ance in guinea pigs26.

Additional hazardous irritants — which include iso-cyanates, ozone, chlorine and cigarette smoke extracts — activate overexpressed TRPA1 and cause pulmo-nary nociceptor activation, respiratory irritation and/or neurogenic inflammation in a TRPA1-dependent manner25,27–29,97. These molecules are broadly toxic, and exposure to them causes marked symptoms and injury in human airways. Perhaps surprisingly, interruption of TRPA1 function in rodents — via gene disruption or pharmacological blockade — nearly abolishes the activation of sensory neurons and/or respiratory reflexes, including coughs produced by acute exposure

to these irritants27–29,98. Thus, emerging data from animal models largely indicate that TRPA1 is the sole effector of sensory neuron activation and respiratory irritation reflexes that are triggered by acute exposure to a range of respiratory irritants. One noteworthy exception is nicotine, which activates sensory neurons by acting at both TRPA1 and nicotinic acetylcholine receptors99. Further clarification is required regarding the com-plex interplay between these two mechanisms in vivo. TRPA1 is necessary for increases in the Penh (enhanced pause) respiratory measurement caused by intrana-sal nicotine99; however, similar provocations pro-duce robust respiratory irritation in TRPA1-deficient mice28.

Although further studies will reveal the contribu-tion of TRPA1 to the overall respiratory pathology that is caused by chronic exposure to hazardous irritants in humans, existing data from mice already demonstrate the role of TRPA1 in allergic airway inflammation. Mice that either lacked TRPA1 or were pretreated with the TRPA1 blocker HC-030031 were protected from air-way inflammation (both in bronchoalveolar lavage and in lung tissue) and bronchial hyperreactivity in response to acetylcholine. Transcription of the gene product for the gel-forming mucin, MUC5AC, was also reduced100. This suggests that HC-030031 prevented gene transcrip-tion, although no measurement of the protein (that is, the gene product) was performed. Importantly, knock-ing out TRPV1 did not alter any of these parameters100, thus providing evidence that TRPA1 has a distinct role in this model of allergic disease. In addition, TRPA1 has been implicated in neurogenic inflammation of the airway caused by the acetaminophen (paracetamol) metabolite N-acetyl-p-benzoquinone imine in sev-eral species of small rodents101. These compelling data demonstrate that TRPA1 can contribute to both res-piratory reflexes and inflammation in laboratory-based animal models.

Box 2 | Emerging functions of TRP channels in cutaneous biology

In the human pilosebaceous unit, capsaicin‑induced activation of transient receptor potential cation channel subfamily V, member 1 (TRPV1) suppresses hair shaft elongation and induces apoptosis‑driven catagen regression84. In this study, TRPV1 activation was accompanied by a considerable alteration in the gene‑expression profiles of the cells and modulation of intrafollicular production of cytokines and growth factors that control human hair growth in vivo84. Interestingly, compared to wild‑type mice Trpv1 gene‑deficient mice exhibited a significant delay in hair follicle cycling — that is, a delay in the onset of the catagen phase158. This supports the concept that TRPV1 may exert functions that are primarily growth‑inhibitory in mammalian skin epithelium. Moreover, stimulation of TRPV1 expressed in human sebaceous gland‑derived SZ95 sebocytes selectively inhibited lipid synthesis (sebum production) and altered the expression profiles of multiple genes that are involved in cellular lipid homeostasis159.

In addition to TRPV1 and TRPV3, other TRP channels (for example, TRP cation channel subfamily C, member 1 (TRPC1), TRPC4, TRPV4, TRPV6, TRP cation channel subfamily A, member 1 (TRPA1) and TRP cation channel subfamily M, member 8 (TRPM8)) have been identified in skin keratinocytes, where they are probably involved in the differentiation — and malignant transformation — of keratinocytes and barrier formation. Intriguingly, several skin pathologies have altered TRP channel expression. For example, basal cell carcinoma cells lack both TRPC1 and TRPC4 expression160. Conversely, TRPC1 is overexpressed in the skin of patients with Darier’s disease161. TRPV4 seems to promote the development and maturation of intercellular junctions in the epidermis85, thus topical TRPV4 agonist preparations may represent a novel approach in the treatment of acne vulgaris. Stimulation of TRPA1 affects the expression profile of genes that are involved in the control of keratinocyte proliferation and differentiation162. TRPV6 participates in the differ‑entiation‑stimulatory effects of 1,25‑dihydroxyvitamin D3 by increasing Ca2+ entry163. At present, it is unclear how specific these effects are, as dramatic increases in calcium concentration are likely to affect the proliferation and differentiation of skin cells. Last, decreased and/or faulty TRPM7 production leads to impaired melanocytic differentiation164, which can result in vitiligo.

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TRPC and TRPV channels in airway structural and inflammatory cells. Several TRP channels are expressed in bronchial and/or vascular smooth muscle. TRP cat-ion channel subfamily C (TRPC) has functional roles in the pulmonary vasculature, particularly with regard to responses to hypoxia. Regional alveolar hypoxia redirects blood flow to well-oxygenated areas. This reflex mecha-nism is impaired in TRPC6-knockout mice, as ex vivo

lungs from these animals have marked deficits in arterial oxygen saturation following airway instillation of saline to produce regional ventillatory failure102. Moreover, a SNP (–254C→–254G) in the promoter region of the gene encoding TRPC6 has been linked to idiopathic pulmonary arterial hypertension103. Consistent with this finding, pulmonary arterial smooth muscle cells from patients with idiopathic pulmonary arterial hyperten-sion had considerably higher levels of the TRPC6 pro-tein, higher resting levels of cytosolic Ca2+ and larger 1-oleyl-2-acetyl-sn-glycerol (OAG)-dependent cationic currents than samples from control subjects103.

Smooth muscle cells in diseased airways typically dis-play abnormalities in contraction and/or proliferation. Although bronchodilators are efficacious therapies that are capable of reversing airflow obstruction, treatments that reduce contractility and pathological remodelling of airway smooth muscle remain targets for active investi-gation. Following incubation with the inflammatory cytokine tumour necrosis factor, human airway smooth muscle cells had marked changes in Ca2+ homeostasis that are accompanied by increased expression of the TRPC3 protein104. Moreover, enhanced acetylcholine-induced elevations in cytosolic Ca2+ in human airway smooth muscle cells are blocked by RNA silencing of TRPC3. These data are consistent with studies in mice with allergen-induced airway inflammation; in these studies TRPC3-specific antibodies inhibited non-selec-tive cation conductances and restored resting membrane potentials of airway smooth muscle cells to values that were more hyperpolarized and consistent with con-trols105. Compensatory elevation of TRPC3 expression occurs in Trpc6–/– mice, which may explain why these mice have enhanced airway reactivity in response to a muscarinic agonist, despite displaying reduced inflam-matory parameters in bronchoalveolar lavage fluid106.

TRPV4 has also been proposed to contribute to Ca2+ mobilization in airway smooth muscle107. This function of TRPV4 is one possible mechanism that may explain the genetic association between multiple SNPs in the gene encoding TRPV4 and chronic obstructive pulmo-nary disease108, although the function of TRPV4 in the respiratory tract extends beyond airway smooth muscle responsiveness. Indeed, the TRPV4 gene containing the chronic obstructive pulmonary disease-associated P19S SNP displays gain-of-function characteristics in human airway epithelial cells, where the disease-associated SNP has been shown to increase Ca2+ influx and secretion of matrix metalloproteinase 1 in response to diesel exhaust fumes109.

