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Transient Receptor Potential Channels As Drug Targets: from the Science of Basic Research to the Art of Medicine

Transient Receptor Potential Channels As Drug Targets: from the Science of Basic Research to the Art of Medicine

1521-0081/66/3/676–814$25.00 http://dx.doi.org/10.1124/pr.113.008268 PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:676–814, July 2014 Copyright © 2014 by The American Society for and Experimental Therapeutics

ASSOCIATE EDITOR: DAVID R. SIBLEY Transient Potential Channels as Targets: From the Science of Basic Research to the Art of Medicine

Bernd Nilius and Arpad Szallasi KU Leuven, Department of Cellular and Molecular Medicine, Laboratory of Research, Campus Gasthuisberg, Leuven, Belgium (B.N.); and Department of Pathology, Monmouth Medical Center, Long Branch, New Jersey (A.S.)

Abstract...... 679 I. Transient Receptor Potential Channels: A Brief Introduction ...... 679 A. Canonical Transient Receptor Potential Subfamily ...... 682 B. Vanilloid Transient Receptor Potential Subfamily ...... 686 C. Melastatin Transient Receptor Potential Subfamily ...... 696 Downloaded from D. Ankyrin Transient Receptor Potential Subfamily ...... 700 E. Mucolipin Transient Receptor Potential Subfamily ...... 702 F. Polycystic Transient Receptor Potential Subfamily ...... 703 II. Transient Receptor Potential Channels: Hereditary Diseases (Transient

Receptor Potential )...... 704 at Open University Library on January 18, 2020 A. Canonical Transient Receptor Potential Channelopathies...... 704 B. Vanilloid Transient Receptor Potential Channelopathies...... 705 C. Melastatin Transient Receptor Potential Channelopathies ...... 708 D. Ankyrin Transient Receptor Potential Channelopathies ...... 710 E. Mucolipin Transient Receptor Potential Channelopathies...... 710 F. Polycystic Transient Receptor Potential Channelopathies ...... 711 III. Transient Receptor Potential Channels: Acquired Diseases ...... 712 A. The Role of Transient Receptor Potential Channels in Nociception, Pain, and Itch ...... 713 1. Basic Concepts ...... 713 a. Differential transient receptor potential channel expression defines functional sensory neuron subtypes: Implications for drug development...... 717 b. Disease-related changes in transient receptor potential channel expression...... 718 2. Transient Receptor Potential Channels in Dental Pain ...... 720 3. Transient Receptor Potential Channels in Visceral Pain...... 722 a. Esophagus and ...... 722 b. Genitourinary tract...... 725 4. Contribution of Transient Receptor Potential Channels to Neuropathic and Cancer Pain . . . . 727 a. Targeting vanilloid transient receptor potential 1 for pain relief: A brief overview of clinical studies...... 727 b. Vanilloid transient receptor potential 3...... 733 c. Melastatin transient receptor potential 8...... 734 d. Ankyrin transient receptor potential 1...... 735 5. The Contribution of Transient Receptor Potentials to -Induced Neuropathic Pain ...... 736 6. Transient Receptor Potential Channels in ...... 738 a. The role of vanilloid transient receptor potential 1 in migraine and cluster .. . . . 738 b. Ankyrin transient receptor potential 1 as a candidate sensor for environmental irritants that provoke migraine attacks...... 740

Address correspondence to: Arpad Szallasi, Department of Pathology, Monmouth Medical Center, 300 Second Avenue, Long Branch, NJ 07740. E-mail: [email protected] dx.doi.org/10.1124/pr.113.008268.

676 TRP Channels as Drug Targets 677

c. Possible Involvement of other transient receptor potential channels in migraine. . . . 740 d. Migraine as a transient receptor potential ? ...... 740

ABBREVIATIONS: 2-APB, 2-aminoethoxydiphenyl borate; 4aPDD, 4a- 12,13-didecanoate; A-1165442, (R)-1-(7-chloro-2,2-bis(fluoro- methyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea; A-784168, 3,6-dihydro-39-(trifluoromethyl)-N-[4-[(trifluoromethyl)sulfonyl]phenyl]-[1(2H),29- bipyridine]-4-carboxamide; A-795614, 1-(1H-indazol-4-yl)-3-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea; A-889425, 1-(3-methylpyridin-2-yl)- N-(4-(trifluoromethylsulfonyl)phenyl)-1,2,3,6-tetrahydropyridine-4-carboxamide; A-967079, (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-pentene-3-one oxime; A-993610, (S)-1-(3-chloropyridin-2-yl)-3-methyl-N-(4-(trifluoromethyl)phenyl)-1,2,3,6-tetrahydropyridine-4-carboxamide; ABT-102, (R)-(5-tert- butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea; ABT-116, urea, N-((2-(3,3-dimethylbutyl)-4-(trifluoromethyl)phenyl)methyl)-N9-(1-methyl- 1H-indazol-4-yl); ADPKD, autosomal dominant polycystic kidney disease; ADPR, ADP-ribose; AKAP, A-kinase–anchoring ; ALS-G, Guamanian amyotrophic lateral sclerosis; AM1241, (2-iodo-5-nitrophenyl)-[1-[(1-methylpiperidin-2-yl)methyl]indol-3-yl]methanone; AM404, N-(4- hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide; AMG517, N-[4-({6-[4-(trifluoromethyl)phenyl]pyrimidin-4-yl}oxy)-1,3-benzothiazol-2-yl]acetamide; AMG9678, (R)-1-(4-(trifluoromethyl)phenyl)-N-((S)-1,1,1-trifluoropropan-2-yl)-3,4-dihydroisoquinoline-2(1H)-carboxamide; AMG9810, (2E)-N-(2,3- dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide;AMI,acutemyocardialinfarction;AMTB,N-(3-aminopropyl)-2-[3- methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride; AP18, 4-(4-chlorophenyl)-3-methyl-3-buten-2-one oxime; Apo, apolipoprotein; ARD, domain; AS1928370, (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-car- boxamide; AZD1386, (S)-N-{1-[4-(t-butyl)-phenyl]-ethyl}-2-(6,7-difluorobenzoimidazol-1-yl)acetamide; BAL, bronchoalveolar lavage; BAT, brown adipose tissue; BayK8644, methyl-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; BCC, basal cell carcinoma; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; BD-I, type I; BDNF, brain-derived neurotrophic factor; BK, ; BTP2, N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide; CaM, calmodulin; CaMKII, Ca2+/CaM-dependent kinase II; CB1, receptor 1; CCI, chronic constriction injury; CCK, cholecystokinin; CDK, cyclin-dependent kinase; CFA, complete Freund’s adjuvant; CGRP, calcitonin -related peptide; CHO, Chinese hamster ovary; CIPN, chemotherapy-induced peripheral neuropathy; CNS, central ; COPD, chronic obstructive pulmonary disease; COX, ; CRP, C-reactive protein; CSE, cigarette smoke extract; D-3263, 3-(2-amino-ethyl)-1(R)-[(2(S)-isopropyl-5(R)-methyl cyclohexanecarbonyl)]-5-methoxy- 1,3-dihydro-benzoimidazol-2-one hydrochloride; DAG, diacylglycerol; DRG, dorsal root ganglion; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EPSP, excitatory postsynaptic potential; ERK, extracellular signal-regulated kinase; FD, functional dyspepsia; FDA, US Food and Drug Administration; FK506, ; FSGS, focal segmental glomerulosclerosis; GBM, glioblastoma multiforme; GERD, gastroesophageal reflux disease; GLP-1, glucagon-like peptide-1; GPCR, G protein–coupled receptor; GRP, gastrin-releasing peptide; GSK1016790A, (N-((1S)-1-{[4- ((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carbox- amide; GSK205, N-(4-(2-(benzyl(methyl)amino)ethyl)phenyl)-5-(pyridine-3-yl)thiazol-2-amine; H2S, hydrogen sulfide; HC-030031, 2-(1,3-dimethyl-2, 6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; HC-067047, 2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoro- methyl)phenyl]-1H-pyrrole-3-carboxamide; HCAEC, coronary artery endothelial cell of origin; hIAP, human islet amyloid polypeptide; HMVEC, human microvascular endothelial cell; HPETE, 12-(S)-5-hydroperoxyeicosatetraenoic acid; HSH, hypomagnesemia with secondary hypocalcemia; IBD, inflammatory bowel disease; IGF, -like growth factor; IL, interleukin; IFN, interferon; iNOS, inducible nitric-oxide synthase; IP3,inositol- 1,4,5-triphosphate; IPAH, idiopathic pulmonary arterial ; IPF, idiopathic pulmonary fibrosis; I-RTX, 5-iodoresiniferatoxin; JNJ17203212, 4-[3-(trifluoromethyl)-2-pyridinyl]-N-[5-(trifluoromethyl)-2-pyridinyl]-1-piperazinecarboxamide; JNJ41876666, 3-[7-trifluoromethyl-5-(2-trifluorome- thyl-phenyl)-1H-benzimidazol-2-yl]-1-oxa-2-aza-spiro[4.5]dec-2-ene hydrochloride; JYL1421, N-(4-tertbutylbenzyl)-N-[3-fluoro-4-(methylsulfonylamino) benzyl]-thiourea; KIF13B, kinesin-3 family member 13B; LA, ; LPS, lipopolysaccharide; LTB4, B4; LTD, long-term ; LTP, long-term potentiation; MCP1, macrophage chemotactic protein-1; MEK, mitogen-activated protein kinase kinase; mGluR, metabotropic glutamate receptor; MHC, major complex; MIA, monoiodoacetate; miR, microRNA; MITF, microphthalmia-associated transcription factor; ML-204, 4-methyl-2-(1-piperidinyl)quinoline; ML-IV, mucolipidosis type IV; MMP1, matrix metalloprotease-1; Mrgpr, Mas-related G protein–coupled receptor; MS, multiple sclerosis; mTOR, mammalian target of rapamycin; NADA, N-arachidonoyldopamine; NFAT, nuclear factor of activated T cells; + + NGF, nerve growth factor; NHERF, Na /H exchanger regulatory factor; NK, neurokinin; NK1R, neurokinin 1 receptor; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOD, nonobese diabetic; NOS, nitric-oxide synthase; Nppb, neuropeptide natriuretic polypeptide b; NSAID, nonsteroidal anti- inflammatory drug; OA, osteoarthritis; OAG, 1-oleoyl-2-acetyl-glycerol; OEA, ; OMIM, Online Mendelian Inheritance in Man; ONO1714, (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride; OTC, over the counter; PAC-14028, (E)-N-((R)-1-(3,5- difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide; PACS, phosphofurin acid cluster sorting protein; PAR-2, proteinase-activated receptor-2; PARP-1, poly(ADP-ribose)polymerase-1; PBMC, 1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl) carbamate; PD-G, parkinsonism- complex of Guam; PGC1a, peroxisome proliferator–activated receptor-g coactivator 1a;PGE2, E2; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, proteinkinaseA;PKC,proteinkinaseC;PKD,polycystickidney disease; PKG, protein kinase G; PLA2, phospholipase A2;PLC,phospholipaseC;PNH,postherpeticneuralgia;PPARg, peroxisome proliferator–activated receptor-g; PSA, prostate-specific antigen; PTH, parathormone; PUFA, polyunsaturated fatty acid; Pyr3, pyrazole compound-3; QX-314, N-(2,6- dimethylphenylcarbamoylmethyl)triethylammonium bromide; RN1734, 2,4-dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide; ROS, reactive species; RT-PCR, reverse-transcription polymerase chain reaction; RTX, ; SA, sinoatrial; SAC, stretch-activated ion channel; SB366791, N-(3-methoxyphenyl)-4-chlorocinnamide; SB705498, 1-(2-bromophenyl)-3-[(3R)-1-[5-(trifluoromethyl)pyridin-2-yl]pyrrolidin-3-yl] urea; SCID, severe combined immunodeficiency; SHR, sensory airway hyper-reactivity syndrome; shRNA, short hairpin RNA; siRNA, small interfering RNA; SK&F-96356, 1H-pyrrolo[3,2-c]quinolin-4-amine,2,3-dihydro-N,6-dimethyl-1-(2-methylphenyl); SNP, single nucleotide polymor- phism; SP, ; SQCC, squamous cell carcinoma; SR140333, (S)1-2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl-4- phenyl-l azonia-bicyclo[2.2.2]octane; Sur1, sulfonylurea receptor 1; T1DM, type 1 diabetes mellitus; T2DM, mellitus; TCS-5861528, 2-(1,3- dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-[4-(1-methylpropyl)phenyl]acetamide; TGF, transforming growth factor; TNBS, trinitrobenzene sulfonic acid; TNF, ; TPC, two-pore channel; TRP, transient receptor potential; TRPA, ankyrin transient receptor potential; TRPC, canonical transient receptor potential; TRPM, melastatin transient receptor potential; TRPML, mucolipin transient receptor potential; TRPP, polycystic transient receptor potential; TRPV, vanilloid transient receptor potential; TSLP, thymic stromal lymphopoietin; UCP, uncoupling protein; UVB, ultraviolet B; UVR, ultraviolet radiation; Va, varitint-waddler; VCAM-1, vascular cell adhesion -1; VEGF, vascular endothelial growth factor; WAT, white adipose tissue; WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone. 678 Nilius and Szallasi

7. Transient Receptor Potential Channels and Pruritus ...... 740 B. Transient Receptor Potential Channels in Airway Disorders ...... 744 1. What Should We Target in Patients with Chronic Cough: Ankyrin Transient Receptor Potential 1, Vanilloid Transient Receptor Potential 1, or Both?...... 744 a. Vanilloid transient receptor potential 1...... 745 b. Ankyrin transient receptor potential 1...... 747 2. Transient Receptor Potential Channels Other than Vanilloid Transient Receptor Potential 1 and Ankyrin Transient Receptor Potential 1 in Airway Structural and Inflammatory Cells: Implications for Therapy ...... 750 C. Transient Receptor Potential Channels as Therapeutic Targets in Bladder Disorders...... 752 1. Vanilloid Transient Receptor Potential 1 and Vanilloid Transient Receptor Potential 4. . . . 752 2. Melastatin Transient Receptor Potential 8 and Ankyrin Transient Receptor Potential 1. . . 754 3. CanonicalTransientReceptorPotential1 andCanonicalTransientReceptorPotential4....755 D. Transient Receptor Potential Channels in Cardiovascular Disorders ...... 755 1. Transient Receptor Potential Channels and Heart Disease ...... 755 2. Transient Receptor Potential Channels and Hypertension ...... 757 3. Transient Receptor Potential Channels and Atherosclerosis ...... 758 4. Transient Receptor Potential Channels and ...... 760 E. Transient Receptor Potential Channels in Obesity and Metabolic Disorders ...... 760 1. Therapeutic Intervention for Appetite Control and Weight Loss: Central versus Peripheral Transient Receptor Potential Targets ...... 761 a. Vanilloid transient receptor potential 1...... 761 b. Vanilloid transient receptor potential 4...... 763 c. Ankyrin transient receptor potential 1...... 764 d. Melastatin transient receptor potential 5...... 765 e. Melastatin transient receptor potential 8...... 766 f. Canonical Transient Receptor Potentials...... 766 2. Obesity as a Low-Grade Inflammatory Disease: Implications for Transient Receptor Potential Channel Targeting ...... 766 3. Targeting Transient Receptor Potential Channels in Metabolic Disorders and Diabetes. . . . 766 4. Targeting Transient Receptor Potential Channels in Sensory Afferents in the Pharmacotherapy of Diabetes ...... 766 a. A role for vanilloid transient receptor potential 1 in autoimmune diabetes...... 767 b. Vanilloid transient receptor potential 1 and type 2 diabetes mellitus: is type 2 diabetes mellitus a low-grade inflammatory disorder? ...... 768 5. Targeting Transient Receptor Potential Channels in b Cells for Controlling Insulin Release...... 769 a. Melastatin transient receptor potential 2...... 770 b. Melastatin transient receptor potential 4...... 770 c. Melastatin transient receptor potential 5 and melastatin transient receptor potential 6...... 770 d. Melastatin transient receptor potential 3, vanilloid transient receptor potential 2, vanilloid transient receptor potential 4, and ankyrint transient receptor potential 1...... 771 F. Transient Receptor Potential Channels in the Brain as Novel Targets for Neurologic and Psychiatric Disorders ...... 771 1. Vanilloid Transient Receptor Potential 1 and Canonical Transient Receptor Potential 5 in Disorders ...... 772 2. Canonical Transient Receptor Potential 3 and Cerebellar Ataxia ...... 773 3. Melastatin Transient Receptor Potential 2, Melastatin Transient Receptor Potential 7, and Canonical Transient Receptor Potential 3: PotentialTargetsinStrokePatients...... 774 4. Transient Receptor Potential Channels in Neurodegenerative Disorders and Mental Retardation ...... 774 5. Transient Receptor Potential Channels and Neuroregeneration...... 775 G. Transient Receptor Potential Channels in Cancer ...... 775 1. Glioblastoma Multiforme ...... 776 2. Breast Cancer...... 777 3. Prostatic Adenocarcinoma ...... 778 TRP Channels as Drug Targets 679

4. Ovarian, Pancreatic, and Gastric Adenocarcinoma ...... 779 5. Melanoma and Nonmelanoma Skin Cancers ...... 779 H. Transient Receptor Potential Channels as Therapeutic Targets in Dermatology ...... 780 I. Transient Receptor Potential Channels and Eye Diseases ...... 783 IV. Conclusions ...... 783 Suggested Reading: Books on TRP Channels: ...... 785 Acknowledgments ...... 786 References ...... 786

Abstract——The large Trp gene family encodes tran- TRP channels, the so-called “TRP channelopathies,” are sient receptor potential (TRP) that form novel responsible for a number of hereditary diseases that cation-selective ion channels. In mammals, 28 Trp affect the musculoskeletal, cardiovascular, genitourinary, channel have been identified. TRP proteins and nervous systems. This review gives an overview of exhibit diverse permeation and gating properties and the functional properties of mammalian TRP channels, are involved in a plethora of physiologic functions describes their roles in acquired and hereditary diseases, with a strong impact on cellular sensing and signaling and discusses their potential as drug targets for pathways. Indeed, in human genes encoding therapeutic intervention.