The most striking manifestation of TRPV4 biology in the lung occurs in the alveolar septae. In isolated, per-fused lungs in mice, activation of TRPV4 by elevated vascular pressure110 or injurious high-pressure mechani-cal ventilation111 caused extravascular leakage of fluid, as reflected by increases in the filtration coefficient (Kf) of lung fluid via extravascular leakage. Consistent with these findings, intravenous administration of the TRPV4 agonist GSK1016790A causes circulatory collapse that is characterized by the failure of the alveolar septal bar-rier112. Although the same study also demonstrated that

Figure 5 | Diverse roles of TRP channels in the pathophysiology of the mammalian respiratory tract. Although transient receptor potential (TRP) channels generally increase intracellular Ca2+ concentrations and/or depolarize membrane potentials, their varied expression patterns and sensitivity to agonists result in considerable functional diversity. For instance, in vagal sensory nerve terminals, noxious chemical and physical stimuli activate TRP cation channel subfamily A, member 1 (TRPA1) and TRP cation channel subfamily V, member 1 (TRPV1) to produce nerve activation, which initiates reflexes and critically regulates sensations, as shown in the figure. In airway smooth muscle cells, functional TRP cation channel subfamily C, member 3 (TRPC3) and TRPV4 channels have been identified and these are thought to contribute to constriction that leads to airflow obstruction, as these channels are Ca2+-permeable and Ca2+ is a necessary mediator of smooth muscle constriction. TRPC6, which is present in airway vascular smooth muscle cells (VSMCs), can mediate vessel constriction to reduce blood flow. Several other TRPCs have been identified in endothelial cells of larger (extra-alveolar) blood vessels within the lung, where their activation can increase vascular permeability and cause fluid to leak into interstitial spaces between vessels. Activation of endothelial TRPV4 also increases vascular permeability, although this increase is specific to the small blood vessels that supply the alveoli of lungs. TRPV4 has also been identified on alveolar macrophages, where its activation triggers the production of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS). TRPV2 channels are also present on alveolar macrophages, as well as several other macrophage populations, where their activation stimulates phagocytosis of foreign material. Although these data have largely been generated in laboratory animal species, clinical data already exist that support a role of TRPA1 and TRPV1 in respiratory sensory nerves, as acute exposure to agonists of either channel (notably, tear gases for TRPA1 and pepper sprays for TRPV1) can cause intense, incapacitating respiratory irritation in humans. CNS, central nervous system.

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GSK1016790A can produce dramatic deformation of cultured human endothelial cells, it was recently dem-onstrated that TRPV4 that is expressed on alveolar mac-rophages can have a crucial role in ventilator-induced injury in isolated lungs of mice113. Although these data suggest that discerning the exact mechanism (or mecha-nisms) of TRPV4-mediated lung injury may be challeng-ing, they highlight the contribution of TRPV4 activation to pulmonary oedema that is caused by aberrant vascu-lar or airway pressures.

TRP channels and human genetic diseasesMutations in at least six of the 28 members of the TRP channel superfamily are associated with genetic dis-eases in humans (reviewed in REFS 3,4). The diversity of pathologies caused by TRP channel dysfunction high-lights the range of roles TRP channels have in normal physiology and underscores the importance of calcium signalling in various systems. Some of the rare muta-tions may also point to a more general role for TRP channels in these pathologies. In situations in which a gain-of-function mutation underlies the pathology of a TRP channelopathy, the potential utility of an antago-nist is evident; however, TRP channel agonists could also have therapeutic benefits in situations in which one allele is intact or the mutation reduces the probabil-ity of the channel being open but leaves TRP channel function intact.

TRPV4. Mutations in TRPV4 cause several divergent heritable diseases that affect diverse systems. Genetic deletion of Trpv4 in mice leads to a substantial increase in bone mass and reduced bone loss owing to lack of weight bearing114. TRPV4 also has an important role in bone resorption and osteoclast differentiation115. In line with this finding, more than 19 autosomal domi-nant human mutations in TRPV4 are associated with skeletal dysplasias116. In two families, TRPV4 mutations (R616Q and V620I) that caused profound increases in levels of current in heterologous systems led to autoso-mal dominant brachyolmia117. Other mutations in TRPV4 lead to additional skeletal disorders of varying severity, including spondylometaphyseal dysplasia118. Both mild and lethal forms of metatrophic dysplasia are also due to mutations in TRPV4 that increase channel activity in vitro116–118.

Mutations in TRPV4 have also been linked to periph-eral neuropathies, including hereditary motor and sensory neuropathy type 2C (also known as Charcot–Marie–Tooth disease type 2C), scapuloperoneal spinal muscular atrophy and congenital distal spinal muscular atrophy119,120. In five families, three separate missense mutations were identified that altered arginine residues in the amino terminal ankyrin repeat domains119,120. These mutations appear to be gain-of-function muta-tions120, although they have also been characterized as loss-of-function mutations that are potentially caused by the decreased expression of functional channels119. These discrepancies need to be resolved by further stud-ies, as the cellular context in which the TRP channel is expressed may influence the function of the mutated

TRP channels. Collectively however, these data suggest a potential value for TRPV4 modulators in the treatment of heritable disorders, including skeletal dysplasias and sensory and motor neuropathies.

TRPP2. Mutations in the genes encoding two proteins, polycystic kidney disease 1 (PKD1) and TRP cation channel polycystin subfamily 2 (TRPP2; also known as PKD2), are causative factors in polycystic kidney dis-ease and are responsible for ~85% and ~15% of cases, respectively121. The proteins encoded by these genes are thought to interact with each other in vivo to form a cation channel–receptor complex that is involved in pressure sensing in the cilia122. The exact role of each protein is unclear, but the ratio of PKD1 expression to TRPP2 expression seems to be crucial for normal pres-sure sensing123. These findings may help to explain some of the cardiovascular abnormalities (for example, hypertension and aortic aneurysm) that are observed in polycystic kidney disease. In addition, they highlight the challenge of restoring normal function in patients via a TRPP2 modulator.

TRPC6. Mutations in TRPC6 also lead to kidney dys-function. A large family was analysed and it was discov-ered that a single point mutation (P112Q) in TRPC6 was sufficient for causing focal segmental glomerular sclero-sis124. Subsequently, additional mutations in TRPC6 that led to nephrosis were identified125, and there is increas-ing interest in TRPC6 as a potential target for the therapy of acquired kidney diseases. When they are expressed in heterologous systems, several of these mutated TRP channels show substantial increases in the amplitude of the current126 and they implicate calcium handling in glomerular disease. One possible downstream effect of overactive TRPC6 is the increased activation of the nuclear factor of activated T cells (NFAT)–calcineurin signalling pathway127.

TRPA1. Studies on a family have revealed an autoso-mal dominant mutation in the fourth transmembrane domain of TRPA1 that underlies familial episodic pain syndrome43: this is the first and as yet only example of a TRP channel mutation that is implicated in a human pain syndrome. The recombinantly expressed familial episodic pain syndrome mutant N855S TRPA1 carries more current than wild-type TRPA1 at negative poten-tials, although maximal current responses to reactive chemical agonists appear to be unaltered43. Not only do these data highlight the relevance of the modulation of TRPA1 at cold temperatures in humans but they also suggest a possible connection between cellular energetics and TRPA1 function.

TRPM6. Like the closely related TRPM7 channel, TRPM6 is distinguished from other TRP channel family members because it has a large carboxyl terminal pro-tein kinase domain (reviewed in REF. 3). Loss-of-function mutations in TRPM6 underlie inherited autosomal recessive hypomagnesaemia with secondary hypocalcae-mia (reviewed in REF. 4). Several mutations, including

Bronchoalveolar lavageA procedure in which inflammatory cells and other materials within the airway lumen are collected via repeated washings.

Alveoli Distal regions of the lung in which a thin barrier between airspaces and capillaries allows for gas exchange.

Filtration coefficient (Kf)A measurement that is used to reflect the permeability of the pulmonary vasculature to fluid.

Autosomal dominant brachyolmiaA disorder that is typified by short stature, a short trunk and curved spine.

Spondylometaphyseal dysplasiaA skeletal disorder that is typified by short stature and abnormalities in the vertebrae and tubular bones.

Charcot–Marie–Tooth diseaseAlso known as hereditary motor and sensory neuropathy. This disease, named after the three doctors who first identified it, is one of the most common inherited neuropathies. Symptoms include weakness, motor atrophy and foot deformities.

Focal segmental glomerulosclerosisA disease that is typified by glomerular scarring, which results in proteinuria, oedema and the eventual need for dialysis.

Familial episodic pain syndromeA rare disorder that is typified by periods of severe pain in the trunk and upper body. Episodes are typically triggered by cold temperatures and/or a low energy state brought about by hunger or fatigue.

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deletion of exons, point mutations, deletion mutations and premature stop codons can lead to the disease128–130. These findings raise the possibility that compounds that increase TRPM6 activity could be useful in the treatment of both inherited and sporadic hypomagnesaemia.

TRPML1. Mutations in TRP cation channel mucolipin subfamily 1 (TRPML1; also known as mucolipin 1) cause mucolipidosis type IV. Subsequent studies on the func-tion of TRPML1 have indicated that it is an intracellular protein that is localized to the endosome and lysosome, and is responsible for iron transport independently of the divalent metal transporter 1 (REF. 131). Disease-associated mutations in TRPML1 compromise its ability to transport iron, and this occurs in a manner that corre-lates well with disease severity131. These data suggest that molecules that improve the function of TRPML1 could be useful treatments for individuals with mucolipidosis type IV. However, screening for such compounds using traditional fluorescent assays and electrophysiological techniques is difficult given the intracellular localiza-tion of the channel.

TRP channels in the brainMany TRP channels are expressed by brain tissue; some are expressed at high levels (for example, TRPC3 and TRPC5), whereas others are expressed at low levels (for example, TRPV1). The function of these channels remains to be elucidated, but there is evidence that sev-eral TRP channels may contribute to neuronal excit-ability and neurotransmitter-mediated signalling in the brain.

TRPV1. There is controversy surrounding the expres-sion and role of TRPV1 in the brain. Some studies report that TRPV1 is widely expressed throughout the whole neuroaxis of the rat (albeit at much lower levels than in sensory neurons; reviewed in REF. 132). However, recent research — that relies on a powerful combination of reporter mice, in situ hybridization, electrophysiological recordings and calcium imaging — suggests that TRPV1 expression is restricted to very few regions of the brain, most notably the caudate nucleus of the hypothalmus133. Consequently, additional research will need to be car-ried out to explain the differences that have been noted in the behavioural responses between wild-type and Trpv1–/– mice.

Trpv1–/– mice adapt more easily than their wild-type littermates to aversive light, and they explore the open arm of the elevated maze more freely; these results are indicative of a reduced unconditional fear response in Trpv1–/– mice134. Furthermore, Trpv1–/– mice exhibit less freezing than wild-type mice in auditory fear con-ditioning assays134. These findings imply that TRPV1 antagonists have therapeutic potential as novel anxio-lytic agents. Notably, TRPV1 is present in dopaminergic neurons in the basal ganglia and it was speculated that malfunction of TRPV1 may be involved in the patho-genesis of Parkinson’s disease135. Some studies have also reported on the presence of TRPV1 in the hippocam-pus136 and nucleus accumbens137; TRPV1 may modulate

the strength of synaptic transmission in these regions. Future behavioural studies that directly compare selec-tive TRPV1 antagonists with differential central nervous system penetrances are needed to elucidate the role of TRPV1 in brain function.

TRPC3. TRPC3 is the most abundant TRP channel of the TRPC subfamily in cerebellar Purkinje cells. Like TRPC6 and TRPC7, TRPC3 is activated by diacyl-glycerol, and Gq-coupled GPCRs are likely to be crucial regulators of channel activity in vivo (reviewed in REF. 3). In addition, receptor tyrosine kinases activate TRPC3 in vitro and brain-derived neurotrophic factor-mediated survival of cerebellar granular cells depends on func-tional TRPC3 expression138.

Trpc3–/– mice show considerably attenuated inward currents in response to activation of metabotropic glu-tamate receptor 1 (mGluR1), which is a GPCR that is thought to be upstream of TRPC3; this finding is consistent with the role of TRPC3 in the cerebellum. Trpc3–/– mice also display impairments in walking behav-iour, although — as predicted — the TRPC3-knockout phenotype is less severe than the ataxia observed in mGluR1–/– animals139.

A screen in chemically mutagenized mice also identified a crucial role for TRPC3 in Purkinje cells. Moonwalker mice — mice with a T635A mutation in Trpc3 — showed progressive Purkinje cell degeneration and ataxia140. In cerebellar slices, this mutation resulted in a higher amplitude of currents in response to low concentration of an mGluR1 agonist140. Taken together, these data indicate that TRPC3 channels have an impor-tant role in synaptic transmission in cerebellar Purkinje cells and in the survival of these cells. Identification of selective pharmacological agents will be useful for deter-mining whether TRPC3 has a broader role in ataxia.

TRPC5. High levels of TRPC5 are expressed in the hip-pocampus and amygdala141. Trpc5–/– mice show less innate fear behaviour than their wild-type littermates in an open-field test, an elevated plus maze test and a nose bumping assay141. In addition, their conditioned fear memory is unaffected in a single fear conditioning paradigm141. These data are consistent with observations made in brain slice recordings in which Trpc5–/– mice showed normal membrane excitability and synaptic function but reduced synaptic responses to activation of mGluRs and cholecystokinin (CCK) receptors141. These data provide a potential link between TRPC5 and the CCK4 signalling pathway, the activation of which induces anxiety behaviours in rodents and humans. This suggests that TRPC5 antagonists might be useful as anxiolytic agents. Additional work will be required to determine whether a pharmacological agent can recapitulate the effects of genetic deletion. Further analysis of the role of TRPC5 in learning and memory is also warranted.

TRPC5 may also have an important role in the cal-cium-mediated guidance of neuronal growth cones142. Further in-depth studies on the brain architecture in developing Trpc5–/– mice and experiments that

Mucolipidosis type IVA lysosomal storage disorder. Symptoms typically present during the first year of life and affected individuals suffer from psychomotor retardation, ophthalmological abnormalities and anaemia.

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examine receptor-activated neurite extension in brain slices should help to clarify the contribution of TRPC5 to axonal pathfinding.

TRPM2 and TRPM7. In the brain, TRPM2 is expressed by neurons and microglia. Consistent with the concept that TRPM2 functions as a redox sensor, Trpm2–/– mice are protected against various pathologies related to oxi-dative stress, including the focal ischaemia model of stroke143. The TRPM2 gene is also a candidate risk fac-tor gene for bipolar disorder144. Knockdown of TRPM7 reduces cell death that is caused by oxygen- and/or glu-cose deprivation in isolated neuronal cultures145, which implies that TRPM7 antagonists may have a role in the treatment of stroke.

Other diseasesIn addition to the therapeutic areas described above, TRP channels have been implicated in various other diseases. Although some are not reviewed here, we dis-cuss the most noteworthy findings, in our opinion, for therapeutic targeting.

Cancer. Several TRP channels have been linked to can-cer, some as markers of biological behaviour (such as aggressive versus indolent phenotypes), whereas oth-ers could be putative therapeutic targets (reviewed in REF. 146). TRPM8 is a prime example of a marker that is also a target. TRPM8 is overexpressed in prostate can-cer, and its level of expression correlates with tumour severity147. At the same time, the TRPM8 agonist men-thol reduces the proliferation and viability of prostate cancer cell lines148. Notably, the synthetic TRPM8 ago-nist D-3263, which reduces benign prostatic hyperplasia in rats, is in clinical trials (ClinicalTrials.gov identifier: NCT00839631). The structure of this compound has not yet been published. As patients with benign prostatic hyperplasia often have prostate cancer, they could con-ceivably benefit from TRPM8 agonist treatment, which would both improve bladder function and reduce the risk of cancer.