I. Transient Receptor Potential Channels: A cell depolarization that can result in activation or Brief Introduction inactivation of voltage-dependent ion channels and modulate the driving force of ion flux through channels In 1969, a Drosophila mutant was discovered that and transporters. TRPs can also function as intracel- was defective in light sensing and exhibited only lular ion channels, mainly as Ca2+ release channels, in transient light-induced receptor potentials (TRPs) in- several cell organelles such as , endosomes, stead of the normal maintained response (Cosens and the Golgi network, the endoplasmic reticulum, and Manning, 1969). This finding was explained by a defect in an ion channel and triggered the discovery of the synaptic vesicles (Fig. 3) (Gees et al., 2010). A functional — large gene family baptized “trp genes” that encode TRP subset of TRP channels vanilloid TRP channels TRPV1, channels (for the history of this discovery, see Minke, TRPV2, TRPV3, and TRPV4; ankyrin TRP channel 2010; Hardie, 2011; Montell, 2011). By the latest count, TRPA1; melastatin TRP channel TRPM8; and canonical — the TRP channel superfamily contains 28 mammalian TRP channel TRPC5 is distinguished by their sensi- members (27 in ; see Fig. 1 for the TRP “family tivity to temperature (reviewed in Szallasi et al., 2007; tree”) and is subdivided into six subfamilies, all of which Voets, 2012). In combination, these channels (collec- permeate cations (reviewed in Clapham et al., 2001; tively referred to as thermoTRPs) cover a broad range of Clapham, 2003; Nilius et al., 2007b; Wu et al., 2010; temperatures, ranging from noxiously hot to danger- Nilius and Owsianik, 2011, Gees et al., 2012). ously cold. As known thus far, all TRP channels share some TRP channels are polymodal channels, that is, they structural similarities (similarities and differences are can be activated by a plethora of physical (voltage, shown in Fig. 2). They contain six transmembrane temperature, force, pressure, and tension) and chem- spanning regions (S1–S6), they have a pore-forming loop ical (both endogenous and exogenous) stimuli (see between the fifth (S5) and sixth (S6) regions, their Nieto-Posadas et al., 2011; Jara-Oseguera and Islas, C termini and N termini are intracellular, and they 2013). The mode of TRP channel activation by these probably function mostly as heterotetramers or homote- stimuli at the molecular level is, however, still poorly tramers (Gaudet, 2008, 2009; Wu et al., 2010; for more understood. “Chemical” activation of TRP channels by detailed information, please see the International Union both endogenous and exogenous compounds will be of Basic and Clinical Pharmacology TRP Channel Data- reviewed in the particular channel chapters. There is base (www.iuphar-db.org/index.jsp). an ongoing controversy about the direct “voltage The function of TRP channels is critically regulated activation” (or “modulation by voltage”) of TRP chan- by a variety of interacting and/or associated proteins, nels such as TRPC5, TRPV1, TRPV3, TRPM3, TRPM8, some of which are described in this work. For more TRPA1, and polycystic TRP channel TRPP3 (and prob- detailed information, interested readers are referred to ably others as well). As opposed to voltage-gated the excellent database on Mammalian Transient Receptor channels that only changes in the transmem- Potential Channel-Interacting Protein (www.trpchannel. brane electrical field, TRP channels are polymodal org). (i.e., integrators of various cellular signals), a property TRP channels are nonselective cation channels that renders them coincidence detectors (Ramsey et al., located in the plasma membrane. Some function as 2006; Nieto-Posadas et al., 2011). The role of TRP Ca2+ entry channels. Upon activation, they generate channels in temperature sensing is well established 680 Nilius and Szallasi

Fig. 1. The phylogenic tree of human TRP channels. On the basis of , all TRP channels fall into seven subfamilies that comprise proteins with distinct channel properties. Note that TRPN1 is only present in lower vertebrates (e.g., zebrafish); TRPC2 is a pseudogene in humans. The bar (0.2) indicates point accepted units, which is the evolutionary distance between two amino acids (1 point accepted mutation unit = 1 point mutation event per 100 amino acids, which is accepted and is passed to progeny). Reprinted with permission from Nilius and Owsianik (2011).

(Baez-Nieto et al., 2011). However, the list of ther- proteins TMC1 and TMC2 (Pan et al., 2013). Although moTRPs is expected to shrink/change in the near future both TRPV4 and TRPA1 have been implicated in the because a number of postulated temperature-sensitive development and maintenance of mechanical hyper- TRP channels (including TRPV2, TRPV3, TRPM4, and algesia during inflammation, their roles in physiologic TRPM5) are probably not functionally involved in ther- mechanosensing remain to be established. Importantly, mosensing. The mechanisms by which TRP channels none of the TRP channels suggested to function as SACs detect changes in temperature are not (for critical in free nerve endings and/or Merkel cell-neurite com- reviews, see Latorre, 2009; Baez-Nieto et al., 2011; Voets, plexes can be directly activated by pressure under 2012; Jara-Oseguera and Islas, 2013). experimental conditions (e.g., fast-pressure clamps Controversy remains with regard to the function of system) that are used to record SAC-like activity. Other TRP channels as direct mechanosensors, especially in putative mechanosensitive TRP channels (e.g., TRPA1, mammals (see Pedersen and Nilius, 2007). Although TRPC1, and TRPC6) could not be activated by mem- TRPN1 (a member of the no mechanoreceptor potential brane stretch in expression systems. It is clear that the C family) is clearly part of the pore-forming a-subunit gamut of the evidence (extensively discussed in Chris- of the native mechanoactivated or stretch-activated ion tensen and Corey, 2007; Nilius and Honoré, 2012; channels (SACs) in ciliated sensory neurons of the Pedersen and Nilius, 2007; Roudaut et al., 2012; Delmas worm and the Johnston organ and Coste, 2013; Eijkelkamp et al., 2013) does not sup- of the fruit fly (together with port a direct role for TRP channels in mechanosensing. the TRP channels Nanchung and Inactive) (reviewed in Indeed, TRPV4 is most likely indirectly force-regulated Wilson and Corey, 2010), the situation in mammals via a mechanosensing phospholipase A2 (PLA2). Possi- remains controversial. For example, the mechanosen- ble exceptions include TRPC1 as a sensor for light touch sitive transducer channel in hair cells of the organ of sensation in cutaneous low-threshold mechanosensory Corti is clearly not TRPA1 (as was originally suggested neurons (Garrison et al., 2012) and TRPA1 as a slowly by Corey et al., 2004) but is probably the newly dis- adapting SAC in sensory neurons (discussed later covered TRP-unrelated transmembrane channel-like in this work). A proteomic network analysis of TRP TRP Channels as Drug Targets 681

Fig. 2. Similarities and differences between the topologic structure of selected TRP channels, representing the ankyrin (TPA1), canonical (TRPC3), melastatin (TRPM2), and vanilloid (TRPV1) subfamilies. TRPA1 is characterized by the unusually high number of ankyrin repeats (after which it was named) at its N terminus, and it lacks the TRP box at its C terminus that is present in the TRPC, TRPV, and TRPM subfamilies. TRPC3 interacts with the scaffolding protein caveolin at its N terminus (that may directly link TRPC3 to the a-subunit of GPCRs); on the C terminus, it has recognition sites for CaM and the IP3 receptor. This binding domain is also referred to as the CaM/IP3R binding site (CIRB). TRPC3 has also been shown to interact with immunophilins (endogenous cytosolic peptidyl-propyl isomerases) as part of the signalplex. TRPM2 possesses four TRPM homology regions (MHR1– MHR4) at its N terminus. At its C terminus, TRPM2 has a nucleoside diphosphate-like moiety X (NUDIX) domain that displays ADPR activity. The ankyrin repeats at the N terminus of TRPV1 are linked to the association of the channel with PI3K, ATP, and CaM. The C terminus carries CaM and PIP2 recognition sites. For further details on TRPV1 regulation, also see Fig. 4. Courtesy of Dr. Rachelle Gaudet (Harvard University). channels may resolve some of the above-described This review first describes the main properties of all controversies by providing novel insights into the TRP channels (Nilius and Owsianik, 2011; Zheng, 2013). functional impact of putative TRP channel b-subunits We then highlight their role in hereditary channelopa- and associated proteins. thies (Nilius and Owsianik, 2010b) and acquired diseases.

Fig. 3. Early endosomes (EEs) derive endocytotic vesicles (EVs) from the plasma membrane. Recycling endosomes (REs) and the late endocytotic pathway via late endosomes (LEs) to the lysosomes (LYs) are indicated. Intermediate transport vesicles (TVs) bud from the endoplasmic reticulum (ER) and trans-Golgi network (TGN). Secretory vesicles (SVs), secretory granules (SGs), and synaptic vesicles (SyVs) are derived from early endosomes. Intracellular locations of TRP channels are indicated by the channel number. Reprinted with permission from Gees et al. (2010). 682 Nilius and Szallasi

Finally, we summarize the current state of the field of caveolae adapted to the scaffolding protein, caveolin-1. and developmental efforts directed at TRP These connections are important for focal adhesions, cell channels (see also Nilius, 2007; Nilius et al., 2007b; migration, and actin stress fiber formation (Ong and Moran et al., 2011; Kaneko and Szallasi, 2013). Ambudkar, 2011; Stiber et al., 2012). Members of the closely related TRPC3/TRPC6/TRPC7 group of channels A. Canonical Transient Receptor Potential Subfamily can be activated by DAG, independent of the stimulation The canonical TRP (TRPC) subfamily was the first of protein kinase C (PKC), suggesting that DAG is the trp gene family to be cloned in mammals (Wes et al., PLC-derived product that mediates the physiologic 1995; Zhu et al., 1995). It contains seven mammalian activation. By contrast, the TRPC1/TRPC4/TRPC5 group members (of note, in humans is only a pseudo- of channels, which are also activated by receptor-induced gene). As a general remark, activation of these channels PLC, are unresponsive to DAG (Venkatachalam is not well understood. Inserted in the plasma mem- et al., 2003). brane, most of the TRPCs are spontaneously active. All The activation mechanism of TRPCs via PLC TRPC channels contain a conserved TRP box, EWKFAR, stimulation is still not completely resolved. All TRPCs in their C-terminal tail and three to four N-terminal exhibit overlapping binding sites for, and interact to ankyrin repeats. All TRPC channels are nonselective a varying extent with, calmodulin (CaM) and the 2+ cation channels and are permeable for Ca . On the basis inositol-1,4,5-trisphosphate (IP3) receptor [the binding of sequence homology and similarities in function, site is referred to as the CaM/IP3R binding site; Fig. 2] TRPCs fall into four groups: TRPC1, TRPC2, TRPC3/ (Trost et al., 2001; Zhu, 2005). Most of the TRPCs are TRPC6/TRPC7, and TRPC4/TRPC5 (Vazquez et al., inhibited by a Ca2+/CaM-dependent mechanism and 2004). The pharmacological characterization of these removal of this inhibition may lead to activation. TRPC channels is hindered by the lack of subtype-specific channels have been reportedly involved in the control and antagonists (Bon and Beech, 2013). of smooth muscle activity (Albert et al., 2009), en- In general, G protein–coupled receptors (GPCRs) dothelial cell function (Morita et al., 2011), regulation play a central role in the regulation of TRPC channels. of the -brain barrier (Brown et al., 2008), control Indeed, TRPCs may be one of the most important of the cell cycle (Madsen et al., 2012), and various downstream targets for GPCRs. In many instances, sig- functions in the brain such as growth cone guidance, nals from GPCRs to TRPCs are mediated via lipid release, synaptic plasticity [long- signals (Kukkonen, 2011). For example, TRPC3, TRPC6, term depression (LTD) and long-term potentiation and TRPC7 are mostly activated by members of the Gq/11 (LTP)], and brain development (Tai et al., 2009; Henle family, which are coupled downstream to phospholipase et al., 2011; Hutchins et al., 2011; Amaral and Pozzo- C(PLC)-b. However, for TRPC4/TRPC5, Gai/o (rather Miller, 2012; Chae et al., 2012). Furthermore, TRPCs than Gq/11) family members are the dominant activators. are involved in the regulation of neurosecretion in MostoftheTRPCsarealsoactivatedviaaPLCg gonadotropin-releasing hormone neurons by stimulat- pathway, downstream of several tyrosine kinase recep- ing peptide release and activating the reproductive tors such as the fibroblast growth factor receptor-1 and axis in mammals (Zhang et al., 2008a). In this context, the brain-derived neurotrophic factor (BDNF). Others kisspeptin, a GPCR (GPR54, also known as Kiss1R), comprise the cytokine receptors morphogenic pro- dominates the excitability of gonadotropin-releasing hor- tein receptor type II and tumor necrosis factor (TNF) mone neurons (Rønnekleiv and Kelly, 2013). This pathway receptor superfamily member 1a,whicharecoupledto seems to be generally involved in the regulation of synaptic the small body size-mothers against decaplentaplegic plasticity and neurotransmitter release. family of proteins that transduce extracellular signals TRPC1, the first mammalian Trp gene, was cloned in from the transforming growth factor (TGF) receptor to 1995 and 1996 (Wes et al., 1995; Zhu et al., 1995, 1996; the nucleus (Zhang et al., 2013a). Further details are Zitt et al., 1996). TRPC1 was defined as a Ca2+-permeable outsidethescopeofthisreview. ion channel. It is widely expressed in heart, brain, A hallmark of TRPC channels is their differential lung, , spleen, kidney, testes, maybe all epithelial modulation by lipids, such as diacylglycerol (DAG), phos- cells, smooth muscle cells, endocrine pituitary cells, phatidyl inositols, lysophospholipids, oxidized phospho- and glial cells (Wang et al., 1999; Strübing et al., 2001). lipids, sphingosine phosphates, and gangliosides, which There are conflicting reports of whether TRPC1 homo- either change channel activity or influence TRPC mers can form functional channels (i.e., whether TRPC1 insertion in the plasma membrane. It is noteworthy is really a pore-forming a-subunit; Strübing et al., 2001; that these lipids are coupled to several proteins, such as Storch et al., 2012). The finding that overexpressed caveolins, GPCRs, and the phospholipid-binding proteins, homomeric TRPC1 channels were described as stretch such as the Saccharomyces cerevisiae 14-like (SEC14) and activated by some (Maroto et al., 2005) but not others spectrin domain 1 (SESTD1) proteins (Beech, 2012). (Gottlieb et al., 2008; Sharif-Naeini et al., 2008) is also TRPCs can be coupled with the cytoskeleton via Homer puzzling. By contrast, there is no doubt that TRPC1 can scaffolds with actin or localized in channelosomes with form functional heteromers with other TRPC channels TRP Channels as Drug Targets 683

(Strübing et al., 2001; Storch et al., 2012), with TRPV4 (Vazquez et al., 2004); therefore, it will not be discussed (Ma et al., 2010, 2011), and probably also with ORAI1 (a here. Ca2+ release-activated channel protein), form- TRPC3 is closely related to TRPC6 and TRPC7, with ing store-operated channels (Ong et al., 2007; Cioffi a sequence identity of approximately 75% (topologic et al., 2010, 2012). In microvessels, TRPC1 together structure shown in Fig. 2). The CaM/IP3R binding site with TRPP1 [polycystic kidney disease (PKD)-2, also region of TRPC3 exhibits higher CaM affinity than referred to as PC2] controls the blood-brain barrier seen in other TRPCs (Wedel et al., 2003; Zhu and Tang, (Berrout et al., 2012). It has been reported that TRPC1 2004). TRPC3 can form homomeric channels but also may play a role in smooth muscle contraction (Xu and heteromerizes with TRPC1/TRPC4/TRPC5/TRPC6/ Beech, 2001), osteoclast formation (Ong et al., 2013), TRPC7 in some cell types (Strübing et al., 2003; Dietrich and control of cell migration (Fabian et al., 2008) and is et al., 2005a). TRPC3 is highly expressed in endothelial involved in BDNF- and netrin-1–induced axon guidance cells (in caveolae) (Adebiyi et al., 2011; Senadheera et al., (Wang and Poo, 2005; for review, see Nilius et al., 2012), in the brain (e.g., cerebellum, cortex, substantia 2007b; Gees et al., 2012). Adding to this complexity, the nigra, and hippocampus) both in neurons and glia (Huang TRPC1 gene has multiple variants. Isoform- et al., 2007; Roedding et al., 2009; Li et al., 2010; specific (a and b,encodinglongandshortTRPC1 Mitsumura et al., 2011; Zhou and Lee, 2011), in the isoforms, respectively) TRPC1/TRPC4 heteromultimers pituitary gland, smooth muscle, and cardiac muscle cells were reported to have distinct activation mechanisms (Watanabe et al., 2008; Gonzalez-Cobos and Trebak, and gating properties (Kim et al., 2014a). 2010), as well as in sensory organs such as the cochlea A unique TRPC1 isoform (TRPC1«) was recently (Tadros et al., 2010). identified in preosteoclasts (Ong et al., 2013). Osteoclasts TRPC3 is constitutively active and is regulated by are formed from a subpopulation of circulating mono- monoglycosylation (Dietrich et al., 2003). TRPC3 is nuclear cells that are recruited from the blood to the bone directly activated by both DAG and DAG analogs surface, where they undergo differentiation. I-mfa–null (Hofmann et al., 1999). It is noteworthy that a very mice have an osteopenic phenotype characterized by selective inhibitor for TRPC3, pyrazole compound-3 increased osteoclast activity (Kraut et al., 1998). This (Pyr3) was recently identified (Kiyonaka et al., 2009). phenotype is normalized in the I-mfa/TRPC1 double- PKC has an inhibitory effect on TRPC3 currents mutant animals (Ong et al., 2013). The authors spec- (Venkatachalam et al., 2003). TRPC3 activation is ulated that osteoclastogenesis is fine-tuned by I-mfa coupled to purine receptors both in endothelial and (positive modulator) and TRPC1« (negative modulator). smooth muscle cells (Kamouchi et al., 1999; Reading If this hypothesis holds true, TRPC1« activators may be et al., 2005). In neurons, TRPC3 is activated by BDNF useful in preventing (or even reversing) bone loss. and is possibly associated with the BDNF receptor It is noteworthy that TRPC1 is also associated with TrK-B. This link appears to be essential for the guidance the b-glucuronidase , a fundamental regulator of of nerve growth cones by BDNF (Li et al., 2005b; Amaral aging. The vascular endothelial growth factor (VEGF) and Pozzo-Miller, 2007, 2012). receptor-2/TRPC1/Klotho complex may cause cointer- In the cerebellum, TRPC3 has been identified as an nalization of these proteins and regulate TRPC1-mediated essential player in LTD (Kim, 2013). In T lymphocytes, Ca2+ entry to maintain endothelial integrity (Kusaba TRPC3 is activated by T-cell receptor–triggered immune et al., 2010). Leptins through a Janus kinase-2/ responses and is probably directly coupled to PLCg1/g2, phosphoinositide 3-kinase (PI3K)/PLCg pathway can which mediates translocation of the channel (Philipp also activate TRPC1. Indeed, TRPC1 is a key channel et al., 2003; van Rossum et al., 2005). TRPC3 is inhibited mediating the depolarizing effects of leptins in hypotha- by cGMP/protein kinase G (PKG); in endothelium, it pro- lamic proopiomelanocortin neurons (Qiu et al., 2010). vides a negative feedback mechanism for the generation TRPC1 also mediates glutamate-induced neuronal death of nitric oxide (NO) (Yao and Garland, 2005). In vascular and may represent a promising target for pharmacolog- smooth muscle cells, this mechanism may induce re- ical intervention to prevent or at least reduce glutamate- laxation (Chen et al., 2009a). In endothelial cells, vascular induced neuronal damage (Narayanan et al., 2008). cell adhesion molecule-1 (VCAM-1) expression and mono- Somewhat unexpectedly, evaluation of channel cyte adhesion depend on TRPC3 (Smedlund et al., 2010). functions in Trpc1 knockout mice has not revealed Cardiac atrial natriuretic peptide binds to the trans- any dramatic defects. For example, no changes in membrane guanylyl cyclase and stimulates a unique vascular responses to pressure and/or synaptic plas- cGMP-independent pathway, including TRPC3 activa- ticity were detected (Freichel et al., 2004). Trpc1 tion, which might be involved in cardiac hypertrophy knockout mice, however, do have a slightly increased (Klaiber et al., 2011). In this context, it is interesting that body weight and impaired salivary fluid secretion (Liu TRPC3 is involved in mechanosensing, probably via the et al., 2007). mechanotransducer zyxin (Suresh Babu et al., 2012). TRPC2 is not expressed in humans, where it is a The genes encoding small C-terminal domain phos- pseudogene, although it is present in all other mammals phatases, which are highly expressed in the heart, 684 Nilius and Szallasi contain microRNAs (e.g., miR-26) that negatively reg- and the spectrin cytoskeleton (Odell et al., 2008). The two ulate TRPC3 expression (Sowa et al., 2012). The splice variants of TRPC4 are differentially regulated. nonreceptor tyrosine kinase Src has a stimulatory TRPC4a and the shorter TRPC4b, which lacks 84 amino effect on TRPC3 (Vazquez et al., 2004). This channel is acids in the cytosolic C terminus, are highly expressed also activated via stimulation of metabotropic gluta- in smooth muscle and endothelial cells. Both TRPC4 mate receptors (mGluRs), which is dependent on variants are activated by Gq/PLC-coupled receptors. phospholipase D but not PLC (Glitsch, 2010). PLCg Importantly, TRPC4a, but not TRPC4b,wasstrongly contains a so-called split pleckstrin-homology domain. inhibited by intracellularly applied phosphatidylino- The C-terminal half of this split pleckstrin-homology sitol 4,5-bisphosphate (PIP2), which binds to the domain has been proposed to interact with an C terminus of TRPC4a. This inhibitory action was de- N-terminal site of TRPC3. This direct interaction may pendent on the association of TRPC4a with actin cy- cause an increased surface expression of TRPC3 upon toskeleton that binds to the PDZ domain. PIP2 breakdown PLCg activation (van Rossum et al., 2005). is a required step in activation of TRPC4a but PIP2 Moreover, TRPC3 is involved in the control of the depletion alone is not sufficient for channel opening, which erythropoietin-dependent survival, proliferation, and additionally requires Ca2+ and pertussis toxin–sensitive differentiation of mammalian erythroid progenitors Gai-proteins. TRPC4a is also strongly voltage dependent (Hirschler-Laszkiewicz et al., 2011). TRPC3 contrib- (Jeon et al., 2008; Otsuguro et al., 2008). Moreover, utes to the constitutive Ca2+ influx in macrophages TRPC4 channels can be directly activated by the trivalent that is required for macrophage survival (Tano et al., cations La3+ and Gd3+ (Schaefer et al., 2000), indepen- 2011). TRPC3 is also involved in the immunoreceptor dently on interference with the Ca2+ sensor. activation in mast cells (Cohen et al., 2009). Orexin Similar to TRPC1, TRPC4 is believed to participate (hypocretin), a neurotransmitter that regulates arousal, in store-operated Ca2+ entry in pulmonary artery en- wakefulness,andappetite,isabletoactivateTRPC3 dothelial cells, leading to the formation of gaps between via orexin type 1 receptor (Peltonen et al., 2009; endothelial cells and subsequent endothelial barrier Louhivuori et al., 2010). Activation of mGluR1 in the disruption. The large molecular weight immunophilin cerebellum initiates slow excitatory postsynaptic FK506-binding proteins FKBP51 and FKBP52 associate potentials (EPSPs), which are mediated by TRPC3 with the store-operated Ca2+ entry/TRPC4 channel pro- (Hartmann et al., 2008, 2011). Finally, it was recently tein complex (Kadeba et al., 2013). From the phenotype suggested that a TRPC3/TRPC6 complex may form of Trpc4 knockout mice, it is deduced that this channel (at least part of) the mechanotransduction chan- can be activated via muscarinic receptors and is essential nel in cochlear hair cells (Quick et al., 2012). Sur- for endothelium-dependent vasorelaxation (Freichel prisingly, Trpc3 knockout mice did not show the et al., 2001) and regulation of the endothelial barrier expected alterations in brain development (Hartmann function (Tiruppathi et al., 2006). et al., 2008). TRPC5 shares approximately 64% sequence identity TRPC4 is a homolog of TRPC5. It contains a PDZ- with TRPC4. The TRPC5 protein also contains a PDZ- binding motif in its C-terminal tail that binds PDZ binding motif in the C terminus that interacts with the domain scaffolding proteins. PLCb1 and PLCb2 asso- Na+/H+ exchanger regulatory factor (NHERF), an adapter ciate with TRPC4 via this domain (Tang et al., 2000). protein involved in signal transduction (Obukhov and TRPC4 can form functional homomultimers, as well as Nowycky, 2004). TRPC5 forms homomultimers or hetero- heteromultimers with TRPC1, TRPC3, TRPC5, and merizes with TRPC1, TRPC3, TRPC4, and TRPC6. TRPC6 (Strübing et al., 2001, 2003). Similar to other TRPC1/TRPC5 is the major heteromer in the brain TRPC channels, TRPC4 is widely expressed, including (Strübing et al., 2003). TRPC5 is widely expressed, es- in endothelium, smooth muscle cells, brain (geniculate pecially in the brain (hippocampus, cortex, cerebellum, nucleus, hippocampus, dentate gyrus), the kidney, substantia nigra, entorhinal cortex, amygdala, the pre- keratinocytes (in the skin and gingiva), as well as in Bötzinger complex, and the olfactory bulb) (De March interstitial cells of Cajal (reviewed in Pedersen et al., et al., 2006; Fowler et al., 2007; Yan et al., 2009), but also 2005). Homotetrameric TRPC4 and heterotetrameric intheheart,liver,lung,spleen, testis, and uterus (Philipp TRPC1/TRPC4 channels are activated after activation et al., 1998; Beech, 2007). TRPC5 shows considerable of PLC-coupled receptors but the mechanisms that link similarities to TRPC4 in its gating properties. Homote- receptor activation to channel gating are still controver- trameric TRPC5 and heterotetrameric TRPC1/TRPC5 sial (Hofmann et al., 1999; Schaefer et al., 2000). TRPC4 channels are activated via PLC-coupled receptors, but the is associated with PKG-phosphorylated vasodilator- mechanism underlying this link is not yet clear (Beech, stimulated phosphoprotein, which inhibits the channel 2007). The muscarinic receptor/Gaq-11/PLCb1cascade (Wang et al., 2007). Tyrosine phosphorylation by epider- seems to play a crucial role in the activation of brain mal growth factor receptor (EGFR) activation is critical TRPC5 (Yan et al., 2009). TRPC5 is modulated by lipids for plasma membrane translocation and activation of (e.g., sphingosine-1-phosphate) and, in general, it TRPC4; this requires a direct interaction between TRPC4 bipolar phospholipids (AL-Shawaf et al., 2011). TRP Channels as Drug Targets 685