Cardiovascular diseases. TRPM4, which is activated by (but not permeable to) Ca2+ ions, regulates many aspects of cardiovascular function (reviewed in REF. 3). Within the cerebral arteries of rats, antisense oligonucleotide deple-tion of TRPM4 reduces the potential depolarization of the myocyte membrane and reduces myogenic vasoconstric-tion caused by elevated intraluminal pressure149. TRPM4 has also been implicated in human cardiac conduction disorders150, as mutations in the channel that resulted in a net gain-of-function have been identified in several fami-lies that had a block of cardiac electrical conduction151. Loss of TRPM4 expression is, however, not categorically cardioprotective as Trpm4–/– mice are hypertensive152. Mechanistic investigations have revealed that this is due to increased adrenal catecholamine release rather than direct cardiac or vascular effects152.

Overexpression of TRPC6 and calcineurin-NFAT signalling are associated with angiotensin II-induced cardiac hypertrophy, and the expression of a dominant

negative Trpc6 transgene renders mice less susceptible to hypertrophy153. Consequently, there is substantial interest in assessing the utility of TRPC6 antagonists in various models of cardiovascular disease. Owing to the dramatic upregulation of TRPC3 following genetic dele-tion of TRPC6, selective TRPC6 antagonists are needed to probe the utility of TRPC6 as a therapeutic target.

Metabolic disorders. Genetic deletion of TRPM5, a known taste sensor, results in impaired glucose toler-ance in mice154. This phenotype may be due to the loss of high-frequency calcium oscillations in pancreatic β-cells155, although the effects of TRPM5 deletion can-not be accounted for by simple changes in membrane potential. In animal models, inactivation of TRPV1 by genetic or pharmacological manipulation has been shown to protect against the development of type 1 dia-betes and improve glucose tolerance in type 2 diabetes (reviewed in REF. 156). In Trpv1–/– mice, both increased13 and decreased157 body fat was reported, therefore the role of TRPV1 in the regulation of body weight remains controversial.

ConclusionsThe recent expansion of research into TRP channels has resulted in the identification of numerous potential drug targets beyond TRPV1, and has elucidated roles for TRP channels in diverse therapeutic areas includ-ing pain, pulmonary indications, oncology, neurology and genetic disorders. Interest is mounting as a result of emerging data from animal models, human genetic dis-orders and, in some cases, compounds entering clinical trials. Indeed, at this early stage, with very limited clini-cal data available regarding the effects of small-molecule blockade of a single TRP channel (TRPV1), it is decep-tively easy to speculate on the therapeutic potential — or the potential to cause mechanism-based toxicological liabilities — of TRP channel modifiers. Several questions remain unanswered, and are listed below.

How predictive are the animal models that are used to test TRP channel modulators? Available data largely suggest that recombinant human TRP channels respond to the same sorts of stimuli as their laboratory animal orthologues (for example, rat, mouse and human TRPM8 are all activated by menthol and cool temperatures). Moreover, acute behavioural effects of known TRP chan-nel modulators tend to be similar between humans and laboratory animals (for example, topical capsaicin causes nocifensive behaviour and swelling when it is applied to rodent or human skin), even if the molecular identity of the protein (or proteins) underlying the response in humans remains unconfirmed. Although most TRP channels have not been studied to this extent, in vitro differences between mammalian orthologues to date are generally quantitative (for example, differences in the potency of activating ligands) rather than qualitative.

Will target validation generated via genetic means be predictive of blocker effects? Potent and selective small-molecule modulators of TRP channels are continuing

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to emerge. These tools are vital for advancing the field, as acute blockade does not always mimic the protec-tive and/or deleterious effects of genetic removal. Although these two methods often correlate well, a number of notable exceptions have occurred. A prime example is TRPV1. Some TRPV1 antagonists cause hyperthermia, which has necessitated the withdrawal of these compounds from clinical trials (reviewed in REF. 14), yet Trpv1–/– mice have normal body tempera-tures9,11. Furthermore, the TRPV1 antagonist GRC-6211 improves bladder capacity65; by contrast, Trpv1–/– mice show spotty incontinence60. Other examples include TRPV4 (for example, the TRPV4 blocker HC-067047 does not alter thermal selection behaviour or water consumption in mice67) and TRPA1 (for example, block-ade but not knockout of TRPA1 inhibits CFA-induced mechanical hyperalgesia34).

Will TRP channel modulators be safe and well toler-ated in humans? As with all novel targets, questions exist regarding the potential mechanism-based toxicological liabilities of TRP channel modulators. Speculation in this area is rampant, owing at least in part to the paucity of empirical evidence regarding the safety and tolerability of modulators of any of these targets other than TRPV1. Even in the case of TRPV1, in which several structur-ally unrelated molecules have been dosed in patients and dose-limiting effects that appear to be mechanism-based

(such as alterations in thermosensation16,17 and/or ther-moregulation10,16,17) have been consistently observed, the magnitude of these effects has varied considerably owing to factors that are only partially understood.

Will an agonist or an antagonist be therapeutically use-ful? The answer to this question is rarely as obvious as it seems: in the pain area, both agonists and antagonists of TRPV1 are being evaluated in clinical trials, and the first approved TRPV1 modulator is capsaicin, which robustly activates the channel (reviewed in REF. 5). Similar ques-tions exist for TRPM8, as both agonists and antagonists are being pursued for the treatment of pain (reviewed in REF. 7). Clearly, the answer to this question will vary depending on the nature of the disease state and the TRP channel (or channels) that are involved.

The answers to the above questions will help to determine the answer to the most pressing question in the TRP channel-related field: will blocking (or other-wise modulating) TRP channels ameliorate established human disease? This answer will only be obtained after long-term clinical studies have been carried out in patients. However, the broad and pronounced links to pathophysiological processes that have been revealed by extensive preclinical target validation and human genetic studies — in addition to the relatively high chemical tractability of the TRP superfamily of ion channels — provide strong cause for optimism.

1. Patapoutian, A., Tate, S. & Woolf, C. J. Transient receptor potential channels: targeting pain at the source. Nature Rev. Drug Discov. 8, 55–68 (2009).

2. Szallasi, A., Cortright, D. N., Blum, C. A. & Eid, S. R. The vanilloid receptor TRPV1, 10 years from channel cloning to antagonist proof‑of‑concept. Nature Rev. Drug Discov. 6, 357–372 (2007).

3. Wu, L. J., Sweet, T. B. & Clapham, D. E. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol. Rev. 62, 381–404 (2010).

4. Nilius, B. & Owsianik, G. Transient receptor potential channelopathies. Pflugers Arch. 460, 437–450 (2010).

5. Szallasi, A. & Blumberg, P. M. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol. Rev. 51, 159–212 (1999).

6. Fanger, C. M., del Camino, D. & Moran, M. M. TRPA1 as an analgesic target. Open Drug Discov. J. 2, 63–69 (2010).

7. McKemy, D. D. Therapeutic potential of TRPM8 modulators. Open Drug Discov. J. 2, 80–87 (2010).

8. Everaerts, W., Nilius, B. & Owsianik, G. The vanilloid transient receptor potential channel TRPV4: from structure to disease. Prog. Biophys. Mol. Biol. 103, 2–17 (2010).

9. Caterina, M. J. et al. The capsaicin receptor: a heat‑activated ion channel in the pain pathway. Nature 389, 816–824 (1997).

10. Gavva, N. R. et al. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain 136, 202–210 (2008).

11. Iida, T., Shimizu, I., Nealen, M. L., Campbell, A. & Caterina, M. Attenuated fever response in mice lacking TRPV1. Neurosci. Lett. 378, 28–33 (2005).

12. Toth, D. M. et al. Nociception, neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice. Cell. Mol. Life Sci. 11 Nov 2010 (doi:10.1007/s00018‑010‑0569‑2).

13. Garami, A. et al. Thermoregulatory phenotype of the Trpv1 knockout mouse: thermoeffector dysbalance with hyperkinesis. J. Neurosci. 31, 1721–1733 (2011).

14. Romanovsky, A. A. et al. The transient receptor potential vanilloid‑1 channel in thermoregulation: a thermosensor it is not. Pharmacol. Rev. 61, 228–261 (2009).

15. Gavva, N. R. et al. Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade. J. Pharmacol. Exp. Ther. 323, 128–137 (2007).

16. Rowbotham, M. C. et al. Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT‑102 in a randomized healthy volunteer trial. Pain 152, 1192–1200 (2011).

17. Krarup, A. L. et al. Randomised clinical trial: the efficacy of a transient receptor potential vanilloid 1 antagonist AZD1386 in human oesophageal pain. Aliment. Pharmacol. Ther. 33, 1113–1122 (2011).This was the first report of a TRPV1 antagonist that had clinical efficacy in a painful disease state without causing significant adverse effects.

18. Lehto, S. G. et al. Antihyperalgesic effects of (R,E)‑N‑(2‑hydroxy‑2, 3‑dihydro‑1H‑inden‑4‑yl)‑3‑(2‑(piperidin‑1‑yl)‑4‑(tri fluoromethyl)phenyl)‑acrylamide (AMG8562), a novel transient receptor potential vanilloid type 1 modulator that does not cause hyperthermia in rats. J. Pharmacol. Exp. Ther. 326, 218–229 (2008).

19. Chizh, B. A. et al. The effects of the TRPV1 antagonist SB‑705498 on TRPV1 receptor‑mediated activity and inflammatory hyperalgesia in humans. Pain 132, 132–141 (2007).

20. Knotkova, H., Pappagallo, M. & Szallasi, A. Capsaicin (TRPV1 agonist) therapy for pain relief: farewell or revival? Clin. J. Pain 24, 142–154 (2008).

21. Noto, C., Pappagallo, M. & Szallasi, A. NGX‑4010, a high‑concentration capsaicin dermal patch for lasting relief of peripheral neuropathic pain. Curr. Opin. Investig. Drugs 10, 702–710 (2009).

22. Li, H., Wang, S., Chuang, A. Y., Cohen, B. E. & Chuang, H. H. Activity‑dependent targeting of TRPV1 with a pore‑permeating capsaicin analog. Proc. Natl Acad. Sci. USA 108, 8497–8502 (2011).

23. Story, G. M. et al. ANKTM1, a TRP‑like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003).

24. Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282 (2006).

25. Bessac, B. F. et al. TRPA1 is a major oxidant sensor in murine airway sensory neurons. J. Clin. Invest. 118, 1899–1910 (2008).

26. Satoh, J. & Yamakage, M. Desflurane induces airway contraction mainly by activating transient receptor potential A1 of sensory C‑fibers. J. Anesth. 23, 620–623 (2009).

27. Bessac, B. F. et al. Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases. FASEB J. 23, 1102–1114 (2009).This was the first demonstration that activation of TRPA1 is both necessary and sufficient to cause nocifensive reflexes in response to inhalation of a broadly reactive respiratory irritant.

28. Taylor‑Clark, T. E., Kiros, F., Carr, M. J. & McAlexander, M. A. Transient receptor potential ankyrin 1 mediates toluene diisocyanate‑evoked respiratory irritation. Am. J. Respir. Cell Mol. Biol. 40, 756–762 (2009).

29. Taylor‑Clark, T. E. & Undem, B. J. Ozone activates airway nerves via the selective stimulation of TRPA1 ion channels. J. Physiol. 588, 423–433 (2010).

30. Cruz‑Orengo, L. et al. Cutaneous nociception evoked by 15‑δ PGJ2 via activation of ion channel TRPA1. Mol. Pain 4, 30 (2008).

31. Trevisani, M. et al. 4‑hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc. Natl Acad. Sci. USA 104, 13519–13524 (2007).

32. Kwan, K. Y. et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair‑cell transduction. Neuron 50, 277–289 (2006).

33. McNamara, C. R. et al. TRPA1 mediates formalin‑induced pain. Proc. Natl Acad. Sci. USA 104, 13525–13530 (2007).

34. Petrus, M. et al. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol. Pain 3, 40 (2007).

35. Eid, S. R. et al. HC‑030031, a TRPA1 selective antagonist, attenuates inflammatory‑ and neuropathy‑induced mechanical hypersensitivity. Mol. Pain 4, 48 (2008).

36. Wei, H., Hamalainen, M. M., Saarnilehto, M., Koivisto, A. & Pertovaara, A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals. Anesthesiology 111, 147–154 (2009).

R E V I E W S



37. Katsura, H. et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats. Exp. Neurol. 200, 112–123 (2006).

38. del Camino, D. et al. TRPA1 contributes to cold hypersensitivity. J. Neurosci. 30, 15165–15174 (2010).

39. da Costa, D. S. et al. The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation. Pain 148, 431–437 (2010).

40. Chen, J. et al. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain 152, 1165–1172 (2011).

41. McGaraughty, S. et al. TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic, and inflamed rats. Mol. Pain 6, 14 (2010).This was the first demonstration that a TRPA1 antagonist is capable of relieving pathological pain in animal models without altering cold sensation in naive animals.

42. Kerstein, P. C., del Camino, D., Moran, M. M. & Stucky, C. L. Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors. Mol. Pain 5, 19 (2009).

43. Kremeyer, B. et al. A gain‑of‑function mutation in TRPA1 causes familial episodic pain syndrome. Neuron 66, 671–680 (2010).This paper was the first to link the activity of a TRP channel to a pain syndrome in humans. It also suggested that potentiation of TRPA1 by cold temperatures is physiologically relevant, as cold is one of the triggers for pain episodes in patients suffering from pain syndromes.

44. Cheng, X. et al. TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation. Cell 141, 331–343 (2010).

45. Xu, H., Delling, M., Jun, J. C. & Clapham, D. E. Oregano, thyme and clove‑derived flavors and skin sensitizers activate specific TRP channels. Nature Neurosci. 9, 628–635 (2006).

46. Gopinath, P. et al. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Womens Health 5, 2 (2005).

47. Facer, P. et al. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol. 7, 11 (2007).This was the first report of disease-related changes in the expression of TRPV1, TRPV3 and TRPV4 in painful disease states in humans.

48. Xiao, R. et al. Calcium plays a central role in the sensitization of TRPV3 channel to repetitive stimulations. J. Biol. Chem. 283, 6162–6174 (2008).

49. Khairatkar Joshi, N., Maharaj, N. & Thomas, A. The TRPV3 receptor as a pain target: a therapeutic promise or just some more new biology? Open Drug Discov. J. 2, 88–95 (2010).

50. Moqrich, A. et al. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 307, 1468–1472 (2005).

51. Okazawa, M. et al. Noxious heat receptors present in cold‑sensory cells in rats. Neurosci. Lett. 359, 33–36 (2004).

52. Dhaka, A. et al. TRPM8 is required for cold sensation in mice. Neuron 54, 371–378 (2007).

53. Bautista, D. M. et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204–208 (2007).

54. Colburn, R. W. et al. Attenuated cold sensitivity in TRPM8 null mice. Neuron 54, 379–386 (2007).

55. Proudfoot, C. J. et al. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr. Biol. 16, 1591–1605 (2006).

56. Parks, D. J. et al. Design and optimization of benzimidazole‑containing transient receptor potential melastatin 8 (TRPM8) antagonists. J. Med. Chem. 54, 233–247 (2011).