TRPC5 is modulated by covalent modification of have interesting and far-reaching implications in arterial reactive cysteines and functions as a redox sensor. It is pressure sensing and mechanotransduction (Jemal et al., activated by extracellular application of the endoge- 2013). nous redox protein, thioredoxin (Xu et al., 2008), by From the phenotype of Trpc5 knockout mice it can be endogenous (Yamamoto et al., 2010; deduced that this channel is involved in fear-related Naylor et al., 2011), oxidized phospholipids (Al-Shawaf behavior (reviewed in Kaczmarek, 2010). The TRPC5- et al., 2010), and NO (Yoshida et al., 2006). As is the null mouse shows no apparent anatomic or develop- case for all TRP channels, currents through TRPC5 are mental defects. However, a reduction in responses modulated by plasma membrane insertion or retrieval mediated by synaptic activation of group I metabo- of the channels. Plasma membrane insertion of TRPC5 tropic glutamate and cholecystokinin (CCK)-2 recep- can be promoted by epidermal growth factor (EGF). tors in amygdala neurons was present in the Trpc5 This effect requires the presence of Rho GTPase, Rac1, knockout animals (Riccio et al., 2009). This nucleus is PI3K, and phosphatidylinositol 4-phosphate 5-kinase involved in fear reactions, which are diminished in the (Bezzerides et al., 2004). Protein kinase A (PKA) knockout mouse (Riccio et al., 2009). inhibits TRPC5 via Ga proteins (Sung et al., 2011). TRPC6 shares a sequence identity of approximately TRPC5 is also inhibited by neuroactive (Majeed 75% with TRPC3 and TRPC7. It likely has four et al., 2011). PIP2 is involved in channel regulation. Its ankyrin repeats, which interact with MxA (a member breakdown is required for desensitization of TRPC5 of the dynamin protein family), and has two glycosyl- current, but PIP2 depletion alone is insufficient for ation sites, which control channel activity and traffick- channel desensitization (Kim et al., 2008a). Extracellular ing (Dietrich and Gudermann, 2007). TRPC6 can form lanthanides (at low concentrations), Gd3+, lead, increased either homomultimers or heteromultimers with TRPC3 2+ [Ca ]i, and also , directly activate TRPC5 (Jung and TRPC7 (Dietrich et al., 2005a). TRPC6 is widely et al., 2003; Zeng et al., 2004; Semtner et al., 2007; Blair expressed, especially in smooth muscle cells, in lung, et al., 2009; Sukumar and Beech, 2010). TRPC5 is in heart, brain, and kidney (glomerular podocytes), as well cross-talk with voltage-activated Ca2+ channels in as in blood and immune cells. TRPC6 expression has also several tissues (Gross et al., 2009). It is noteworthy been reported in the adrenal gland, bone, epididymis, that TRPC5 is also activated by cold; this observation intestine, ovary, pancreas, and prostate (see Dietrich and adds TRPC5 to the list of thermoTRPs (Zimmermann Gudermann, 2007). In contrast with TRPC3, TRPC6 is et al., 2011). (almost) inactive under unstimulated conditions, which The activation pattern of TRPC5 becomes complex might be attributed to the double glycosylation (Dietrich 2+ because of its striking Ca dependence (Blair et al., et al., 2003). TRPC6 is activated by Ga/q11-coupled 2009), and especially because of its voltage depen- GPCRs and forms the essential part of the vascular a1- dence. Indeed, TRPC5 I–V curves change during the adrenoreceptor–activated Ca2+-permeable cation channel activation-deactivation cycle, shifting between outwardly complex in smooth muscle (Inoue et al., 2001; Jung et al., rectifying and doubly rectifying shapes. Depolarization 2002b). Endotoxin lipopolysaccharide (LPS) activates activates the channel (Obukhov and Nowycky, 2008). It is TRPC6 in endothelial cells in a Toll-like receptor-4– noteworthy that TRPC5 is also a redox sensor: it senses dependent manner (Tauseef et al., 2012). oxidative stress and induces adaptation reactions. There is increasing evidence to suggest that TRPC6 Finally, TRPC5 is activated by cell swelling, which hints plays important functions in smooth muscle and en- to some mechanosensing properties (Gomis et al., 2008). dothelial cells. TRPC6 is directly activated by DAG and TRPC5 is reportedly involved in various cellular DAG analogs (Hofmann et al., 1999; Estacion et al., functions such as cell migration, contribution to the work- 2004). PKCd exerts an inhibitory effect on TRPC6 ing memory establishment, growth cone guidance and through direct phosphorylation (Bousquet et al., 2010). dendrite extension, cardiac pacemaking, and mitosis. Tyrosine kinases also activate TRPC6. This is thought TRPC5 is also involved in erythropoietin effects, motility to play an important role in VEGF-mediated angio- of gastrointestinal smooth muscle, and much more (Lee genesis that requires TRPC6 activation (Hamdollah et al., 2003c; Ju and Allen, 2007; Kaczmarek, 2010; Tian Zadeh et al., 2008; Ge et al., 2009). TRPC6 is activated et al., 2010; Liu et al., 2011b; Zhang et al., 2011b; by cAMP, possibly via the cAMP-PI3K-PKB-MEK- Kaczmarek et al., 2012). extracellular signal-regulated kinase (ERK) 1/ERK2 An intriguing new modulation mechanism describes cascade (Shen et al., 2011a). TRPC6 plays a key role the activation of TRPC5 by hypo-osmotic cell swelling in excitatory synaptic transmission, plasticity, and via activation of Gq-coupled receptors, which, in turn, neural development. It inhibits N-methyl-D-aspartate enhances the activation of TRPC5 by regulating the (NMDA)–induced currents probably via activation of membrane insertion of this channel. Because Gq-coupled calcineurin and functions as a negative modulator of receptors and TPRC5 are coexpressed in several tissues NMDA receptors (Shen et al., 2013). TRPC6 channels (e.g., vascular system, glia cells, and neurons), the Gq- can be negatively regulated by the NO/cGMP/PKG coupled receptor mechanism of TRPC5 activation may pathway (Takahashi et al., 2008; Koitabashi et al., 686 Nilius and Szallasi

2010). PI3K and its antagonistic phosphatase, phos- Morris water maze test, suggesting a role for the phatase and tensin homolog, are involved in the channel in synaptic and behavioral plasticity (Zhou activation of TRPC6 and promote its plasma mem- et al., 2008). The important role of TRPC6 in smooth brane insertion (Monet et al., 2012). Insertion is also muscle cells is supported by data from Trpc6 knockout promoted by Rab11, a member of the Ras superfamily mice, which unexpectedly exhibit a slightly elevated of monomeric G proteins (Cayouette et al., 2010). Of blood pressure associated with enhanced basal and note, TRPC6 couples to the Na+/Ca2+ exchanger and -induced cation entry. This effect is due to thus might contribute to any signaling mechanism a compensatory upregulation of TRPC3 and TRPC7 that depends on localized cytosolic subplasmalemmal (Dietrich et al., 2005b). Na+ elevation (Poburko et al., 2008). TRPC6 plays a unique and indispensable role in At least in endothelial cells, cAMP/PKA induces acute hypoxic pulmonary vasoconstriction (also known intracellular translocation of TRPC6 channels in as the Euler–Liljestrand mechanism), a local vasocon- caveolin-1–rich areas, which potentiate its activation striction in the lung that shifts blood flow from hypoxic (Fleming et al., 2007). The metabolite to normoxic areas, thereby maintaining effective gas 11,12- potentiates the contrac- exchange. Trpc6 knockout mice lack the hypoxic pulmo- tile responses in pulmonary arteries via TRPC6 activa- nary vasoconstriction reflex and develop arterial hypox- tion (Loot and Fleming, 2011). -activating factor emia during conditions of regional hypoventilation also activates TRPC6 in endothelial cells. On the basis (Weissmann et al., 2006). It is noteworthy that TRPC6 of these observations, it was suggested that TRPC6 is is expressed in podocytes, where it is required for involved in the control of the endothelial barrier. For establishing the glomerular filtration barrier (Reiser example, activation of TRPC6 disturbs the barrier et al., 2005). TRPC6 also plays an activating role in cell function and translocates it in caveolae of endothelial migration downstream of the CXC-type, Gq protein– cells (Samapati et al., 2012). coupled chemokine receptors and TRPC6 downregula- As shown now for many TRP channels, TRPC6 is tion strongly impairs the chemotaxis of neutrophils activated by reactive oxygen species (ROS; e.g., H2O2) (Damann et al., 2009; Lindemann et al., 2013). (Graham et al., 2010; Ding et al., 2011). TRPC6 activity TRPC7 occurs in two different splice variants. It shares is potentiated by flufenamate and the 20- an 81% and 75% overall identity with TRPC3 and TRPC6, hydroxyeicosatetraenoic acid (Inoue et al., 2001; Jung respectively. It forms homomultimers, but also heteromul- et al., 2002b). It is also is activated by thrombin and timers with TRPC1, TRPC3, and TRPC6, and is widely platelet-activating factor and plays an important role expressed in tissues from brain, eye, heart, intestine, in platelet activation (Hassock et al., 2002; Dionisio kidney, lung, and pituitary gland. It is constitutively et al., 2011, 2012). Remarkably, TRPC6 has been also active and can be further activated by GPCRs or DAG identified as a “candidate” mechanosensing TRP chan- analogs. It is negatively regulated by extracellular Ca2+ nel that is activated by stretch and may contribute to and PKC (see Numaga et al., 2007). TRPC7 is also ne- mechanosensing in the heart (Spassova et al., 2006; gatively regulated by the cGMP-regulated protein kinase I Dyachenko et al., 2009). pathway; that is, cGMP-regulated protein kinase I – (structure shown in Fig. 6), one of the mediated phosphorylation inhibits the channel (Yuasa main bioactive compounds that underlie the antide- et al., 2011). The functional role of TRPC7 is still not well pressant actions of the medicinal St. John’s wort understood and phenotypic alterations in Trpc7 knockout ( perforatum), is a potent TRPC6 activator mice have not yet been described. and promotes channel expression (Leuner et al., 2007; Griffith et al., 2010; Harteneck and Gollasch, 2010). B. Vanilloid Transient Receptor Potential Subfamily As with all TRPCs, TRPC6 is a nonselective cation The TRPV subfamily comprises six members that, channel with Ca2+ selectivity similar to its closest based on homology, fall into four groups: TRPV1/ homologs, TRPC3 and TRPC7. However, although TRPV2, TRPV3, TRPV4, and TRPV5/TRPV6. This TRPC6 is Ca2+ permeable, under physiologic condi- vanilloid subfamily was named after its founding tions TRPC6 induces depolarization and mainly Na+ member, the vanilloid () receptor VR1 enters the cell via this channel. It is only at very (Szallasi and Blumberg, 1999; Nilius and Owsianik, negative potentials that Ca2+ entry through TRPC6 2011). Members of the TRPV subfamily function as becomes significant (Estacion et al., 2006). TRPC6 is tetrameric complexes with each subunit containing also a Zn2+-permeable channel (Gibon et al., 2011). six N-terminal ankyrin repeats (Gaudet, 2008, 2009). With regard to its functional impact, TRPC6 has All members have a TRP box in their C terminus (Fig. 2). been identified as an important player in dendritic TRPV1, TRPV2, TRPV3, and TRPV4 are modestly growth, neuronal survival, and synapse formation (Jia permeable to Ca2+, whereas TRPV5 and TRPV6 are et al., 2007; Zhou et al., 2008). Strikingly, mice over- the only highly Ca2+ selective channels in the TRP expressing TRPC6 show enhanced spine formation and family. These latter channels are tightly regulated by 2+ improved spatial learning and memory formation in the [Ca ]i. TRP Channels as Drug Targets 687

Remarkably, TRPV1 has a dynamic pore, meaning capsaicin than rats or humans. Remarkably, point that it shows pore dilation during stimulation (Chung mutations in only two key residues (547 and 550; Fig. et al., 2008) (Fig. 4). TRPVs seem to be also permeable 4) are responsible for most of these differences in for large cations (Chung et al., 2008). The physiologic capsaicin potencies (Jordt and Julius, 2002; Gavva significance of this dilation is unclear. TRPV channels et al., 2004). show a complex pharmacology including very selective Most recently, the first high-resolution (3.4 Å) agonists for some TRPVs, such as capsaicin (structure structure of TRPV1 was described in the presence of shown in Fig. 5) for TRPV1, but mostly less selective vanillotoxin, double-knot toxin (from Psalmopoeus activators such as 2-aminoethoxydiphenyl borate (2- cambridgei), and resiniferatoxin (RTX) (Cao et al., APB; see Fig. 6 for structure) for TRPV3 and, to 2013a; Liao et al., 2013). As expected, S1–S4 bundles a lesser extent, also TRPV2 and TRPV1 (for a compre- are located in the periphery of the channel and are hensive review, see Vriens et al., 2009). rather “rigid” (which is unusual for a putative voltage blocks all TRPV channels with variable potencies (IC50 sensor). The four S5 to S6 bundles tetramerize and values within the range of 0.1–9.0 mM) (for a review, form the ion pore. Importantly, the TRPV1 pore features see Vennekens et al., 2008). two constrictions: a funnel-like extracellular pore forms TRPV1 has been cloned from different species the selectivity filter and functions as an upper gate, (including humans, guinea pigs, rabbits, mice, chick- whereas the lower gate is located in the middle of the S6 ens, and pigs), revealing marked species-related differ- helix. Both gates must be simultaneously open to allow ences in pharmacological profiles. It has long been ion permeation. Double-knot toxin opens the extracellu- known that birds (unlike rodents) are essentially lar pore, whereas RTX (and also capsaicin) operates the insensitive to the pungent action of capsaicin, forming lower gate and also interacts with the S5 to S6 linker. the experimental foundation of “-free” bird feed Interestingly, the TRP box is involved in capsaicin (reviewed in Szallasi and Blumberg, 1999). Rabbits are binding and leads to opening of the lower gate in S6. also much less sensitive (approximately 100-fold) to Obviously, there is cooperativity between the two pores