57. Andersson, K. E., Gratzke, C. & Hedlund, P. The role of the transient receptor potential (TRP) superfamily of cation‑selective channels in the management of the overactive bladder. BJU Int. 106, 1114–1127 (2010).

58. Avelino, A. & Cruz, F. TRPV1 (vanilloid receptor) in the urinary tract: expression, function and clinical applications. Naunyn Schmiedebergs Arch. Pharmacol. 373, 287–299 (2006).

59. Everaerts, W. et al. Functional characterization of transient receptor potential channels in mouse urothelial cells. Am. J. Physiol. Renal Physiol. 298, F692–F701 (2010).

60. Birder, L. A. et al. Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nature Neurosci. 5, 856–860 (2002).

61. MacDonald, R., Monga, M., Fink, H. A. & Wilt, T. J. Neurotoxin treatments for urinary incontinence in subjects with spinal cord injury or multiple sclerosis: a systematic review of effectiveness and adverse effects. J. Spinal Cord Med. 31, 157–165 (2008).

62. Cruz, C. D. et al. Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals. Exp. Neurol. 214, 301–308 (2008).

63. Cruz, F. & Dinis, P. Resiniferatoxin and botulinum toxin type A for treatment of lower urinary tract symptoms. Neurourol. Urodyn. 26, 920–927 (2007).

64. Sculptoreanu, A., de Groat, W. C., Buffington, C. A. & Birder, L. A. Protein kinase C contributes to abnormal capsaicin responses in DRG neurons from cats with feline interstitial cystitis. Neurosci. Lett. 381, 42–46 (2005).

65. Charrua, A. et al. GRC‑6211, a new oral specific TRPV1 antagonist, decreases bladder overactivity and noxious bladder input in cystitis animal models. J. Urol. 181, 379–386 (2009).

66. Gevaert, T. et al. Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding. J. Clin. Invest. 117, 3453–3462 (2007).

67. Everaerts, W. et al. Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide‑induced cystitis. Proc. Natl Acad. Sci. USA 107, 19084–19089 (2010).

68. Mochizuki, T. et al. The TRPV4 cation channel mediates stretch‑evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J. Biol. Chem. 284, 21257–21264 (2009).

69. Thorneloe, K. S. et al. N‑((1S)‑1‑{[4‑((2S)‑2‑ {[(2,4‑dichlorophenyl)sulfonyl]amino} ‑3‑hydroxypropanoyl)‑1‑piperazinyl]carbonyl} ‑3‑methylbutyl)‑1‑benzothiophene‑2‑carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: part I. J. Pharmacol. Exp. Ther. 326, 432–442 (2008).

70. Mukerji, G. et al. Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol. 6, 6 (2006).

71. Lashinger, E. S. et al. AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome. Am. J. Physiol. Renal Physiol. 295, F803–F810 (2008).

72. Paus, R., Schmelz, M., Biro, T. & Steinhoff, M. Frontiers in pruritus research: scratching the brain for more effective itch therapy. J. Clin. Invest. 116, 1174–1186 (2006).

73. Biro, T. et al. TRP channels as novel players in the pathogenesis and therapy of itch. Biochim. Biophys. Acta 1772, 1004–1021 (2007).

74. Bodo, E. et al. Vanilloid receptor‑1 (VR1) is widely expressed on various epithelial and mesenchymal cell types of human skin. J. Invest. Dermatol. 123, 410–413 (2004).

75. Stander, S. et al. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures. Exp. Dermatol. 13, 129–139 (2004).

76. Shim, W. S. et al. TRPV1 mediates histamine‑induced itching via the activation of phospholipase A2 and 12‑lipoxygenase. J. Neurosci. 27, 2331–2337 (2007).

77. Weisshaar, E., Heyer, G., Forster, C. & Handwerker, H. O. Effect of topical capsaicin on the cutaneous reactions and itching to histamine in atopic eczema compared to healthy skin. Arch. Dermatol. Res. 290, 306–311 (1998).

78. Alenmyr, L., Hogestatt, E. D., Zygmunt, P. M. & Greiff, L. TRPV1‑mediated itch in seasonal allergic rhinitis. Allergy 64, 807–810 (2009).

79. Wilson, S. R. et al. TRPA1 is required for histamine‑independent, Mas‑related G protein‑coupled receptor‑mediated itch. Nature Neurosci. 14, 595–602 (2011).This study showed that TRPA1 not only mediates pain and airway irritation but is also required for histamine-independent itch.

80. Carrillo, P. et al. Cutaneous wounds produced by capsaicin treatment of newborn rats are due to trophic disturbances. Neurotoxicol. Teratol. 20, 75–81 (1998).

81. Lagerstrom, M. C. et al. VGLUT2‑dependent sensory neurons in the TRPV1 population regulate pain and itch. Neuron 68, 529–542 (2010).

82. Liu, Y. et al. VGLUT2‑dependent glutamate release from nociceptors is required to sense pain and suppress itch. Neuron 68, 543–556 (2010).

83. Bodo, E. et al. A hot new twist to hair biology: involvement of vanilloid receptor‑1 (VR1/TRPV1) signaling in human hair growth control. Am. J. Pathol. 166, 985–998 (2005).This study was the first to show that TRPV1 expressed on non-neuronal skin cells is involved in the regulation of cell growth.

84. Toth, B. I. et al. Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor‑1 and transient receptor potential Vanilloid‑1. J. Invest. Dermatol. 131, 1095–1104 (2011).

85. Denda, M., Sokabe, T., Fukumi‑Tominaga, T. & Tominaga, M. Effects of skin surface temperature on epidermal permeability barrier homeostasis. J. Invest. Dermatol. 127, 654–659 (2007).

86. Lee, Y. M., Kim, Y. K. & Chung, J. H. Increased expression of TRPV1 channel in intrinsically aged and photoaged human skin in vivo. Exp. Dermatol. 18, 431–436 (2009).

87. Lee, Y. M. et al. A novel role for the TRPV1 channel in UV‑induced matrix metalloproteinase (MMP)‑1 expression in HaCaT cells. J. Cell Physiol. 219, 766–775 (2009).

98. Peier, A. M. et al. A heat‑sensitive TRP channel expressed in keratinocytes. Science 296, 2046–2049 (2002).

89. Asakawa, M. et al. Association of a mutation in TRPV3 with defective hair growth in rodents. J. Invest. Dermatol. 126, 2664–2672 (2006).

90. Yoshioka, T. et al. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice. J. Invest. Dermatol. 129, 714–722 (2009).These experiments demonstrated that a gain-of-function mutation in the Trpv3 gene results in severe dermatitis in mice; the TRPV3 protein is abundant in keratinocytes.

91. Borbiro, I., Geczy, T., Paus, R., Kovacs, L. & Biro, T. Activation of transient receptor potential vanilloid‑3 (TRPV3) inhibits human hair growth. J. Invest. Dermatol. 128, S151 (2008).

92. Mazzone, S. B. & Undem, B. J. Cough sensors. V. Pharmacological modulation of cough sensors. Handb. Exp. Pharmacol. 187, 99–127 (2009).

93. Carr, M. J. & Lee, L. Y. Plasticity of peripheral mechanisms of cough. Respir. Physiol. Neurobiol. 152, 298–311 (2006).

94. Fujimura, M. et al. Prostanoids and cough response to capsaicin in asthma and chronic bronchitis. Eur. Respir. J. 8, 1499–1505 (1995).

95. Blom, H. M. et al. Intranasal capsaicin is efficacious in non‑allergic, non‑infectious perennial rhinitis. A placebo‑controlled study. Clin. Exp. Allergy 27, 796–801 (1997).

96. Matta, J. A. et al. General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. Proc. Natl Acad. Sci. USA 105, 8784–8789 (2008).