Fig. 4. TRPV1 channel topology highlighting key residues and amino acids (hot spots) involved in gating function in response to different stimuli (e.g., capsaicin, protons, and heat). The hot spots represent cumulative evidence from site-directed mutagenesis experiments; they provide evidence for distinct vanilloid (capsaicin/RTX), , alkaline (base), heat, and recognition sites. Black circles designate phosphorylation sites for PKA and PKC that are involved in channel sensitization. LPA, . Reprinted with permission from Szolcsányi and Sándor (2012). 688 Nilius and Szallasi

Fig. 5. Selected TRPV1 agonists. Capsaicin is responsible for the piquancy of hot chili peppers. Capsiate is a nonpungent capsaicin analog. Olvanil is a synthetic capsaicin congener. RTX is an ultrapotent capsaicin analog isolated from the latex of resinifera Berg. is an endocannabinoid that also acts as an endovanilloid. Oleamide (cis-9,10-octadecenoamide) is a fatty acid amide discovered in the cerebrospinal fluid of starving rats that also activates CB1 receptors. is a major alkaloidal principle extracted from Evodia fruits that has been shown to reduce fat accumulation in mice. Courtesy of Dr. Giovanni Appendino. to control TRPV1 gating. The determination of the high- Several TRPV1 splice variants have been identified. resolution structure of TRPV1 marks a breakthrough in A short N-terminal splice variant was isolated from TRP channel research. supraopticus nucleus neurons where it might be in- TRPV1 predominantly forms homotetramers. Al- volved in osmosensing (Sharif Naeini et al., 2006). though heteromers with TRPV2, TRPV3, and TRPV4 Another truncated form seems to be present in have been reported, their existence remains debated cells, mediating amiloride insensitive salt taste (Pingle (Hellwig et al., 2005). (TRPA1/TRPV1 heteromers will et al., 2007; Lyall et al., 2010). be discussed later as potential targets.) The exact tissue expression pattern of TRPV1 is still under discussion. Historically, capsaicin sensitivity was considered to be the “functional signature” of pri- mary sensory neurons (reviewed in Szallasi and Blumberg, 1999; Szolcsányi, 2004). Indeed, expression of TRPVR1/TRPV1 initially seemed to be restricted to sensory neurons (Szallasi and Blumberg, 1990; Caterina et al., 1997). Subsequently, a wide variety of tissues and cell types were reported to express TRPV1 (albeit at much lowerlevelsthaninsensoryganglia), including both neurons (throughout the whole neuroaxis of the rat, from the olfactory bulb through the cortex and basal ganglia to the cerebellum; see Szallasi and Di Marzo, 2000) and nonneuronal cells (in kidney, pancreas, testes, uterus, spleen, stomach, small intestine, liver, lung, bladder, skin, , mast cells, macrophages, and leu- kocytes) (reviewed in Tominaga and Tominaga, 2005; Szallasi et al., 2007). These reports are puzzling and Fig. 6. Representative structures of compounds that modulate TRP channels. (from peppermint) is a TRPM8 agonist. (a difficult to reconcile with the well characterized spectrum monoterpenoid phenol from ) acts as an agonist for TRPA1 and of capsaicin actions. Indeed, a recent article utilizing TRPV3 but as an antagonist for TRPM7. (from oil) a TRPV1 reporter mouse (Cavanaugh et al., 2011) found activates both TRPA1 and TRPV1. 2-APB (a synthetic chemical) is an activator of TRPV2 and TRPV3 but a blocker of TRPC3, TRPC6, TRPC7, only minimal TRPV1 expression in a few discrete brain TRPV6, and TRPM7. Hyperforin (an antidepressive regions (e.g., caudal hypothalamus). produced by , St. John’s wort) stimulates TRPC6. Naringenin (a citrus fruit flavanone) blocks TRPM3 channels. Courtesy of Outside of the central nervous system (CNS), TRPV1 Dr. Delia Preti. expression was mainly found in arteriolar smooth muscle TRP Channels as Drug Targets 689 cells (i.e., in tissue that are involved in thermoregulation) the HPETE [12-(S)-5-hydroperoxyeicosate- (Cavanaugh et al., 2011). This observation may provide traenoic acid], 15-(S)-HPETE, 5-(S)-hydroxyeicosatetrae- an alternative mechanistic explanation for the opposing noic acid, 20-hydroxyeicosatetraenoic acid, leukotriene actions of TRPV1 agonists (hypothermia) and antago- B4 (LTB4), N-arachidonoyldopamine (NADA), N-oleoyl- nists (hyperthermia) on body temperature regulation. , and polyamines (reviewed in Morales-Lázaro Capsaicin and its ultrapotent analog RTX (structure et al., 2013). Negatively charged intracellular lipids can showninFig.5)cause“reddening” (vasodilatation) when also activate TRPV1 (Lukacs et al., 2013). Generally applied to rodent or (reviewed in Szallasi speaking, TRPV1 activation by these compounds re- and Blumberg, 1999). This effect was traditionally quires such high concentrations that are unlikely to attributed to neurogenic inflammation. Indeed, the mouse occur under physiologic conditions. Other endogenous ear erythema assay was used as a surrogate marker to TRPV1 activators include ROS and reactive nitrogen detect capsaicin-like bioactivity (Szallasi and Blumberg, species (Tang et al., 2010a; Takahashi and Mori, 2011; 1989). The mechanism by which capsaicin evokes hy- Linley et al., 2012) and oxidized linoleic acid (LA) pothermia is essentially unknown, but it was speculated metabolites such as 9-hydroxyoctadecadienoic acid and that it might be a direct effect on preoptic neurons in- 13-hydroxyoctadecadienoic acid (Patwardhan et al., volved in body temperature regulation (Hori, 1984). 2009; Alsalem et al., 2013). NO can downregulate the Conversely, most (but not all) TRPV1 antagonists evoke function of TRPV1 through activation of the cGMP/PKG a febrile reaction, implying an essential role for TRPV1 in pathway in peripheral sensory neurons (Jin et al., the regulation of body temperature (see Gavva, 2008). 2012b). It is noteworthy that oleoylethanolamide (OEA), (For a comprehensive and critical state-of-the-art review an endogenous lipid that regulates appetite and body on TRPV1 and thermoregulation, see Romanovsky et al., weight, can directly activate TRPV1 (Ahern, 2003). 2009.) If capsaicin can directly constrict (and TRPV1 TRPV1 is a shared target for a number of structur- antagonist dilate) arteriolar smooth muscles via TRPV1 ally unrelated irritant compounds in nature. For to control blood flow, this effect may have profound example, TRPV1 is activated by capsaicin (responsible consequences on body temperature. Of note (as discussed for the piquancy of hot chili peppers), RTX (from the later), both TRPV1 agonists and antagonists dramatically soap of ), (in Piper nigrum), differ in their ability to influence body temperature. For (from ), curcumine (in example, the small molecule TRPV1 antagonist PHE377 Curcuma longa), and [6]-, a major constituent of blocks capsaicin- and proton-evoked currents in rat (see Szallasi and Blumberg, 1999; Vriens et al., sensory neurons, and is active in various rodent models 2009; Nilius and Appendino, 2011, 2013). For dietary of inflammatory and neuropathic pain (see Trevisani and modulation of TRPV1 to control appetite, food supple- Szallasi, 2010). Yet unlike other TRPV1 antagonists, mentation by nonpungent capsinoids such as capsiate PHE377 does not elevate body temperature. To explain (Fig. 5) has been advocated (Ludy et al., 2012). Capsiate these unexpected findings, one may speculate that is, however, not selective for TRPV1: Indeed, it can also TRPV1 channels in sensory neurons and arterial smooth activate TRPA1 (Shintaku et al., 2012). Conversely, some muscle cells differ in their ligand-recognition properties. TRPA1-selective compounds such as allicin (from ) TRPV1 was first identified as the vanilloid receptor and allyl- (in oil; structure shown VR1 by its highly selective binding of capsaicin and the in Fig. 7) can also activate and/or sensitize TRPV1 even more selective RTX (Szallasi and Blumberg, 1990). (Macpherson et al., 2005; Everaerts et al., 2011; Alpizar The binding domain of TRPV1 for capsaicin and RTX et al., 2014). Indeed, allyl-isothiocyanate increases has been located to the binding pocket between TM3 and TM4 (see Fig. 4) (Gavva et al., 2004; Lee et al., 2011). Structurally related are also potent activators of the channel. Furthermore, TRPV1 is activatedbyheataswellasdivalent(e.g.,Ni2+)and trivalent cations (e.g., Gd3+) (Tousova et al., 2005). It is noteworthy that TRPV1 is activated both by acidifica- tion (protons) and alkalinization (Dhaka et al., 2009). + [Na ]e negatively regulates the gating and polymodal sensitization of the TRPV1 channel (Ohta et al., 2008). To note, molecular mechanisms of TRPV1 activation were recently reviewed in Jara-Oseguera et al. (2010). Despite extensive research, the physiologic regula- Fig. 7. Representative TRPA1 agonists. is from tion of TRPV1 by “endovanilloids” is poorly understood . Allyl-isothiocyanate is from mustard. 4-Hydroxynonenal is (Di Marzo et al., 2002; Palazzo et al., 2013). Endoge- a diffusible product of lipid peroxidation. is from the Californian headache tree Umbellularia californica. is nous activators include the cannabinoid receptor ligands a major nonpsychoactive constituent in . Courtesy of Dr. anandamide and oleamide (structures shown in Fig. 5), Giovanni Appendino. 690 Nilius and Szallasi carbohydrate oxidation (Mori et al., 2011), and thereby mice have an apparent affinity for PIP2 indistinguish- reduces hyperglycemia after challenge (Mori able from the wild types (Ufret-Vincenty et al., 2011). et al., 2013), in TRPA1-null mice, whereas these effects The previous view on PIP2 as a negative regulator of are absent in the TRPV1 knockout animals. Of note, TRPV1 recently again received attention. Purified certain venoms and toxins from spiders (e.g., Tarantula), TRPV1 reconstituted into artificial liposomes is fully jelly fish, and snakes cause pain by directly activating functional in the absence of phosphoinositides, thus TRPV1 (reviewed in Kumar et al., 2014). PIP2 has likely no obligatory role in channel activation, TRPV1 is a voltage-gated channel that is activated and, even more striking, introduction of PIP2 inhibits by depolarization. Most of the TRPV1 activators induce the channel (Cao et al., 2013a). a leftward shift in voltage dependence, resulting in channel A more dynamic regulation TRPV1 activity is activation (Voets et al., 2004; Nilius et al., 2005b). It is mediated by various scaffolding proteins. For example, noteworthy that TRPV1 shows a rapid and a slow de- TRPV1 activity is enhanced by interaction of TRPV1 sensitization. This phenomenon is a Ca2+-mediated pro- with the A-kinase anchoring protein (AKAP) 79/150. cess: it is abolished in the absence of extracellular Ca2+ AKAP79/150 forms a multiprotein complex with TRPV1/ or by intracellular Ca2+ chelation. Ca2+ entry (al- PKA/PKC (Zhang et al., 2008d; Efendiev et al., 2013). though the fractional Ca2+ current through TRPV1 is b-Arrestin-2, another scaffolding protein, promotes only 5%) results in a sufficiently high Ca2+ concentration TRPV1 desensitization (Por et al., 2012). TRPV1 is to trigger desensitization (a negative feedback mecha- variably N-glycosylated (glycosylation sites are shown in nism). Desensitization is now mainly considered to be a Fig. 4), and glycosylation is a key determinant of ca- dephosphorylation event. Indeed, it is inhibited by in- psaicinregulationofTRPV1desensitizationandperme- hibitors of calcineurin, a Ca2+-dependent phosphatase. ability(Veldhuisetal.,2012).Inaddition,TRPV1is Dephosphorylation events include sites that have been functionally coupled to the sensory neuron-specific Mas- phosphorylated by PKA, PKC, and Ca2+/CaM-dependent related G protein–coupled receptor (Mrgpr)-X1, which is kinase II (CaMKII) (summarized in Fig. 4). Molecular important for nociception (Solinski et al., 2012). Mrgpr-X1 events that promote channel phosphorylation sensitize sensitizes TRPV1 to heat and protons in a PKC- the TRPV1 channel; this is often due to a leftward shift of dependent manner. Direct Mrgpr-X1–mediated TRPV1 the voltage dependence (i.e., in a depolarizing direction at activation is independent of Mrgpr-X1–induced Ca2+ the channel protein) (Nilius et al., 2005b; Tominaga and release and PKC activity or other TRPV1 affecting Tominaga, 2005). , such as lipoxygenase and PI3K. Mutations in The complexity of TRPV1 channel regulation is the DAG or PIP2 binding sites in TRPV1 decreased further increased if we take into account the modulator Mrgpr-X1–induced TRPV1 activation. The functional effects via GPCRs that, in turn, increase cAMP [e.g., interaction between Mrgpr-X1 and TRPV1 results in (PGE2) receptors], DAG, and IP3 [e.g., a PKC-dependent TRPV1 sensitization and DAG/PIP2- angiotensin, bradykinin (BK), proteinase-activated mediated activation (Solinski et al., 2012). receptor-2 (PAR-2), nerve growth factor (NGF) recep- Surface expression is an efficient regulator of TRPV1 tors], which all indirectly target PKA, PKC, and activity. One important player that promotes surface CaMKII (Fig. 12). In addition, PIP2 is changed. Al- expression of TRPV1 is NGF, which activates a signal- though PIP2 was initially considered as a tonic in- ing pathway in which PI3K plays the crucial role and hibitor of TRPV1 (Prescott and Julius, 2003), it is now a downstream Src kinase phosphorylates the channel accepted that PIP2 is required for channel activation, (Zhang et al., 2005). Cyclin-dependent kinase (CDK)-5 whereas PIP2 depletion conversely causes desensitization positively regulates TRPV1 surface localization. TRPV1- (Stein et al., 2006; Rohacs, 2007; Klein et al., 2008). PIP2 containing vesicles bind to the forkhead-associated do- binds to a proximal C-terminal region of the TRPV1 main of the kinesin-3 family member 13B (KIF13B) and protein (Ufret-Vincenty et al., 2011). The enhancement of are thus delivered to the cell surface. Overexpression of TRPV1 activity by PIP2 may require the presence of Pirt, CDK5 (or its activator, p35) promoted, whereas the in- a phosphoinositide-binding protein (Kim et al., 2008a). hibition of CDK5 activity prevented, the KIF13B–TRPV1 Indeed, in Pirt-deficient dorsal root ganglion (DRG) association, indicating that CDK5 promotes TRPV1 neurons both noxious heat- and capsaicin-evoked currents surface expression (Xing et al., 2012). Surface expres- are significantly attenuated. Furthermore, Pirt-null mice sion of TRPV1 is also regulated by the ubiquitin ligase exhibit deficits in behavioral responses to histamine, MYCBP2 (Holland et al., 2011). a pruritogenic compound that activates TRPV1+ sensory Insertion of TRPV1 into the plasma membrane is an afferents (Patel et al., 2011). important regulatory mechanism. Growth factors such However, this view has been challenged because Pirt as NGF and insulin-like growth factor (IGF)-I activate does not alter the phosphoinositide sensitivity of TRPV1 PI3K and/or CDK5, which, in turn, mediate TRPV1 in HEK293 cells. In addition, there is no fluorescence plasma membrane insertion signaling (Zhang et al., 2005; resonance energy transfer signal between TRPV1 and Lilja et al., 2007; Canetta et al., 2011; Xing et al., 2012). Pirt, and dissociated DRG neurons from Pirt knockout TRPV1-containing vesicles bind to the forkhead-associated TRP Channels as Drug Targets 691 domain of the KIF13B, which mediates the delivery to constitutively active (Kanzaki et al., 1999; Iwata et al., the cell surface (Xing et al., 2012). Intracellular traffick- 2003; Hisanaga et al., 2009). The key for plasma ing of TRPV1 also depends on microtubules (Goswami membrane insertion for many TRP channels is PI3K, et al., 2006). which probably activates TRPV2 without changes in It is noteworthy that TRPV1 is the major channel for plasma membrane insertion (Penna et al., 2006). Other the detection and integration of nociceptive chemical reports, however, describe an increased TRPV2 traffick- and thermal stimuli in sensory nerve fibers (thin- ing to the plasma membrane (Nagasawa et al., 2007). In myelinated Ad-fibers and unmyelinated C fibers) in- pancreatic b cells, plasma membrane insertion of TRPV2 nervating most of our organs (see Szallasi et al., 2007; is regulated by PI3K after insulin treatment and this Julius, 2013). This concept is supported by the Trpv1 mechanism accelerates the exocytotic response during the knockout mouse, which shows markedly reduced glucose-induced insulin secretion (Aoyagi et al., 2010). thermal hyperalgesia after inflammation and injury TRPV2 associates with recombinase gene activator, (Caterina et al., 2000; Davis et al., 2000). As discussed which probably plays a role in the maturation of the later, this makes TRPV1 an obvious target for novel channel and promotes surface expression (Stokes analgesic compounds. et al., 2005). The antiaging Klotho also promotes TRPV2 shows a 50% sequence similarity to TRPV1. plasma membrane insertion (Lin and Sun, 2012). TRPV2 mostly forms homomers, but heteromers with Moreover, TRPV2 was found to be directly phosphor- TRPV1, TRPV3, and TRPV4 have also been reported ylated by PKA. Of note, TRPV2 forms a complex with (Hellwig et al., 2005; Cheng et al., 2007b). TRPV2 is the AKAP-like protein ACBD3, which appears to link probably the least understood member of the TRPV TRPV2 to PKA (Stokes et al., 2004). Depletion of PIP2 subfamily. Cloned as a “capsaicin receptor homolog causes Ca2+-dependent desensitization of the channel with a high threshold for noxious heat,” TRPV2 is (Mercado et al., 2010). expressed in a subset of DRG neurons (Caterina et al., Although it is highly expressed in the brain, the 1999). In addition, it is present in certain hypothalamic functional role of TRPV2 is unclear. Reportedly, TRPV2 brain nuclei and the magnocellular neurons of the promotes growth cone guidance (Shibasaki et al., 2010) paraventricular and supraoptic nucleus (Nedungadi and negatively controls glioma cell survival and pro- et al., 2012). In the brain, TRPV2 was also detected in liferation in an ERK-dependent manner (Nabissi et al., astrocytes (Shibasaki et al., 2013). The function of 2010). TRPV2 is believed to be a major player in TRPV2 in the CNS is essentially unknown. Further- macrophage migration (Nagasawa et al., 2007). In mac- more, TRPV2 is expressed in the heart, gastrointesti- rophages, it is localized abundantly in the podosomes, nal tract, lung (alveolar cells), pancreas, retina, and which are adhesion structures required for cell migra- both smooth and skeletal muscle cells (Muraki et al., tion. TRPV2 functions as the Ca2+ entry channel in 2003; Beech et al., 2004). TRPV2 has also been de- these structures (Nagasawa and Kojima, 2012). TRPV2 scribed as an intracellular ion channel residing in the mediates the gastric adaptive relaxation in the stomach membranes of early endosomes, where it may act as (Mihara et al., 2013). It is required for the LPS-induced aCa2+ release channel (Saito et al., 2007). cytokine production in macrophages (Yamashiro et al., TRPV2 is activated by extremely noxious heat (.53°C) 2010). TRPV2 also plays a role in mast cell degranula- (Caterina et al., 1999). Growth factors such as IGF-1 tion (Zhang et al., 2012a). It was widely assumed that (and also insulin) transfer the channel to the plasma TRPV2 would be responsible for (at least part of) the membrane, where it seems to be constitutively active residual heat sensitivity in Trpv1 knockout mice. How- (Kanzaki et al., 1999; Iwata et al., 2003). TRPV2 has ever, the effect of Trpv2 knockout in mice on thermal been also described as a candidate SAC (Muraki et al., responsiveness has not yet been established and pre- 2003; Beech et al., 2004). The channel probably plays a liminary findings actually indicate that TRPV2 has no role in skeletal muscle and cardiac muscle degeneration, obvious role in heat sensing. Increasing evidence links in innate immunity, as well as in the pain pathway. 2- TRPV2 to the innate . Indeed, zymosan-, APB, , (via the TXA2 receptor), IgG-, and complement-mediated particle binding and and lysophospholipids all can activate TRPV2. Impor- phagocytosis are impaired in mice lacking the TRPV2 tantly, such as D9-, receptor (Link et al., 2010). cannabidiol (structure shown in Fig. 7), and TRPV3 shares a 43% sequence similarity with are TRPV2 activators (Qin et al., 2008). However, all of TRPV1. It is still controversial whether TRPV3 can these activators are nonselective and act in a species- form heteromers with TRPV1, TRPV2, or TRPV4 dependent manner, and the regulation of human TRPV2 (Cheng et al., 2007b). Expression of TRPV3 was is essentially unknown (Neeper et al., 2007). reported both in neurons and non-neuronal tissues, It is likely that the most important mechanism for including the tongue, testis, skin keratinocytes, and TRPV2 regulation is its triggered plasma membrane the cells surrounding the hair follicles (Peier et al., insertion. Growth factors (e.g., IGF-I) facilitate TRPV2 2002; Xu et al., 2002; Moqrich et al., 2005; Borbíró transfer to the plasma membrane, where it seems to be et al., 2011). 692 Nilius and Szallasi

TRPV3 is activated by innocuous warm temperatures A ligand binding pocket is located in TM4 and shows above 30°C–33°C. Unlike TRPV1, TRPV3 is sensitized some homology with the capsaicin site in TRPV1 by repeated stimulations with temperature (Xu et al., (Nilius and Owsianik, 2011). TRPV4 is widely ex- 2002). Furthermore, sensitization of TRPV3 to warm pressed in neurons of the peripheral and central temperatures is influenced by different endogenous nervous systems. It is also detectable in a variety of non- proinflammatory agents such as BK, histamine, ATP neuronal tissues, including chondrocytes, osteoclasts/ (binding to ankyrin repeats), PKC, and PGE2 (Huang osteoblasts, insulin-secreting b cells, salivary gland, et al., 2008; Mandadi et al., 2009; Phelps et al., 2010). hair cells of the inner ear, lung, keratinocytes, urothe- TRPV3 is activated by protons and is highly expressed lium, and placenta. Furthermore, TRPV4 is expressed in keratinocytes. Interestingly, a-hydroxyl acids from in the smooth muscle cells of the respiratory system and natural sources, which function as proton donors when cerebral arteries. Finally, it is a highly expressed in applied to the skin and are used in the cosmetic vascular endothelial cells and in a multitude of epi- industry, activate TRPV3 (Cao et al., 2012a,b). 2-APB thelial cell types in lungs, trachea, oviducts, and kidneys (already mentioned as a TRPV2 agonist) also activates (Everaerts et al., 2010a). TRPV3 most likely by binding to two cytoplasmic sites, TRPV4 is activated by moderate temperatures (.24°C); one of which is close to the TRP box (Hu et al., 2009b). therefore, it is constitutively active at normal body TRPV3 is activated by an array of natural com- temperatures. It is considered to be a mechanosensor pounds, including camphor, , carvacol (see Fig. 6 and osmosensor, probably via functional coupling to for structure), carveol, , cresols, eugenol (struc- a mechanosensitive PLA2 (Nilius et al., 2001; Vriens ture shown in Fig. 6), and Boswellia resin, which are all et al., 2005). Direct mechanoactivation of TRPV4 was sensitizers for temperature activation of the channel recently demonstrated (Loukin et al., 2010b). TRPV4 (Vriens et al., 2009). These natural compounds are, interacts (or even forms heteromers) with TRPP1 however, not selective for TRPV3. Indeed, camphor also (Köttgen et al., 2008), 4 (Benfenati et al., activates TRPV1 (Xu et al., 2005) and TRPM8 channels 2011), (Galizia et al., 2012), TRPC1 (Ma (Selescu et al., 2013). In contrast with other TRP et al., 2010, 2011), and/or calcium-activated potas- channels, PIP2 hydrolysis (and/or pharmacological in- sium channel (KCa2.3 cells) (Ma et al., 2013). hibition of phosphatidylinositol-4 kinase to block PIP2 TRPV4 can be activated by a number of endogenous synthesis) potentiates TRPV3. In excised patches, agonists. The endocannabinoid anandamide (which is TRPV3 is potentiated by PIP2 depletion. PIP2 directly also an endogenous activator of TRPV1), its metabolite interacts with a specific protein motif and reduces , and epoxygenase- TRPV3 channel open probability (Doerner et al., 2011; derived epoxyeicosatrienoic acids are potent TRPV4 for review, see Nilius et al., 2014). activators (Watanabe et al., 2003; Vriens et al., 2005). As anticipated from its high expression in keratino- Furthermore, TRPV4 is activated by bisandrographo- cytes, TRPV3 is required for several skin functions, lide A (Fig. 8), a compound isolated from Andrographis including skin barrier formation and hair morphogen- paniculata extracts, a plant used in traditional Chinese esis. In the skin, TRPV3 forms a signaling complex medicine as an anti-inflammatory agent and immunos- with TGF-a and EGFR. Activation of EGFR increases timulant (Vriens et al., 2007). Chemicals that are potent TRPV3 channel activity to induce an increased release activators of TRPV4 comprise synthetic of TGF-a (Cheng et al., 2010a). such as 4a-phorbol 12,13-didecanoate (4aPDD; Fig. 8), The role of TRPV3 in heat sensing is supported by the 4a-phorbol 12,13-dihexanoate, and lumi-4aPDD, as phenotype of the Trpv3 knockout mouse, which shows well as GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2, strong deficits in responses to innocuous and noxious 4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)- heat but not in other sensory modalities (Moqrich et al., 1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene- 2005). Heating of keratinocytes causes ATP release, 2-carboxamide; Fig. 8] (Everaerts et al., 2008; Klausen which, in turn, activates sensory nerves fibers; this et al., 2009). HC-067047 [2-methyl-1-[3-(4-morpholinyl) mechanism is defective in Trpv3 knockout mice (Mandadi propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole- et al., 2009). It came as a surprise that TRPV3 is 3-carboxamide] was recently described as a potent and functionally connected to the M-type selective TRPV4 antagonist (Figs. 9, 10, and 11) (Everaerts Kv7.2 in keratinocytes. M-channel activators dramati- et al., 2010a). TRPV4 gating by hypotonic and heat stimuli cally enhance the release of ATP induced by TRPV3 requires PIP2 binding. This binding is combined with agonists (Reilly et al., 2013). TRPV3 is also involved in a rearrangement of the N terminus. Downstream of the the hippocampal LTD; therefore, it was suggested to Pacsin3 binding site, a cluster of positive charges has been play a role in synaptic plasticity (Brown et al., 2013; for identified as phosphoinositide-binding site. PIP2 facilitates recent comprehensive reviews on TRPV3, see Nilius and TRPV4 activation by osmotransducing messengers, cyto- Bíró, 2013; Nilius et al., 2014). solic epoxyeicosatrienoic acids, and allows the activation of TRPV4 has a proline-rich region and six ankyrin the channel by heat in inside-out patches (Garcia-Elias repeat domains (ARDs) in its cytosolic N-terminal part. et al., 2013). TRP Channels as Drug Targets 693

Everaerts et al., 2008). Moreover, TRPV4 is activated in endothelial cells with the consequences of activation of endothelial IK(Ca) channels, , and reduction of the myogenic tone that underpins tissue blood flow autoregulation (Bagher et al., 2012). Finally, TRPV4 is involved in the regulation of the synaptic inputtoneuroendocrinecellsinthehypothalamicpara- ventricular nucleus (Boychuk et al., 2013). TRPV5 exhibits a low homology to TRPV1 to TRPV4 (,30%) but shares 74% identity with TRPV6. TRPV5 can form homomers but also heteromers with TRPV6. TRPV5 expression, which is influenced by estrogen, parathormone (PTH), 1,25 dihydroxyvitamin D3, and dietary Ca2+ intake, is high in the placenta, kidney (predominantly in the distal convolute and connecting tubules), and bone, but also in pancreatic b cells and the inner ear (Hoenderop et al., 2005). TRPV5 and TRPV6 are the only highly Ca2+ selective TRP channels. It is only in the absence of extracellular Ca2+ that they transport monovalent cations. TRPV5 is a constitutively active channel. Repeated stimulation of the channel results in current decay and this process 2+ 2+ 2+ seems to be Mg and Ca dependent. [Ca ]i acts as an important feedback inhibitor for Ca2+ entry via TRPV5. Both intracellular and extracellular acidic pH Fig. 8. Chemical structures of selected TRPV3 and TRPV4 agonists. 4a- block this channel (see Vennekens et al., 2008). TRPV5 Phorboldidecanoate (from croton oil), bisandrographolide (from Andror- is effectively regulated by plasma membrane insertion aphis paniculata), and GSK1016790A (GlaxoSmithKline, Uxbridge, and retrieval, which is controlled by many interacting Middlesex, UK) selectively activate TRPV4. (an intermediate metabolite in the mevalonate pathway) functions as an proteins such as CaM, S100A10-annexin II, the PKC activator of TRPV3. Courtesy of Dr. Delia Preti. substrate 80K-H, Calbindin 28K, and the sodium- hydrogen exchange regulatory cofactor 2 (NHERF2). NHERF2 stabilizes TRPV5 when inserted to the plasma Because TRPV4 is activated in many different membrane. Interaction of TRPV5 with the Ca2+-binding tissues and is involved in very different cellular pro- proteins S100A10 and annexin II is required for plasma cesses, it might become an important, but difficult, membrane insertion. The antiaging hormone Klotho drug target (see Vincent and Duncton, 2011). Because regulates TRPV5 activity by modifying its glycosylation Trpv4 knockout mice only show only very minor status and increases the plasma membrane-bound deficits in temperature sensing, TRPV4 is possibly fraction of the channel (Chang et al., 2005). An important not a functional thermoTRP (Lee et al., 2005). TRPV4 regulatory pathway involves the tissue serine protease, is important for the maintenance of skin barrier func- kallikrein. Kallikrein activates the BK receptor-2, which, tion (Denda et al., 2007). It is noteworthy that TRPV4 in turn, activates TRPV5 DAG-dependently via PLCg is highly expressed in chondrocytes (Muramatsu et al., and PKC (de Groot et al., 2008; Boros et al., 2009). 2007); indeed, it is important for the normal develop- Phosphorylation of the Ca2+ reabsorption channels in- ment of the bone growth plates. In the skin, TRPV4 creases TRPV5 membrane insertion and delays retrieval. activation strengthens the epidermal tight junction Binding to calbindin D28K functionally results in local (which includes occludin, claudin-4, and tight junction buffering of entering Ca2+ and inhibits channel inactiva- regulatory proteins), and is thus important for the in- tion (for review, see Hoenderop et al., 2005). tegrity of the skin barrier (Akazawa et al., 2013). TRPV5 is effectively regulated by WNK4, a protein Deduced from the Trpv4 knockout phenotype, TRPV4 serine/threonine kinase, which also promotes plasma might function as a mechanosensor and chemosensor, membrane insertion of the channel (Jiang et al., such as sensing for osmoreceptive neurons of the circum- 2008b). TRPV5 is also coupled with the extracellular ventricular organ and for sensing luminal tonicity in Ca2+-sensing receptor. Activation of the Ca2+-sensing cholangiocyte cilia, which are required for bile formation. receptor stimulates TRPV5, but not TRPV6 (Topala TRPV4 also plays an important role in the bladder et al., 2009). Interaction of TRPV5 with 80K-H, (mostly in urothelial cells), where it is involved in sensing a substrate for PKC, inhibits Ca2+ entry and increases of the intravesicular pressure and regulation of the the TRPV5 sensitivity to intracellular Ca2+, thereby detrusor function (for reviews, see Gevaert et al., 2007; accelerating the feedback inhibition of the channel. 694 Nilius and Szallasi

Fig. 9. Representative structures of potent, small molecule TRPV1 antagonists.

The parathyroid hormone PTH activates the cAMP- and Gd3+. It is abundantly expressed in the intestine, PKA signaling cascade, which rapidly phosphorylates placenta, uterus, pancreas, epididymal epithelium, TRPV5, thereby activating the channel (de Groot et al., brain, and stomach (Hoenderop et al., 2005). TRPV6 2009). Rab11a, which is colocalized with TRPV5 (and is localized to the brush-border membrane of intestinal also TRPV6) in vesicular structures underlying the enterocytes and functions there as the main Ca2+entry apical plasma membrane, also promotes channel in- channel. TRPV6 is also expressed in kidney, where it is sertion in the plasma membrane (for reviews, see de mainly found in cortical and medullary collecting Groot et al., 2008; Boros et al., 2009). ducts. TRPV6 is a constitutively open ion channel The main function of TRPV5 is Ca2+ reabsorption in and shows a striking negative feedback inhibition by 2+ 2+ 2+ the kidney. Trpv5 knockout mice show impaired Ca Ca entry (e.g., an increase in [Ca ]i) (Nilius et al., reabsorption in the distal convoluted and connecting 2002). Expression of TRPV6 is upregulated by tubules, resulting in calciuria and polyuria with D (Balesaria et al., 2009). Both PIP2 and cytoplasmic significantly more acidic urine than that seen in wild- ATP are important for maintaining TRPV6 activity. type mice (Hoenderop et al., 2003). Mg2+ATP is the substrate for the lipid kinase type III, TRPV6 is the closest relative of TRPV5. It is a highly phosphatidylinositol 4-kinases, which allows the resyn- 2+ 2+ Ca -selective TRP channel that is also permeable to thesis of PIP2.Ca -dependent inactivation of TRPV6 Ba2+,Sr2+,Mn2+,Zn2+, and Cd2+, as well as for La3+ (and also TRPV5) is mediated by a Ca2+-dependent TRP Channels as Drug Targets 695

Fig. 10. Examples of selective TRPA1 antagonists.

activation of a PLC, inducing PIP2 depletion (Zakharian also interacts with S100A10 and annexin II, which et al., 2011). TRPV6 can also bind to CaM. Over- promotes plasma membrane insertion. The PDZ expression of CaM reduces currents through TRPV6 proteins NHERF2 and NHERF4 form scaffolds for and accelerates inactivation (Derler et al., 2006). TRPV6 TRPV6 and stabilize plasma membrane insertion

Fig. 11. Representative examples of compounds that block TRPM8 (AMTB, JNJ41876666, PBMC, and AMG9678), TRPV4 (HC-067047), and TRPC3 (Pyr3) channels. 696 Nilius and Szallasi

(van de Graaf et al., 2006). In the small intestine, the tumor suppressor activities of MITF and/or TRPM1 TRPV6 binds to calbindin-D(9k); this interaction is may at least partially be due to miR-211 (Mazar et al., required for Ca2+ buffering close to the channel and 2010; Margue et al., 2013). delays inactivation. Nipsnap1 is an associated protein At least five different TRPM1 isoforms have been that inhibits TRPV6 possibly via a direct action at the reported, which makes the functional analysis difficult. plasma membrane (Schoeber et al., 2008). Nedd4 These isoforms are expressed in melanocytes/melanoma regulates ubiquitination of TRPV6 and reduces the cells, as well as in the brain and retina (Oancea et al., plasma membrane abundance of the channel (Zhang 2009). Initially, expression data revealed only very small et al., 2010). An opposite (positive) effect on plasma currents through TRPM1. Therefore, TRPM1 was first membrane abundance of TRPV6 was demonstrated thought to be an ion channel whose function was critical for WNK3 (Zhang et al., 2008c). It is noteworthy that to normal melanocyte pigmentation. If so, TRPM1 may TRPC1 might have an inhibitory effect on TRPV6 be a potential target for pigmentation disorders (Oancea (Schindl et al., 2012). et al., 2009). TRPM1 can be weakly activated by the TRPV6 is the main channel for intestinal trans- , sulfate. By contrast, TRPM1 is epithelial Ca2+ absorption (Hoenderop et al., 2005). inhibited by Zn2+ (Lambert et al., 2011). Trpv6 knockout mice show decreased Ca2+ absorption In the retina, TRPM1 is expressed on ON bipolar and, subsequently, decreased serum Ca2+ levels (Bianco cells, which invert the signal of light responses from et al., 2007). More strikingly, TRPV6 contributes to hyperpolarizing to depolarizing before passing them on intestinal calcium transport only under conditions when to ganglion cells. TRPM1 colocalizes with mGluR6, the dietary calcium supply is limited. In such situations, aGao and Gb3 protein. Light responses are generated TRPV6 regulates bone formation and mineralization when TRPM1 is activated. Removal of glutamate from (Lieben et al., 2010). TRPV6 controls the extracellular its receptor, mGluR6, activates TRPM1. Glutamate is Ca2+ concentration toward the distal segments of the released from the adjacent rod photoreceptor cells. epididymal duct, which is essential for survival of Binding to mGluR6 closes the channel. After activa- spermatozoa. Loss of functional channels causes se- tion, TRPM1 desensitizes and the light response verely impaired fertility due to a reduction in motility decays (Morgans et al., 2009; Koike et al., 2010). The and fertilization capacity of sperms (Weissgerber et al., synaptic efficiency of the rod-bipolar cells is regulated 2012). In the skin, TRPV6 is a key element in Ca2+/1,25- via modulation of a block by Mg2+ of TRPM1 via DAG, dihydroxyvitamin D3–induced keratinocyte develop- PIP2, and PKCa (Rampino and Nawy, 2011). ment (Lehen’kyi et al., 2007). TRPM1 associates with nyctalopin, a small leucine- rich repeat proteoglycan. Its absence causes a complete C. Melastatin Transient Receptor Potential Subfamily inhibition of TRPM1 (Bojang and Gregg, 2012). Nycta- The melastatin TRP (TRPM) family comprises eight lopin has been shown to interact with TRPM1, and the members. All have a 25- TRP box, the expression of TRPM1 on the dendritic tips of the bipolar functional impact of which is still uncertain. The cells is dependent on nyctalopin expression. TRPM1 is N terminus of all eight members is much longer than in also expressed on a subset of cells in the ganglion cell other TRP channels; it contains a TRPM homology layer, including melanopsin-expressing photosensitive region shared by all TRPM proteins (Fig. 2). TRPM ganglion cells (Hughes et al., 2012). channels lack the N-terminal ankyrin repeats. Mem- The functional role of TRPM1 in the retina is to provide bers of the TRPM family, on the basis of sequence theONresponseofbipolarcells. It is also involved in the homology, fall into three subgroups: TRPM1/TRPM3, pupillary reflex and plays a role in skin pigmentation TRPM4/TRPM5, and TRPM6/TRPM7 with TRPM2 and (i.e., in the function of melanocytes). In neonatal human TRPM8 representing structurally distinct channels. epidermal melanocytes, TRPM1 expression correlates TRPM channels exhibit highly variable permeability to with melanin content, suggesting that TRPM1 is critical Ca2+ and Mg2+,rangingfromCa2+ impermeable (TRPM4 to normal melanocyte pigmentation and thus represents and TRPM5) to highly Ca2+ and Mg2+ permeable (TRPM6 a potential target for pigmentation disorders (Oancea and TRPM7) (for review, see Nilius et al., 2007b). et al., 2009; Hughes et al., 2012). TRPM1 regulates TRPM1 is the least studied member of the mela- pigmentation by controlling melanin synthesis in skin family. It was first identified as a tumor sup- melanocytes (Devi et al., 2009, 2013). pressor protein expressed at high levels in melanocytes TRPM2 (Fig. 2) is a Ca2+-permeable cation channel but at very low levels in metastatic melanoma cells characterized by a functional nudix hydrolase domain (i.e., it shows an inverse correlation with melanoma that is highly homologous to the ADP-pyrophosphatase progression) (Duncan et al., 1998). Microphthalmia- NUDT9 that cleaves ADP-ribose (ADPR) into ribose-5 associated transcription factor (MITF), a melanocytic phosphate and AMP. TRPM2 forms functional tetra- marker used in diagnostic surgical pathology, is a major homomers (see Eisfeld and Lückhoff,2007).AC-terminal regulator of TRPM1 expression (Miller et al., 2004). MITF CC domain plays an important role in the TRPM2 is also required for miR-211 expression, suggesting that channel assembly. TRP Channels as Drug Targets 697