97. Andrè, E. et al. Cigarette smoke‑induced neurogenic inflammation is mediated by α,β‑unsaturated aldehydes and the TRPA1 receptor in rodents. J. Clin. Invest. 118, 2574–2582 (2008).

98. Birrell, M. A. et al. TRPA1 agonists evoke coughing in guinea pig and human volunteers. Am. J. Respir. Crit. Care Med. 180, 1042–1047 (2009).

99. Talavera, K. et al. Nicotine activates the chemosensory cation channel TRPA1. Nature Neurosci. 12, 1293–1299 (2009).

100. Caceres, A. I. et al. A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma. Proc. Natl Acad. Sci. USA 106, 9099–9104 (2009).

101. Nassini, R. et al. Acetaminophen, via its reactive metabolite N‑acetyl‑p‑benzo‑quinoneimine and transient receptor potential ankyrin‑1 stimulation, causes neurogenic inflammation in the airways and other tissues in rodents. FASEB J. 24, 4904–4916 (2010).

102. Weissmann, N. et al. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc. Natl Acad. Sci. USA 103, 19093–19098 (2006).

R E V I E W S



103. Yu, Y. et al. A functional single‑nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension. Circulation 119, 2313–2322 (2009).

104. White, T. A. et al. Role of transient receptor potential C3 in TNF‑α‑enhanced calcium influx in human airway myocytes. Am. J. Respir. Cell. Mol. Biol. 35, 243–251 (2006).

105. Xiao, J. H., Zheng, Y. M., Liao, B. & Wang, Y. X. Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 43, 17–25 (2010).

106. Sel, S. et al. Loss of classical transient receptor potential 6 channel reduces allergic airway response. Clin. Exp. Allergy 38, 1548–1558 (2008).

107. Jia, Y. et al. Functional TRPV4 channels are expressed in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L272–L278 (2004).

108. Zhu, G. et al. Association of TRPV4 gene polymorphisms with chronic obstructive pulmonary disease. Hum. Mol. Genet. 18, 2053–2062 (2009).This was the first study to suggest that TRPV4 can regulate lung function in humans.

109. Li, J. et al. TRPV4‑mediated calcium‑influx into human bronchial epithelia upon exposure to diesel exhaust particles. Environ. Health Perspect. 119, 784–793 (2011).

110. Jian, M. Y., King, J. A., Al‑Mehdi, A. B., Liedtke, W. & Townsley, M. I. High vascular pressure‑induced lung injury requires P450 epoxygenase‑dependent activation of TRPV4. Am. J. Respir. Cell Mol. Biol. 38, 386–392 (2008).

111. Hamanaka, K. et al. TRPV4 initiates the acute calcium‑dependent permeability increase during ventilator‑induced lung injury in isolated mouse lungs. Am. J. Physiol. Lung Cell. Mol. Physiol. 293, L923–L932 (2007).

112. Willette, R. N. et al. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: part 2. J. Pharmacol. Exp. Ther. 326, 443–452 (2008).

113. Hamanaka, K. et al. TRPV4 channels augment macrophage activation and ventilator‑induced lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 299, L353–L362 (2010).

114. Mizoguchi, F. et al. Transient receptor potential vanilloid 4 deficiency suppresses unloading‑induced bone loss. J. Cell Physiol. 216, 47–53 (2008).

115. Masuyama, R. et al. TRPV4‑mediated calcium influx regulates terminal differentiation of osteoclasts. Cell. Metab. 8, 257–265 (2008).

116. Dai, J. et al. Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family. J. Med. Genet. 47, 704–709 (2010).

117. Rock, M. J. et al. Gain‑of‑function mutations in TRPV4 cause autosomal dominant brachyolmia. Nature Genet. 40, 999–1003 (2008).

118. Krakow, D. et al. Mutations in the gene encoding the calcium‑permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia. Am. J. Hum. Genet. 84, 307–315 (2009).

119. Auer‑Grumbach, M. et al. Alterations in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C. Nature Genet. 42, 160–164 (2010).

120. Landoure, G. et al. Mutations in TRPV4 cause Charcot‑Marie‑Tooth disease type 2C. Nature Genet. 42, 170–174 (2010).

121. Feng, S. et al. Identification and functional characterization of an N‑terminal oligomerization domain for polycystin‑2. J. Biol. Chem. 283, 28471–28479 (2008).

122. Nauli, S. M. et al. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nature Genet. 33, 129–137 (2003).

123. Sharif‑Naeini, R. et al. Polycystin‑1 and ‑2 dosage regulates pressure sensing. Cell 139, 587–596 (2009).

124. Winn, M. P. et al. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 308, 1801–1804 (2005).

125. Moller, C. C. et al. Induction of TRPC6 channel in acquired forms of proteinuric kidney disease. J. Am. Soc. Nephrol. 18, 29–36 (2007).

126. Reiser, J. et al. TRPC6 is a glomerular slit diaphragm‑associated channel required for normal renal function. Nature Genet. 37, 739–744 (2005).

127. Wang, Y. et al. Activation of NFAT signaling in podocytes causes glomerulosclerosis. J. Am. Soc. Nephrol. 21, 1657–1666 (2010).

128. Chubanov, V. et al. Hypomagnesemia with secondary hypocalcemia due to a missense mutation in the putative pore‑forming region of TRPM6. J. Biol. Chem. 282, 7656–7667 (2007).

129. Schlingmann, K. P. et al. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nature Genet. 31, 166–170 (2002).

130. Schlingmann, K. P. et al. Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia. J. Am. Soc. Nephrol. 16, 3061–3069 (2005).

131. Dong, X. P. et al. The type IV mucolipidosis‑associated protein TRPML1 is an endolysosomal iron release channel. Nature 455, 992–996 (2008).

132. Szallasi, A. & Di Marzo, V. New perspectives on enigmatic vanilloid receptors. Trends Neurosci. 23, 491–497 (2000).

133. Cavanaugh, D. et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle. J. Neurosci. 31, 5067–5077 (2011).This careful study highlighted the challenges of determining the expression pattern for a target of interest, and the need to combine multiple approaches.

134. Marsch, R. et al. Reduced anxiety, conditioned fear, and hippocampal long‑term potentiation in transient receptor potential vanilloid type 1 receptor‑deficient mice. J. Neurosci. 27, 832–839 (2007).

135. Mezey, E. et al. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1‑like immunoreactivity, in the central nervous system of the rat and human. Proc. Natl Acad. Sci. USA 97, 3655–3660 (2000).

136. Kauer, J. A. & Gibson, H. E. Hot flash: TRPV channels in the brain. Trends Neurosci. 32, 215–224 (2009).

137. Grueter, B. A., Brasnjo, G. & Malenka, R. C. Postsynaptic TRPV1 triggers cell type‑specific long‑term depression in the nucleus accumbens. Nature Neurosci. 13, 1519–1525 (2010).

138. Jia, Y., Zhou, J., Tai, Y. & Wang, Y. TRPC channels promote cerebellar granule neuron survival. Nature Neurosci. 10, 559–567 (2007).

139. Hartmann, J. et al. TRPC3 channels are required for synaptic transmission and motor coordination. Neuron 59, 392–398 (2008).

140. Becker, E. B. et al. A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc. Natl Acad. Sci. USA 106, 6706–6711 (2009).

141. Riccio, A. et al. Essential role for TRPC5 in amygdala function and fear‑related behavior. Cell 137, 761–772 (2009).This study was the first to implicate TRPC5 in anxiety. The neuronal recordings obtained in the study suggest that there is a potential link between TRPC5 and the CCK4 pathway.

142. Greka, A., Navarro, B., Oancea, E., Duggan, A. & Clapham, D. E. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nature Neurosci. 6, 837–845 (2003).