TRPM2 is highly expressed in the brain (both in TRPM3 is closely related to TRPM1 and is the TRP neurons and glial cells, especially in the substantia channel with the most splice variants (Oberwinkler nigra and hippocampus), in monocytes, as well as in et al., 2005). TRPM3 expression is most prominent in spleen, heart, lung, eye, vascular endothelium, thymo- brain, kidney (human), and pituitary cells. Expression cytes, pancreatic b cells, neutrophils, and leukocytes was also demonstrated in liver, pancreatic b cells, (reviewed in Eisfeld and Lückhoff, 2007; Sumoza-Toledo ovaries, testes, spinal cord, pulmonary endothelium, and Penner, 2011). In pancreatic b cells, TRPM2 has sensory (trigeminal and DRG) neurons, been shown to function not only as a plasma membrane cells, iris, and retinal pigmented cells (Lee et al., channel but also as an intracellular channel mediating 2003a). TRPM3 shows some basal activity that in- release of Ca2+ from intracellular lysosomal stores creases with decreasing intracellular Mg2+ concentra- (Lange et al., 2009). tions (Oberwinkler et al., 2005). This channel is rapidly TRPM2 is directly activated by both NAD and and reversibly activated by extracellular pregnenolone ADPR: this depends on hydrolyzation after binding sulfate, a neuroactive steroid (Wagner et al., 2008). By with the NUDT9 motif. It is noteworthy that TRPM2 is contrast, inhibits the channel (Majeed a redox-sensitive channel. Products of oxidative stress et al., 2012). Other TRPM3-activating stimuli (depend- (e.g., H2O2) activate TRPM2. In contrast, inhibitors of ing on the splice variants) are D-erythro-sphingosine oxidative stress (e.g., glutathione, catalase, dimethylth- and . TRPM3 is activated by heat and functions iourea, antioxidants, and mannitol) close the channel. as a voltage-dependent channel that is activated by Sirtuins (NAD+-dependent deacetylases) synthesize nic- depolarization (Vriens et al., 2011). Some splice variants otinamide and ADPR, which, in turn, activate TRPM2 are inhibited by extracellular Na+.Thenaturalcom- and promote cell death (Grubisha et al., 2006). Oxidative pounds, naringenin (structure shown in Fig. 6) and DNA damage activates poly(ADP-ribose)polymerase-1 hesperetin (which belong to the citrus fruit flavanones), (PARP-1);the poly(ADPR) then is rapidly degraded to and the deoxybenzoin are potent blockers of ADPR by poly(ADPR)glycohydrolase. PARP-1 and poly TRPM3 (Straub et al., 2013a,b). (ADPR)glycohydrolase can, therefore, indirectly activate TRPM3 is thought to play important roles in TRPM2, leading to Ca2+ entry. This may initiate a cell pancreatic islets, oligodendrocytes, and vascular smooth death signaling pathway (Blenn et al., 2011). Activation of muscle. Importantly, it provides an entry pathway for 2+ 2+ TRPM2 is sensitized by an increase of [Ca ]i (Du et al., Zn , which might be especially important for pancre- 2009a). This activation is probably due to a Ca2+-dependent atic b cells (Wagner et al., 2010). Indeed, TRPM3 might association of CaM with the TRPM2 N terminus (Tong be involved in insulin release. In insulinoma cells, et al., 2006). External and internal protons inhibit TRPM2 induces, via TRPM3, an enhanced (Du et al., 2009b). Heat (.35°C) potentiates channel expression of the zinc finger transcription factor, Egr, activation (Togashi et al., 2006). In contrast with other which drives Pdx-1, a major regulator of insulin gene TRP channels, 2-APB inhibits TRPM2. transcription (Mayer et al., 2011). Data from Trpm3 TRPM2 is now defined as a sensor for intracellular knockout mice clearly show that TRPM3 plays a role in oxidation. TRPM2 is implicated in cell death by oxidative heat perception and mediates pain responses (Vriens stress (Takahashi et al., 2011a; Wyrsch et al., 2012). et al., 2011). Finally, TRPM3 is involved in glutaminer- Furthermore, TRPM2 is believed to be a major player in gic signaling and synaptic plasticity in cerebellar LPS-dependent cytokine production, which is inhibited by Purkinje cells (Zamudio-Bulcock et al., 2011). TRPM2 downregulation. Therefore,TRPM2 is a main A novel permeation pathway was recently proposed player in monocyte functions (Wehrhahn et al., 2010; for TRPM3 (Vriens et al., 2014). In addition to the Kashio et al., 2012). It also plays a critical role in che- canonical S5/S6 permeation pore, a secondary, in- motaxis in dendritic cells (Sumoza-Toledo et al., 2011). In dependent permeation pathway may exist in the S1– pancreatic b cells, TRPM2 activation by cyclic ADPR is S4 module (related to the “omega-pore” in voltage- involved in insulin secretion (Togashi et al., 2006). activated ion channels). This secondary TRPM3 pore TRPM2 channels residing in lysosomes act as ADPR- may be gated by combined application of endogenous activated calcium release channels, which are critically neurosteroids and exogenous chemicals such as clomi- linked to b cell death induced by oxidative stress (Lange trazole. This alternative pathway is still functional et al., 2009). TRPM2 is also involved in NMDA receptor– even if the canonical pore is blocked, and this enables dependent LTD (Xie et al., 2011). TRPM2 has been im- massive Na+ influx at negative voltages and enhances plicated in neuropathic pain (Haraguchi et al., 2012). excitation of sensory neurons, worsening the TRPM3- Finally, megakaryocytes are stimulated via ADPR- dependant pain response. dependent TRPM2 activation (Nazıroglu, 2011a,b). TRPM4 shares a 40% sequence homology with TRPM5. A splice variant of TRPM2 has been reported as Both channels are activated by Ca2+; this is interesting a cell-shrinking or hypertonicity-induced cation chan- because these channels are Ca2+ impermeable and they nel, which is functionally involved in apoptosis and cell function as monovalent cation permeating/depolarizing proliferation (Numata et al., 2012). ion channels. TRPM4 contains a number of binding sites 698 Nilius and Szallasi for CaM in the C-terminal end, and for ATP in the excitable cells following action potentials. TRPM4 has N-terminal end. The channel also has phosphorylation also been implicated in cerebral blood flow regulation and sites for PKC. All of these domains modulate the Ca2+ causes smooth muscle contractions. In cerebral arteries, it sensitivity of the channel (Nilius et al., 2005a). Two is involved in myogenic constrictions. Its activation gen- different splice variants, TRPM4a and TRPM4b, have erates transient inward currents that modulate smooth been identified but only TRPM4b is a functional channel, muscle as well as cardiac muscle contractions (Guinamard whereasTRPM4aactsasanegative regulator (Nilius and et al., 2010). This channel negatively regulates catechol- Vennekens, 2006; Gees et al., 2012). amine secretion from chromaffine cells. Indeed, Trpm4- Expression of TRPM4 is ubiquitous, with high deficient mice develop hypertension due to an increased expression levels found in kidney, heart [abundant in release of catecholamines (Mathar et al., 2010). In pan- 2+ sinoatrial (SA) node and pacemaker tissue], placenta, creatic a cells, an increase in [Ca ]i causes glucagon brain [cerebellum, hippocampus, striatum], and pan- secretion. Depolarizing currents generated by TRPM4 creas. Significant TRPM4 expression was also reported constitute an important component in the control of this in the human gastrointestinal tract, prostate, colon, response (Marigo et al., 2009). The physiologic role of bladder (mostly in detrusor muscle), testes, lymphocytes, TRPM4 in the pancreas is, however, still unclear. spleen, lung, pituitary, skeletal muscle, adipose tissue, TRPM5 and TRPM4 are closely related channels and bone (reviewed in Ullrich et al., 2005; Nilius and with 40% sequence homology. TRPM5 is a monovalent- Vennekens, 2006). selective cation channel with negligible Ca2+ perme- 2+ TRPM4 is directly activated by an increase in [Ca ]i, ability. TRPM5 expression was shown mainly in the which also leads to desensitization of the channel. large intestine, pancreatic b cells, duodenum, stomach, Desensitization is due to a Ca2+-induced breakdown of lung, testis, brain, and the vomeronasal organ (Fonfria PIP2 (Nilius et al., 2006). TRPM4 is a voltage-dependent et al., 2006b; Colsoul et al., 2010). It is noteworthy that channel, activated by depolarization. The channel is TRPM5 is expressed in cells and other inhibited by intracellular ATP and several other nucleo- chemosensory cells of the taste signaling cascade in the tides, as well as by polyamines (Nilius and Vennekens, vomeronasal organ, olfactory epithelium, brainstem, 2006). PKCd, CaM, and decavanadate all activate enterocrine cells in stomach, duodenum, large intestine, TRPM4 probably via an increase of its sensitivity to respiratory tract, and pancreatic b cells (see Nilius and 2+ elevated [Ca ]i. ROS also activate TRPM4, as does the Appendino, 2013). Furthermore, TRPM5 expression pyrazole derivative, BTP2 [N-{4-[3,5-bis(trifluoromethyl)- was reported in the testis, where it is believed to be 1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole- involved in spermatogenesis (Li and Zhou, 2012). 5-carboxamide]. The channel protein may associate In solitary chemosensory cells, TRPM5 mainly with the sulfonylurea receptor 1 (Sur1) which renders functions as a regulator of the release of several it sensitive to the KATP channel blocker glibenclamide endocrine messengers, including the two incretin (Woo et al., 2013). hormones, glucagon-like peptide-1 (GLP-1) and gas- In nonexcitable cells, activation of TRPM4 acts as a trin inhibitory polypeptide, as well as ghrelin, CCK, break on Ca2+ entry following receptor-mediated Ca2+ and the peptides b-endorphin, Met-enkephalin, signalsbyreducingthedrivingforceforCa2+ entry and uroguanylin (Kinnamon, 2012). Some TRPM5- through store-operated channels. This mechanism un- containing taste receptor cells are coupled (as in the derlies the increased Ca2+ signal that follows IgE- gastric groove) to neuronal nitric-oxide synthase dependent activation of mast cells. In Trpm4 knockout (nNOS), suggesting paracrine interactions (Eberle mice, mast cell activation leads to enhanced allergic et al., 2013). TRPM5 in taste receptor cells of the response (Vennekens et al., 2007). Via the same mec- tongue is important for the transduction of sweet, hanism, TRPM4 is involved in nuclear factor of activated amino acid, and bitter stimuli that bind to GPCRs T cells (NFAT) signaling, cytokine production, and that are coupled to PLCb.This,inturn,causesCa2+ motility in T lymphocytes (Weber et al., 2010). release from the endoplasmic reticulum via IP3 and In magnocellular cells of the supraoptic nucleus and resultant IP3R activation (Chaudhari and Roper, the paraventricular nucleus of the hypothalamus, TRPM4 2010; Nilius and Appendino, 2013). TRPM5 is directly plays a role in magnocellular cells of the supraoptic activated by intracellular Ca2+, but has a higher 2+ 2+ nucleus and the paraventricular nucleus of the hypothal- affinity for [Ca ]i.Ca also desensitizes TRPM5 amus in the generation of fast after depolarization, which currents (i.e., it functions as a negative feedback determines the distinct firing properties of vasopressin- mechanism for channel activation). Desensitization by 2+ releasing neurons in this region (Teruyama et al., 2011). Ca is attributed to PIP2 depletion (Nilius et al., These neurons also express TRPV1 and TRPV4, with 2007a). Similar to TRPM4, TRPM5 is activated by TRPV1 being essential for their spontaneous activity heat. Heat activation results in a leftward shift of the (Sudbury and Bourque, 2013). In general, TRPM4 seems voltage dependence of activation (Nilius et al., 2005b). to act as a pacemaking channel in several neuronal TRPM5 is directly activated by arachidonic acid, and networks. It also generates spontaneous depolarization in indirectly by monoglyceride lipase, cyclooxygenase TRP Channels as Drug Targets 699

(COX)-2, and PLA2, suggesting that arachidonic acid can form both homomers and heteromultimers with is a native endogenous TRPM5 modulator (Oike et al., TRPM6. TRPM7 is ubiquitously expressed. High ex- 2006). pression levels were found in human heart, pituitary TRPM5 acts as a positive regulator of glucose-induced gland, bone, and adipose tissue (Fonfria et al., 2006b). insulin release. Glucose generates bursts of action TRPM7 can also function as an intracellular channel. potentials that subsequently trigger a Ca2+ transient. For example, in cholinergic synaptic vesicles, TRPM7 The depolarization during the interburst interval which regulates neurotransmitter release. TRPM7 forms determines the frequency of the Ca2+ transients (and, a complex with synapsin I and synaptotagmin I, and therefore, the efficiency of insulin release) is sensitively it directly binds to snapin and controls the EPSP controlled by the depolarizing Ca2+-activated currents amplitude in a pH-dependent fashion (Krapivinsky through TRPM5. In addition, there seems to be a role et al., 2006). for TRPM5 in glucose-induced insulin secretion beyond TRPM7 is permeable to divalent cations: it conducts membrane depolarization (reviewed in Colsoul et al., Mg2+ (but also protons). Similar to TRPM6, when 2011, 2013). permeated predominantly by monovalent cations, the TRPM6 contains a functional serine/threonine protein current-voltage relationship of the channel exhibits kinase in the C-terminal end of the protein, which pronounced outward rectification that results from resembles members of the a-kinase family, a family voltage-dependent block by extracellular divalent with no sequence homology to any other conventional cations. TRPM7 is also a channel permeable for trace protein kinases (Drennan and Ryazanov, 2004). TRPM6 metals such as Zn2+,Co2+, and Mn2+. TRPM7 is tightly 2+ functions as a homotetramer, but it can also form a regulated by [Mg ]i and also by intracellular Mg-ATP, functional heterotetramer with TRPM7. TRPM6 ex- which causes inhibition. Inward currents through pression is found predominantly in epithelial cells of the TRPM7 are activated by low pH, which decreases kidney (distal convoluted tubule) and small intestine, block by Ca2+ and Mg2+ of monovalent currents the colon, the mammary gland, and the testes. through the channel. Hypoxia and ROS production TRPM6 forms an Mg2+-permeable channel. The ex- induce activation of the channel in brain neuronal cells pression of TRPM6 is regulated by multiple factors, with concomitant Ca2+ overload and Mg2+ deregulation including dietary Mg2+, magnesiotropic hormones, and (Zhang et al., 2011a). NGF and its receptor, TrkA, . TRPM6 is constitutively active. Low pH poten- reduce this hypoxic overexpression of TRPM7 via tiates the activation of TRPM6. Intracellular Na+-and a PI3K pathway (Jiang et al., 2008a). In this respect, Mg2+-ATP decrease currents through TRPM6. ATP it is interesting that many exogenous compounds (e.g., regulates TRPM6 channel activity via its a-kinase do- ginsenoside and carvacrol) block TRPM7 (Parnas et al., main independently of the a-kinase activity. TRPM6 is 2009). 5-Lipoxygenase inhibitors are also potent block- activated by low 2-APB concentrations (whereas TRPM7 ers of the channel. TRPM7 is believed to be a mecha- is inhibited) and currents through TRPM6/TRPM7 nosensor channel. It is activated by stretch. TRPM7 is heteromers are only slightly enhanced (Dimke et al., thought to be involved in volume regulation. Increased 2011). EGF is a magnesiotropic hormone that increases shear stress induces a translocation of TRPM7 to the TRPM6 activity. It upregulates TRPM6 expression via plasma membrane and increases currents through the activation of an ERK/activator protein-1–dependent path- channel (Oancea et al., 2006; Numata et al., 2007). way. Expression of the channel is also upregulated by the Most important, TRPM7 is critical for regulating PI3K/Akt/mammalian target of rapamycin (mTOR) path- cellular growth and embryonic development. In vivo, way, and rapamycin (an immunosuppressant drug that global disruption of TRPM7 in mice results in embry- blocks mTOR) reduces TRPM6-mediated Mg2+ transport. onic lethality before embryonic day 7 (Jin et al., 2008, The receptor for activated C-kinase 1 was identified as 2012a). , pluripotent stem cells and neural stem the first associated protein of the TRPM6 a-kinase do- cells with TRPM7 disruption show a defect in the main. The intracellular Mg2+ concentration plays a feed- formation of the stem cell monolayer that is required for back role in controlling TRPM6-mediated Mg2+ influx, organogenesis. The TRPM7 channel kinase is required preventing Mg2+ overload during epithelial Mg2+ for embryogenesis (Jin et al., 2012a; for a review, see transport (Cao et al., 2008). TRPM6 is downregulated Paravicini et al., 2012). During early embryogenesis, by oxidative stress, which is attenuated by methionine TRPM7 is critical for heart development (myocardial sulfoxide reductase B1, an antioxidant enzyme coex- proliferation), although this mechanism seems to be pressed with TRPM6 (van der Wijst et al., 2009; Cao dispensable after birth (Sah et al., 2013). In adult et al., 2010). In summary, TRPM6 is an essential for tissues, TRPM7 may play a role as a pacemaker intestinal Mg2+ absorption and renal Mg2+ reabsorption; channel in Cajal cells and thus affect intestinal thus, it plays a critical role in Mg2+ homeostasis (Ferrè motility (Kim et al., 2011a). The channel is also et al., 2011). present in human atrial myocytes, where its expres- TRPM7 is 50% homologous to TRPM6. It also con- sion is upregulated during atrial fibrillation (Zhang tains a C-terminal a-kinase domain (Fig. 13). TRPM7 et al., 2012c). 700 Nilius and Szallasi

TRPM7 increases NOS expression in endothelial stable complexes with intracellular polyphosphates and cells and thereby facilitates NO production in an ERK their enzymatic breakdown inhibits the channel. The G pathway-dependent manner. Therefore, TRPM7 was protein subunit Gaq binds to TRPM8. Activation of a Gq- postulated to be an important player in vasomotor coupled receptor directly inhibits TRPM8 (Zhang et al., control (Inoue and Xiong, 2009). High-calcium micro- 2012b). TRPM8 is also inhibited by the Gi protein/ domains (“calcium flickers”), which are essential for adenylate cyclase/cAMP/PKA signaling cascade (Bavencoffe activation of cell migration, are generated by TRPM7 et al., 2010). activation. The channel interacts at the posterior The role of TRPM8 as a cold sensor in the somato- appendix in many migrating cells with the calcium- sensory system is well established. It is also involved in activated potassium channel, KCa3.1. TRPM7 effects somatosensation and nociception (Dhaka et al., 2006). on migration play a role in angiogenesis (Wei et al., TRPM8 channels play a role in the regulation of body 2009a) and in osteoclastogenesis (Yang et al., 2013) temperature. Agonists of this “cold temperature sensor” and regulate the function of vascular endothelial cells cause a transient increase in body temperature by (Baldoli and Maier, 2012; Baldoli et al., 2013). stimulating BAT thermogenesis; this could constitute TRPM8 shares an approximately 42% sequence id- a promising way to treat obesity (Gavva et al., 2012). entity with TRPM2, its closest relative. TRPM8 functions Furthermore, as discussed later, TRPM8 is required for as a homotetramer. It is highly expressed in pain- and lacrimation and contributes to the regulation of basal cold-sensitive C fibers, as well as in the bladder, prostate, tear flow (Parra et al., 2010). Unexpectedly, TRPM8 is hippocampus, skin, brown adiposetissue(BAT),vascular involved in the regulation of tonic GABAergic inhibition smooth muscle cells, macrophages, and sperm (reviewed in the hippocampus (Zhang et al., 2008e). in Voets et al., 2007). TRPM8 is activated by cooling (,22°C) and pharmacological agents [e.g., menthol D. Ankyrin Transient Receptor Potential Subfamily (structure shown in Fig. 6), , , and TRPA1 is the only mammalian member of this family. ] and chemicals [e.g., and carboxamides] Its name derives from the unusually high number (14– that evoke a cooling sensation. Extracellular protons 19) of ankyrin repeats in the N terminus (Fig. 2). TRPA1 suppress the activity of TRPM8 probably via surface further contains an N-terminal Ca2+-binding EF hand charge screening and an intracellular pH drop. TRPM8 domain and has at least 11 (of 31) reactive cysteines for is inhibited by an increase in extracellular Ca2+,because covalent modification, but lacks the typical 25-amino of a surface charge screening (Mahieu et al., 2010). acid TRP domain (Nilius and Owsianik, 2011; Nilius Structurally diverse agents function as TRPM8 an- et al., 2012). tagonists, including the first-generation TRPV1 blockers In Drosophila, TRPA1 plays a crucial role in the BCTC [N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2- temperature control of the circadian rhythm: loss of yl)tetrahydropyrazine-1(2H)-carboxamide; structure TRPA1 alters the expression of the circadian clock shown in Fig. 9], and , and the generic TRP protein period (per) in pacemaker neurons (Lee and channel blocker ruthenium red (reviewed in Nilius Montell, 2013). In mammals, TRPA1 is predominantly et al., 2007b; DeFalco et al., 2011). Interestingly, the expressed in nociceptive neurons (DRG as well as tri- “endovanilloid” anandamide and NADA inhibit TRPM8 geminal and nodose ganglia) and the organ of Corti, and activation by both icilin and menthol. Thus, ananda- is also highly expressed in the dental pulp. However, the mide and NADA might represent the first endogenous channel is also widely expressed in brain, heart, small TRPM8 inhibitors (De Petrocellis et al., 2007). Arach- intestine, lung (both in fibroblasts and epithelial cells), idonic acid also inhibits TRPM8. This inhibition is skeletal muscle, skin (keratinocytes), bladder, prostate, mediated by a cytosolic PLA2 (Bavencoffe et al., 2011). vascular endothelial cells, and pancreas (for a review By contrast, TRPM8 seems to be activated via iPLA2- see Nilius et al., 2012). It is also expressed on taste cells, dependent synthesis of lysophosphatides. TRPM8 is where it associates with the bitter taste receptor downregulated by PKC, probably via an indirect path- TAS2R60 (Knaapila et al., 2012). way. Ca2+ activates PKC, which, in turn, activates pro- TRPA1 is a voltage-dependent, Ca2+-permeable cation tein phosphatase 1, causing dephosphorylation and channel with a relatively high fractional Ca2+ current. desensitization of TRPM8 (Premkumar et al., 2005). The voltage dependence of TRPA1 has been linked to PIP2 is critically involved in the activation of TRPM8 by a conserved leucin residue in the putative pore helix both cold and menthol. It is likely that positively (Wan et al., 2013). Gating of the channel induces charged residues in the highly conserved proximal a dilation of the pore (Chen et al., 2009b) which also C-terminal TRP domain of TRPM8 act as interaction increases its Ca2+ permeability (Karashima et al., 2010). sites for PIP2. Depletion of PIP2 desensitizes the channel TRPA1 was initially identified as a noxious cold sensor viaaCa2+-dependent activation of PLCd (Rohacs, 2007). and a mechanosensor (Story et al., 2003). Indeed, at 2+ Ca -CaM reverses the activating effect of PIP2,switch- least in some species, TRPA1 can be directly gated by ing channel gating to a low open probability state with cold temperatures (Karashima et al., 2009; Wang et al., long closed times. Similar to TRPA1, TRPM8 forms 2013a). Cold activation of TRPA1 occurs in rodents, but TRP Channels as Drug Targets 701 not in humans or monkeys because of a single amino includes an initial potentiation/activation and a sub- acid variation in TM5 (Chen et al., 2013a). In contrast, sequent inactivation after Ca2+ entry through the heat suppresses agonist-dependent TRPA1 activation channel (Wang et al., 2008b). PIP2 is a TRPA1 mo- (Brierley et al., 2011; Wang et al., 2012a). TRPA1 may dulator; however, the net effect of PIP2 is unclear since also function as a mechanosensor and contributes to both activating and inhibiting properties were described a complex that mediates transient, mechanically acti- (Karashima et al., 2008). vated currents in C fibers (Vilceanu and Stucky, 2010). TRPA1 is expressed in skin melanocytes, where it is The major activation mode of TRPA1 is, however, by activated by long-wavelength ultraviolet radiation covalent modification of N-terminal cysteines or lysines (UVR, type A), the main component of sunlight UVR by electrophilic compounds (Bandell et al., 2004), which (Bellono et al., 2013). TRPA1 activation is required for makes TRPA1 an ideal chemosensor. Indeed, TRPA1 is the UVR-induced early increase in cellular melanin con- activated by a plethora of pungent and irritant com- tent (“suntan”); that is, TRPA1 is an essential component pounds, including allyl-isothiocyanate (the pungent of the extraocular phototransduction pathway in human compound in mustard oil and ; see Fig. 7 for melanocytes (Bellono and Oancea, 2013). structure), allicin (from garlic), cinnamaldehyde (from TRPA1 is upregulated under inflammatory condi- cinnamon; structure shown in Fig. 7), thymol (from tions (reviewed in Bautista et al., 2013). Artemin, a ), carvacrol (from oregano), and other electrophilic neuronal survival and differentiation factor of the glial compounds in our food, as well as by industrial cell line–derived neurotrophic factor family, sup- isocyanates, tear gases, (an irritant in vehicle presses TRPA1 expression (Yoshida et al., 2011), but exhaust fumes and ), and hypochlorite (see increases TRPM8 expression (Lippoldt et al., 2013). Bessac and Jordt, 2008; Bautista et al., 2013; Radresa Trafficking and plasma membrane insertion of TRPA1 et al., 2013). Most recently, bacterial endotoxins have is regulated by the ubiquitin hydrolase and the tumor been added to the long list of TRPA1 activators suppressor protein CYCL, which binds to TRPA1 to (Meseguer et al., 2014). increase the cellular pool of the functional protein Furthermore, TRPA1 is activated by endogenous (Stokes et al., 2006). TRPA1 is functionally coupled substances such as 4-hydroxynonenal (Fig. 7), H2O2, with PAR-2. Both proteins are colocalized in DRG. 12,14 15-deoxy-D -prostaglandin J2, and electrophilic lipid Stimulation of PAR-2 activates TRPA1 (Dai et al., acids such as nitrooleic acid, which are released after 2007; Terada et al., 2013). As detailed later, there is inflammation, oxidative stress, or tissue damage (for evidence that TRPV1 and TRPA1 are not only coex- details, see Nilius et al., 2012; Radresa et al., 2013). pressed but also functionally coupled in DRG neurons, In another gating mode, TRPA1 can be activated by where they may also form functional heterotetramers. nonelectrophilic compounds that do not induce co- A major function of TRPA1 is its role in chemical valent modification. Among such compounds are D9- nociception. It initiates pain behavior and avoidance tetrahydrocannabinol (the psychoactive compound in when the organism is exposed to irritant TRPA1 marijuana), two nonpsychoactive cannabinoids (canna- agonists (see Fanger et al., 2010). TRPA1 also plays bidiol and ), as well as BK, menthol, an important role in the respiratory system in sensing , (, propofol, general anes- harmful airborne irritants (Bessac and Jordt, 2008; thetics isoflurane, desflurane), four different 1,4-dihy- Facchinetti and Patacchini, 2010). TRPA1 activation dropyridines (nifedipine, nimodipine, , and leads to the release of the vasoactive peptides sub- nitrendipine), the structurally related L-type calcium stance P (SP) and calcitonin gene-related peptide channel agonist BayK8644 [methyl-2,6-dimethyl-5- (CGRP) (Bautista et al., 2013). CGRP release leads to nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine- vasodilation, whereas SP increases vascular perme- 3-carboxylate], polyunsaturated fatty acids (PUFAs), ability during inflammation. In addition to its role as amphipathic such as trinitrophenol, chlor- a chemosensor, thermosensor, and (possibly) mechano- promazine, pinacidil (a KATP channel opener), non- sensor, TRPA1 plays an important role in the vascular pungent capsinoids, and many more. For some of these bed by causing endothelium-dependent smooth muscle compounds, a bimodal effect was reported, changing cell hyperpolarization and vasodilatation that requires from activation at low concentrations to block at high the activity of small and intermediate conductance concentrations (Alpizar et al., 2013). TRPA1 can be Ca2+-activated K+ channels (Earley, 2012). activated by gasotransmitters such as NO, hydrogen TRPA1 also has an influence on gastrointestinal sulfide (H2S), CO, CO2, and ozone. It is interesting that motility (reviewed in Blackshaw et al., 2010, 2013). TRPA1 is also activated by both increased O2 concen- TRPA1 agonists delay in vivo gastric emptying through tration and hypoxia (Takahashi et al., 2011b). TRPA1 is pathways. TRPA1 appears to be function- 2+ 2+ directly activated by an increase in [Ca ]i,byin- ally coupled with the L-type voltage-dependent Ca tracellular Zn2+ and intracellular alkalinization. Ele- channel in pancreatic b cells and mediates insulin 2+ vated [Ca ]i also causes a striking feedback inhibition secretion. In epidermal keratinocytes, TRPA1 affects 2+ 2+ that may depend on Ca entry. Thus, elevation of [Ca ]i the expression of proinflammatory cytokines, including 702 Nilius and Szallasi interleukin (IL)-1a and IL-1b (Bautista et al., 2013). In potentiation by acidic pH (Dong et al., 2009). Under the CNS, TRPA1 modulates glycinergic transmission resting conditions, the basal activity of TRPML1 (and in synapses of the medullary dorsal horn neurons probably all TRPML channels) is most likely very low. (Nilius, 2012). In the presynaptic terminals of magno- Stimulation of channel activity occurs by variations in cellular neurosecretory cells that produce vasopressin luminal pH or synthesis of specific phosphoinositides in the supraoptic nucleus, TRPA1 activation enhances (e.g., PIP2) that bind to a polybasic domain in the N glutamate release and plays a role in the hippocampus terminus. Activation of TRPML1 mediates release of (for a review, see Nilius, 2012; Vennekens et al., 2012). Ca2+ and (maybe also Fe2+) from the lumen of endo- somes and lysosomes. TRPML1 regulates the activity of E. Mucolipin Transient Receptor Potential Subfamily other membrane proteins such as TPCs. The mucolipin TRP subfamily (TRPML) contains Efflux of luminal Ca2+ promotes the recruitment of three members that are mostly expressed in intracel- specific effectors, such as a-1,3-mannosyltransferase, lular vesicles of the endolysosome system. The name is that interact with endosomal proteins that are implicated derived from mucolipin, a protein responsible for the in trafficking of endosomes/lysosomes [e.g., proteins of the neurodegenerative disease mucolipidosis type IV (ML- endosomal sorting complex required for the transport IV). TRPML1 and TRPML3 are relatively small machinery, Alix and the product of the tumor suscepti- proteins consisting of fewer than 600 amino acid bility gene 101 (TSG10)]. TRPML1 also associates with residues. They contain targeting motifs to the endo- members of the -associated protein transmem- plasmic reticulum and lysosomes/endosomes in the brane family, which is important for the organelle N termini and C termini and are characterized by a large remodeling (Spooner et al., 2013). TRPML1 might be loop with multiple N-glycosylation sites between the also coupled to transducer of regulated CREB activity-1, two first transmembrane segments that are located in which supports a role in organelle fusion (Venkatachalam the lumen of the organelles. TRPML functions are et al., 2013). After Ca2+ release, TRPML1 tethers to the difficult to study because of their localization in cell soluble N-ethylmaleimide–sensitive factor attachment organelles (Fig. 3). They play a crucial role in the receptor protein receptor, a complex required for the final trafficking and differentiation of early endosomes to steps of endolysosomal fusion. TRPML1 also regulates multivesicular bodies, late endosomes, lysosomes, and membrane retrieval from hybrid organelles, thus allowing recycling endosomes (Dong et al., 2010). Similar to organelle reformation (Shen et al., 2011b). TRPML1 TRPML channels in the endolysosomal pathway, two- activation is inhibited by PKA. pore channels (TPCs), namely TPC1, TPC2, and TPC3, In general, TRPML1 is important for endosome/ are found in intracellular organelles, in particular in lysosome function, the fusion of amphisomes (autophagic endosomes and lysosomes, and interact with TRPMLs. vacuoles) with lysosomes, endosome differentiation (bio- TRPML1 is a Ca2+-, Fe2+-, and Zn2+-permeable non- genesis), and trafficking (Fig. 3; see Colletti and Kiselyov, selective cation channel that is impermeable for protons 2011). TRPML1 also plays a role in membrane trafficking. and shows a strong, inwardly rectifying IV curve (see Abnormal lipid accumulation resulting in enlarged late Colletti and Kiselyov, 2011). TRPML1 homomultimer- endosomes occurs under conditions when TRPML1 is izes as well as heteromultimerizes with TRPML2 and downregulated. TRPML1 is required for the formation of TRPML3 (Dong et al., 2010). lysosomes from autolysosomes, and for the Ca2+-dependent TRPML1 is ubiquitously expressed, with the highest lysosomal (Fig. 3). TRPML1 is not involved (as expression found in the brain, heart, kidney, liver, and was previously assumed) in the H+ homeostasis of en- spleen. It is an intracellular channel that is mainly dolysosomes. However, as a Fe2+-release channel, it is located in late endosomal and lysosomal compartments involved in Fe2+ homeostasis (Dong et al., 2008). A dif- (Dong et al., 2010; Gees et al., 2010). Targeting of ferent function of TRPML1 is implicated in the reg- TRPML1 to lysosome (i.e., trafficking from the trans- ulation of gastric acid secretion from apical-membrane Golgi network) requires dileucine motifs in both the trafficking in parietal cells (Chandra et al., 2011). N-terminal and C-terminal cytosolic tails that directly TRPML2 was identified together with TRPML3. It interact with the clathrin adaptor proteins AP1 and interacts with TRPML1 and TRPML3 (see Flores and AP3. Another sorting motif is the stretch of polar and Garcia-Anoveros, 2011). TRPML2 forms inwardly recti- hydrophobic amino acids that bind to a-1,3-mannosyl- fying channels and is permeable to Ca2+,Na+,andFe2+. transferase in a Ca2+-dependent manner (Abe and It is present in lysosomes, late endosomes, recycling Puertollano, 2011). endosomes, and, at a lower level, in the plasma TRPML proteins are functionally difficult to access membrane. The location of TRPML2 in recycling en- because of their localization in intracellular organelles. dosomesisdifferentfromthatofTRPML1.TRPML2 TRPML1 was first studied in the varitint-waddler (Va)– is widely expressed in immune cells as well as the like mutant TRPML1 channel (V432P) that exhibits heart, kidney, liver, spleen, thymus, and lymphocytes. plasma membrane localization. This mutant TRPML1 TRPML2 shows a low basal activity and is slightly channel is constitutively active and displays a small potentiated by acidic pH (Dong et al., 2010). It colocalizes TRP Channels as Drug Targets 703 with major histocompatibility complex (MHC) protein which causes autosomal dominant polycystic kidney class I, glycosylphosphatidylinositol-anchored proteins, disease (ADPKD); 2) TRPP2 was previously known as and ADP-ribosylation factor-6; TRPML2 contributes to TRPP3, polycystin-like, PKD-like 2, and PKD2-like 1; the regulation of sorting of glycosylphosphatidylinositol- and 3) TRPP3 was previously referred to as TRPP5 or anchored proteins. TRPML2 is involved in the regulation PKD2-like 2 (Wu et al., 2010). [Please note that this of the trafficking between recycling endosomes and the new nomenclature introduced by Clapham and col- cell surface. This function requires ADP-ribosylation leagues in their recent Pharmacological Reviews article factor-6 but is clathrin independent (Karacsonyi et al., (Wu et al., 2010) differs from the still official HUGO 2007). Similar to TRPML1, it plays a role in lysosomal .] The members possess a coiled-coil storage of fragmented cell organelles and membrane structure in their C terminus, form polymodal multiprotein/ debris. TRPML1/TRPML2 heteromultimers also play a ion channels complexes, and have a Ca2+ binding EF role in membrane trafficking. Moreover, TRPML2 is hand motif in the C terminus. involved in regulation of Fe2+ , especially TRPP1 is a Ca2+-permeable, nonselective cation during maturation of B and T lymphocytes (Dong et al., channel. TRPP1 is widely, if not ubiquitously, ex- 2010). pressed. It is abundant in the kidney (both motile and TRPML3 has been cloned from the Va mouse, which nonmotile cilia), in bone, lacrimal glands, retina, heart, shows deafness and pigmentation defects due to a and especially in vascular smooth muscle cells (Delmas, mutation in the Trpml3 gene (i.e., an A419P substitution 2005; Patel and Honoré, 2010). It contains an in the linker region between S4 and S5). TRPML3 is N-terminal endoplasmic reticulum retention signal that found in the cochlea, eye, kidney, lung, skin (melano- locks the channel in intracellular compartments. The cytes), spleen, and thymus, as well as in somatosensory plasma membrane location of TRPP1 is maintained via neurons (Di Palma et al., 2002; Samie et al., 2009; a C-terminal coiled-coil domain. TRPP1 interacts with reviewed in Noben-Trauth, 2011). TRPML3 is found in PKD1 (also know as ), a large 11 trans- early and late endosomes, early lysosomes, and also in the membrane segment plasma membrane adhesion pro- plasma membrane. TRPML3 accumulates in the plasma tein. Probably one PKD1 forms a complex with three membrane if endocytosis is inhibited, and is highly TRPP1 molecules. A coiled-coil domain in the C ter- expressed in autophagosomes when is induced minus of TRPP1 is critical for the formation of this (Kim et al., 2009). complex (Yu et al., 2009c). Phosphofurin acidic cluster TRPML3 is a Ca2+-permeable channel that is H+ sorting proteins PACS-1 and PACS-2, which interact impermeable. Its activity is regulated by extracytosolic with an acidic amino acid cluster within the C-terminal Na+. In vitro, Na+-free medium is required for channel domain of the TRPP1, regulate its distribution between activation. The channel slowly inactivates after such the endoplasmic reticulum and cell surface (Köttgen activation and subsequent readministration of Na+ et al., 2005). Of note, both TRPP2 and TRPP3 lack this dramatically increases the current. TRPML3 is also PACS domain. regulated by Ca2+ and the luminal pH. The C-terminal region of TRPP1 also binds to the TRPML3 is a prominent regulator of endocytosis (see endoplasmic reticulum stress-inducible proteins Herp Zeevi et al., 2009), membrane trafficking, and autophagy, and protein kinase C substrate 80K-H complex (PKCSH/ likely by controlling the Ca2+ in the vicinity of cellular 80K-H), which are involved in carbohydrate processing, organelles. Overexpression of TRPML3 causes enlarged folding, and translocation of newly synthesized glycopro- endosomes and leads to an increase in autophagy. Down- teins. Both proteins stimulate retention in the endoplas- regulation causes enhanced endocytosis (Kim et al., mic reticulum and protect the channel from endoplasmic 2009). In the Va mouse, the TRPML3 mutant results in reticulum–associated protein degradation (Chapin a constitutively active channel that causes increased Ca2+ and Caplan, 2010). Surface localization of TRPP2 is influx and cell death, followed by a variegated pigmen- regulated by phosphorylation via glycogen synthase tation due to melanocyte dysfunction and a loss of hair kinase 3 at a site in its N-terminal domain ( cells in the cochlea and vestibulum, which induces et al., 2006). deafness (Di Palma et al., 2002). Finally, TRPML3 is For activation, TRPP1 requires the activity of PLC expressed in the tongue, where it may function as a salt and PI3K. Formation of PIP2 suppresses EGF-induced sensor in taste buds (Moyer et al., 2009). currents and acts as negative regulator of TRPP1 (Ma et al., 2005). TRPP1 also interacts with the mamma- F. Polycystic Transient Receptor Potential Subfamily lian diaphanous-related formin 1, a downstream effector The nomenclature of polycystin subfamily of TRP of Ras homolog gene family, member A. Importantly, this channels is somewhat confusing, especially because the interactionconfersvoltagedependencetothechannel.At old terminology appears to be firmly entrenched in the negative potentials, the TRPP1 channel activity is spe- literature. The TRPP family only contains three cifically blocked by mammalian diaphanous-related for- members: 1) TRPP1 was previously known as TRPP2, min 1 that switches in a Ras homolog gene family, member PKD2, or PC2, a name derived from the second gene, A–induced manner from an autoinhibited state at negative 704 Nilius and Szallasi potentials to an activated state at positive potentials (Bai Ca2+-permeable cation channel in primary cilia. It is et al., 2008). activated by the hedgehog pathway proteins GLI2 TRPP1 functions as a Ca2+ entry channel in the and smoothened (SMO). This channel is thought to be plasma membrane, and a Ca2+-activated intracellular an important regulator of the Ca2+ concentration in calcium release channel in the endoplasmic reticulum. a subciliary compartment (DeCaen et al., 2013; Delling It is Ca2+ and pH dependent. A reduction in the cy- et al., 2013), with little or no contribution to the global tosolic pH inhibits the channel (Koulen et al., 2002). As intracellular Ca2+ concentration in primary cilia. It is aCa2+ release channel in the endoplasmic reticulum, intriguing to speculate that TRPP2 may play an TRPP1 interacts with the IP3R and probably also with important role in ciliopathies, in particular in neuro- stromal interacting molecule-1. Initially, TRPP1 was developmental disorders (for an exhaustive review, see described as a mechanosensitive channel in the pri- Valente et al., 2014). mary cilia of renal epithelial cells and vascular TRPP3 shares 59% sequence homology with TRPP1. endothelial cells. It regulates responses to shear stress It forms Ca2+-permeable nonselective cation channels in blood vessels and in the tubular system of the and is widely expressed in fetal tissues (Guo et al., kidney. This function requires interaction with PKD1 2000). The activation mechanism of TRPP3 remains to and/or TRPV4 and results in a flow-induced increase in be determined. 2+ [Ca ]i (Patel and Honoré, 2010). In addition to TRPV4, TRPP1 may associate with TRPC1 and TRPC4. The II. Transient Receptor Potential Channels: adhesion protein PKD1 is supposed to form a flow- Hereditary Diseases (Transient Receptor sensitive ion channel complex in the primary cilium of Potential Channelopathies) both epithelial and endothelial cells. Flow sensing via TRPP1 in primary cilia is required for establishing the In the past few years, several hereditary diseases left-right asymmetry in the body manifested in the caused by defects in genes encoding TRP channel heart, lungs, and gut (Yoshiba et al., 2012). proteins have been described (Fig. 14; for comprehen- It was recently shown that TRPP1 inhibits SACs sive reviews, see Abramowitz and Birnbaumer, 2007, (similar to Piezo1). TRPP1 interacts with filamin A. This 2009; Nilius, 2007; Nilius and Owsianik, 2010b; Everett, cross-linking protein is critical for SAC regulation (Sharif- 2011): collectively, these diseases are referred to as Naeini et al., 2009; Nilius and Honore, 2013; Peyronnet TRP channelopathies. TRP channels also play a role in et al., 2013). TRPP1 is involved in angiogenesis via mod- cancer development and, thus, may represent novel ulation of VEGF and type-1 interferons (IFNs), including anti-cancer drug targets. The evidence for hereditary IFNa/b, and plays a role in -induced neurito- TRP involvement in cancer is, however, weak and genesis (Italia et al., 2011; Zheng et al., 2011). inconclusive (see Santoni and Farfariello, 2011). Here TRPP2 has 71% sequence homology with TRPP1. It we describe the hereditary TRP channelopathies and is a Ca2+-permeable nonselective cation channel. It is mention a few representative examples of pathogenic expressed in the brain (e.g., hypothalamus), heart, TRP dysfunctions. Interested readers are also referred retina, skeletal muscle, spleen, tongue (circumvallate, to the Online Mendelian Inheritance in Man (OMIM) foliate, and fungiform papillae), and testis. It seems to database, a frequently updated database on genetic be highly expressed in embryonic tissues (Wu et al., diseases (www.ncbi.nlm.nih.gov/OMIM). 1998; Ishimaru et al., 2006). TRPP2 is coexpressed (and associates) with PKD1-like 2. A. Canonical Transient Receptor TRPP2 was first described as a channel activated by Potential Channelopathies both low extracellular pH and citric acid (Ishimaru TRPC channel dysfunction has been linked to many et al., 2006). Activation by external pH requires PKD1- acquired diseases, such as cardiovascular, pulmonary, like 3. In the absence of PKD1-like 3, TRPP2 shows skin diseases, and inflammation (Abramowitz and constitutive basal activity and is inhibited by extracel- Birnbaumer, 2009), but there are only very few examples lular citric acid (or low pH) and is potentiated by of hereditary channelopathies. TRPC1 is thought to be alkalization. It is activated by Ca2+ and cell swelling involved in Gorlin syndrome, also known as basal cell and shows clear voltage dependence (e.g., depolariza- nevus syndrome (OMIM 109400). It is a rare, autosomal tion) (Shimizu et al., 2009). dominant disorder with complete penetrance and vari- TRPP2 probably functions as a sour taste receptor. It able expressivity. The syndrome is characterized by also acts as a pH sensor in the cerebrospinal fluid, odontogenic keratocysts of the mandible and postnatal expressed in specific neurons aligning the spinal canal tumors including multiple basal cell carcinomas (BCCs). (Huang et al., 2006). TRPP2 seems also to be essential Mutations in the tumor suppressor gene PTCH1 (a for the development of the kidney and retina (Nomura member of the patched gene family and a receptor for et al., 1998). sonic hedgehog) and in the TRPC1 gene seem to be It is noteworthy that TRPP2 has now been identi- responsible for the development of the tumors (Sauer fied as component (together with PKD1-like 1) of a et al., 2011). A novel spliced isoform of TRPC1 with TRP Channels as Drug Targets 705 exon 9 deletion was suggested to play a role in human show a gain-of-function phenotype. TRPC6 in the ovarian adenocarcinomas (Zeng et al., 2013). In a podocytes associates with the slit protein nephrin that genome-wide association study, single nucleotide is coupled to the nephrin-interacting adapter protein polymorphisms (SNPs) in TRPC1 were discovered CD2AP and to podocin (reviewed in Möller et al., 2009). which are associated with type 2 diabetes mellitus This complex forms the glomerular filter (Reiser et al., (T2DM) (Chen et al., 2013c). 2005). Downstream targets of TRPC6 are still under TRPC3 might be indirectly involved in the patho- investigation. Activation of NFAT in TRPC6 mutants genesis of autosomal dominant spinocerebellar ataxia is blocked by inhibitors of calcineurin, CaMKII, and type 14, caused by mutations in PKCg, which nega- PI3K, independently of Src, Yes, or Fyn. Therefore, it is tively regulates the channel. Mutant PKCg in cerebel- very likely that the calcineurin-NFAT pathway plays lar Purkinje cells of patients with spinocerebellar an important role in mediation of FSGS. The immu- ataxia type 14 could differentially impair TRPC3 nosuppressive agent FK-506 may also inhibit TRPC6 function and disrupt synapse pruning, synaptic plas- (for a review, see Schlöndorff et al., 2009; Wang et al., ticity, and synaptic transmission (Shuvaev et al., 2010; El Hindi and Reiser, 2011). As shown in the rat, 2011). Interestingly, a gain-of-function mutant in knockdown by nephrin and/or TRPC6 small interfering TRPC3 causes cerebellar ataxia in mice, the so-called RNA (siRNA) substantially reduces the protein levels Moonwalker (Mwk) mouse (Becker et al., 2009). of nephrin and/or TRPC6 and counteracts the perme- However, a genetic screen for TRPC3 mutations in ation defect (Hauser et al., 2010). patients with late-onset cerebellar ataxia does not TRPC6 is also involved in the steroid-resistant support a contribution of TRPC3 mutants to this nephrotic syndrome (OMIM 600995). In Turkish disease (Becker et al., 2011). TRPC3 might also be children, three mutations and an intronic nucleotide indirectly involved in Williams–Beuren syndrome, a substitution were described causing the sporadic form neurodevelopmental disorder associated with distinc- of this disease (Mir et al., 2012). A SNP in the promoter tive “elfin” facial appearance, unusually cheerful per- region of TRPC6 and a missense variant in exon 4 of sonality, heart or blood vessel problems (e.g., TRPC6 might be putative causal gene variants for supravalvular aortic stenosis), and hypercalcemia. The infantile hypertrophic pyloric stenosis (OMIM 179010) main genetic defect lies in the transcription factor IIi (Everett et al., 2009). Mutations in TRPC6 may also gene (encoding TFII-I), which normally suppresses cell contribute to idiopathic pulmonary arterial hyperten- surface accumulation of TRPC3; consequently, TFII-I sion (IPAH) (Yu et al., 2004), in which SNPs were mutations cause a TRPC3 gain-of-function phenotype described in a cohort of patients. The abnormal TRPC6 (Letavernier et al., 2012). transcription is linked to nuclear factor-kB, an in- Excessive Ca2+ influx mediates many cytotoxic pro- flammatory and carcinogenic transcription factor (Yu cesses, including those associated with autoimmune et al., 2009b). inflammatory diseases such as acute pancreatitis, and Sjögren syndrome (also known as sicca syndrome), B. Vanilloid Transient Receptor a systemic autoimmune disease in which immune cells Potential Channelopathies attack and destroy the exocrine glands that produce Somewhat unexpectedly, TRPV1, the best charac- tears and saliva. The hallmark symptom of the disorder terized TRP channel, is not yet linked to any is a generalized dryness, typically involving dry mouth hereditary disease (Fig. 12). However, it was suggested (xerostomia) and dry eyes (xerophthalmia). It has been that in the TRPV1 gene may play a role hypothesized that a gain of function of TRPC3 may be in the development and maintenance of chronic pain: involved in the pathogenesis of these diseases, and the indeed, genetic variants of human TRPV1 with M315I TRPC3-selective inhibitor Pyr3 might be beneficial in and I585V channel mutations were found in Caucasian these patients (Kim et al., 2011c). patients with an increased individual susceptibility to TRPC6 is involved in glomerular renal diseases. It is neuropathic pain (Armero et al., 2012). SNPs in TRPV1 required for normal function of podocytes, highly were also identified in a Spanish population with in- specialized epithelial cells that line the urinary surface creased genetic susceptibility to migraine (Carreño of the glomerular capillary tuft. Dysfunction or death of et al., 2012). Interestingly, the M315I variant of the podocytes impairs glomerular permeability and filtra- TRPV1 gene also conferred increased susceptibility tion. This channel is mutated in cases of focal segmental (odds ratio of 1.6) to type 1 diabetes in Ashkenazi Jews glomerulosclerosis (FSGS), type 2 (OMIM 603965) (Sadeh et al., 2013). (Henique and Tharaux, 2012). Of note, the Miller–Dieker lissencephaly syndrome, FSGS is functionally characterized by proteinuria an autosomal dominant congenital disorder character- and progressive decline of renal function. At least 14 ized by a developmental defect in the brain caused by mutations in the N terminus (n = 7) and C terminus incomplete neuronal migration, is due to a (n =6)oftheTrpc6 gene are linked to FSGS type 2 (for 17p13.3 deletion syndrome that includes deletion of a recent update, see Hofstra et al., 2013). All mutations TRPV1 (Laurito et al., 2011). 706 Nilius and Szallasi