143. Miller, B. A. & Zhang, W. TRP channels as mediators of oxidative stress. Adv. Exp. Med. Biol. 704, 531–544 (2011).

144. Xu, C. et al. TRPM2 variants and bipolar disorder risk: confirmation in a family‑based association study. Bipolar Disord. 11, 1–10 (2009).

145. Aarts, M. et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863–877 (2003).

146. Lehen’kyi, V. & Prevarskaya, N. Oncogenic TRP channels. Adv. Exp. Med. Biol. 704, 929–945 (2011).

147. Tsavaler, L., Shapero, M. H., Morkowski, S. & Laus, R. Trp‑p8, a novel prostate‑specific gene, is up‑regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res. 61, 3760–3769 (2001).

148. Thebault, S. et al. Novel role of cold/menthol‑sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store‑operated channels in LNCaP human prostate cancer epithelial cells. J. Biol. Chem. 280, 39423–39435 (2005).

149. Reading, S. A. & Brayden, J. E. Central role of TRPM4 channels in cerebral blood flow regulation. Stroke 38, 2322–2328 (2007).

150. Kruse, M. et al. Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J. Clin. Invest. 119, 2737–2744 (2009).

151. Liu, H. et al. Gain‑of‑function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ. Cardiovasc. Genet. 3, 374–385 (2010).

152. Mathar, I. et al. Increased catecholamine secretion contributes to hypertension in TRPM4‑deficient mice. J. Clin. Invest. 120, 3267–3279 (2010).

153. Onohara, N. et al. TRPC3 and TRPC6 are essential for angiotensin II‑induced cardiac hypertrophy. EMBO J. 25, 5305–5316 (2006).

154. Brixel, L. R. et al. TRPM5 regulates glucose‑stimulated insulin secretion. Pflugers Arch. 460, 69–76 (2010).

155. Colsoul, B. et al. Loss of high‑frequency glucose‑induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5–/– mice. Proc. Natl Acad. Sci. USA 107, 5208–5213 (2010).This study identified TRPM5 as a potential target for antidiabetic drugs.

156. Suri, A. & Szallasi, A. The emerging role of TRPV1 in diabetes and obesity. Trends Pharmacol. Sci. 29, 29–36 (2008).

157. Motter, A. L. & Ahern, G. P. TRPV1‑null mice are protected from diet‑induced obesity. FEBS Lett. 582, 2257–2262 (2008).

158. Biro, T. et al. Hair cycle control by vanilloid receptor‑1 (TRPV1): evidence from TRPV1 knockout mice. J. Invest. Dermatol. 126, 1909–1912 (2006).

159. Toth, B. I. et al. Transient receptor potential vanilloid‑1 signaling as a regulator of human sebocyte biology. J. Invest. Dermatol. 129, 329–339 (2009).

160. Beck, B. et al. TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma. Cell Calcium 43, 492–505 (2008).

161. Pani, B. et al. Up‑regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier’s disease. Mol. Biol. Cell 17, 4446–4458 (2006).

162. Atoyan, R., Shander, D. & Botchkareva, N. V. Non‑neuronal expression of transient receptor potential type A1 (TRPA1) in human skin. J. Invest. Dermatol. 129, 2312–2315 (2009).

163. Lehen’kyi, V. et al. TRPV6 is a Ca2+ entry channel essential for Ca2+‑induced differentiation of human keratinocytes. J. Biol. Chem. 282, 22582–22591 (2007).

164. McNeill, M. S. et al. Cell death of melanophores in zebrafish trpm7 mutant embryos depends on melanin synthesis. J. Invest. Dermatol. 127, 2020–2030 (2007).

165. Kiyonaka, S. et al. Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc. Natl Acad. Sci. USA 106, 5400–5405 (2009).

166. Venkatachalam, K. et al. Motor deficit in a Drosophila model of mucolipidosis type IV due to defective clearance of apoptotic cells. Cell 135, 838–851 (2008).

167. Lambert, S. et al. Transient receptor potential melastatin 1 (TRPM1) is an ion‑conducting plasma membrane channel inhibited by zinc ions. J. Biol. Chem. 286, 12221–12233 (2011).

168. Bellone, R. R. et al. Differential gene expression of TRPM1, the potential cause of congenital stationary night blindness and coat spotting patterns (LP) in the Appaloosa horse (Equus caballus). Genetics 179, 1861–1870 (2008).

169. Audo, I. et al. TRPM1 is mutated in patients with autosomal‑recessive complete congenital stationary night blindness. Am. J. Hum. Genet. 85, 720–729 (2009).

170. Li, Z. et al. Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans. Am. J. Hum. Genet. 85, 711–719 (2009).

171. van Genderen, M. M. et al. Mutations in TRPM1 are a common cause of complete congenital stationary night blindness. Am. J. Hum. Genet. 85, 730–736 (2009).

172. Uchida, K. et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice. Diabetes 60, 119–126 (2011).

173. Harteneck, C., Frenzel, H. & Kraft, R. N‑(p‑amylcinnamoyl)anthranilic acid (ACA): a phospholipase A(2) inhibitor and TRP channel blocker. Cardiovasc. Drug Rev. 25, 61–75 (2007).

174. Walder, R. Y. et al. Mice defective in Trpm6 show embryonic mortality and neural tube defects. Hum. Mol. Genet. 18, 4367–4375 (2009).

R E V I E W S



175. Jin, J. et al. Deletion of Trpm7 disrupts embryonic development and thymopoiesis without altering Mg2+ homeostasis. Science 322, 756–760 (2008).

176. Hermosura, M. C. et al. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc. Natl Acad. Sci. USA 102, 11510–11515 (2005).

177. Chen, H. C. et al. Blockade of TRPM7 channel activity and cell death by inhibitors of 5‑lipoxygenase. PLoS ONE 5, e11161 (2010).

178. Higashi, Y., Kiuchi, T. & Furuta, K. Efficacy and safety profile of a topical methyl salicylate and menthol patch in adult patients with mild to moderate muscle strain: a randomized, double‑blind, parallel‑group, placebo‑controlled, multicenter study. Clin. Ther. 32, 34–43 (2010).

179. Garcia‑Gonzalez, M. A. et al. Pkd1 and Pkd2 are required for normal placental development. PLoS ONE 5, e12821 (2010).

180. Clapham, D. E. TRP channels as cellular sensors. Nature 426, 517–524 (2003).

181. Scholz, J. & Clifford, J. W. Can we conquer pain? Nature Neurosci. 5, 1062–1067 (2002).

AcknowledgementsWe would like to thank B. Nilius for reading the manuscript and providing useful comments, and M. Trevisani for his help in com-piling the TRPV1 antagonist clinical trials database.

Competing interests statementThe authors declare competing financial interests: see Web version for details.

FURTHER INFORMATIONClinicalTrials.gov website: http://www.clinicaltrials.govDaewoong Pharmaceutical website: http://www.daewoong.co.kr/www_pharm/english_new/aboutus/whatsnews_view.asp?idx=60652Drugs.com website: http://www.drugs.com/clinical_trials/anesiva-phase-3-trial-adlea-meets-primary-endpoint-significantly-reduce-pain-after-total-knee-6536.htmlDrugs.com website: http://www.drugs.com/newdrugs/neurogesx-receives-fda-approval-qutenza-capsaicin-8-patch-postherpetic-neuralgia-phn-1772.htmlGlenmark Pharmaceuticals website: http://www.glenmarkpharma.com/GLN_NWS/pdf/GRC_6211.pdfJapan Tobacco — Clinical Development of Pharmaceuticals (29 July 2010): http://www.jt.com/investors/results/pharmaceuticals/pdf/P.L.20100729_E.pdfPharmEste website: http://www.pharmeste.com/home.asp?op=interna&id=2&id_pag=10&tit=PipelineSanofi website: http://en.sanofi.com/research_innovation/rd_key_figures/rd_key_figures.asp

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