Fig. 12. Simplified, schematic representation of the complex regulation of the vanilloid (capsaicin) receptor TRPV1. The upper part of the figure shows the modular structure of TRPV1. The N-terminal intracellular region starts with six ankyrin repeats (dark brown segment) and contains phosphorylation sites for different protein kinases (pale yellow dots; Ser117 for PKCm and PKA, Thr145 and Thr371 for PKA, and Tyr200 for Src kinase). It is followed by the six transmembrane domains (TM1–TM6) with a pore-forming region between TM5 and TM6. Mutational studies revealed the importance of the third and fourth TM domains in vanilloid binding (for more details, see Fig. 4). The lower part summarizes the downstream sensitization of TRPV1 by different mediators released at the site of injury or inflammation. For details and abbreviations, see section I.B. Reprinted with permission from Lázár et al. (2009).

An I585V variant of TRPV1 (that shows decreased (Paltser et al., 2013). Indeed, TRPV1 activators (e.g., channel activity) reportedly might confer decreased arvanil) provide symptomatic relief in a rat model of MS genetic risk for painful knee osteoarthritis (OA) (Valdes (Cabranes et al., 2005). et al., 2011). The same genetic variant of TRPV1, I585V, TRPV2 has been linked to Duchenne muscular is also protective against childhood asthma (Cantero- myopathy, cancer, and diabetes (Nilius et al., 2007b; Recasens et al., 2010). A selective risk association of the Harisseh et al., 2013). However, thus far, no TRPV2 missense TRPV1 SNP, associated with the primary pro- channelopathy has been detected. gressive disease type, was recently discovered in patients TRPV3 is expressed in keratinocytes and in cells with multiple sclerosis (MS) (Paltser et al., 2013). This is surrounding hair follicles. In mice, two gain-of-function all the more interesting because TRPV1 seems to control mutations at a single site cause an autosomal do- the severity and progression of experimental autoimmune minant hairless phenotype with dermatitis (Asakawa encephalomyelitis in mice and may indicate that TRPV1 et al., 2006). In humans, three gain-of-function muta- is a critical disease modifier in experimental autoimmune tions (G573S, G573C, W692G), one of which is identical encephalomyelitis and a novel target for MS therapy to the mouse mutation, cause Olmsted syndrome (also TRP Channels as Drug Targets 707 known as “mutilating palmoplantar keratoderma with and incontinence (Nishimura et al., 2010). A mild form periorificial keratotic plaques” or “polykeratosis of of skeletal dysplasia was recently described as familial Touraine”), a rare disorder characterized by the com- digital arthropathy-brachydactyly (OMIM 606835), bination of periorificial keratotic plaques, bilateral which appears in the first decade of life, characterized palmoplantar keratodermas (horn-like skin growth), by short fingers and deviations in finger , and alopecia, and severe itching (Lai-Cheong et al., 2012; involves mainly irregularities in the articular surfaces Lin et al., 2012; reviewed in Nilius and Biró, 2013). A new (Lamandé et al., 2011). TRPV3 mutant, G573A, was recently identified in patient Thus far, more than 50 TRPV4 mutations (which with Olmsted syndrome that can also cause multiple scatter over the whole protein) have been identified, immune dysfunctions, such as hyper-IgE, elevated follic- causing the six skeletal diseases (Fig. 15). Most of the ular T cells, and persistent eosinophilia (Danso-Abeam mutations are gain-of-function mutations, and the degree et al., 2013). Supporting the role of TRPV3 as an im- of channel overactivity seems to determine the severity of portant channel in skin disorders,rosacea,afrequent the disease (Loukin et al., 2010a,b) (for reviews, see chronic inflammatory skin disease that causes redness Nilius and Owsianik, 2010a; Nishimura et al., 2012; and pimples in the face and forehead, shows upregulation Nilius and Voets, 2013). of TRPV3 (Sulk et al., 2012). A second big surprise was the discovery of the role TRPV4 causes at least nine different channelopa- that TRPV4 plays in autosomal dominant distal thies. The first to be described was the autosomal neuropathies; these involve mainly motoric defects in dominant brachyolmia type 3 (OMIM 113500), a rela- the distal limbs but also in the respiratory system and tively mild skeletal dysplasia characterized by short the vocal cord, and are sometimes associated with stature, platyspondyly (flattened vertebral bodies), sensory defects (for a review, see McEntagart, 2012). reduced intervertebral spaces, scoliosis, or kyphosis The main symptom is muscle atrophy caused by (Rock et al., 2008). Triggered by this surprising finding, degeneration of the motoneurons in the spinal ventral a number of new, TRPV4-caused skeletal dysplasias horn. Most of spinal muscle atrophies are phenotypi- were discovered, all of which show (to various degrees) cally similar and all lead to muscle weakness and short stature, platyspondyly, defects in bone ossifica- wasting. Furthermore, they are often combined with tion, and abnormalities (see Nilius and Owsianik, multiple pulmonary and orthopedic symptoms. Con- 2010a; Nilius and Voets, 2013). These diseases are genital distal spinal muscle atrophy (OMIM 600175) is probably due to a combination of functional defects and a nonprogressive lower motor neuron disorder, re- differentiation abnormalities in chondrocytes of the stricted to the lower part of the body. It is sometimes bone growth plate. associated with arthrogryposis (joint contractures), The spondyloepimetaphyseal dysplasia of Maroteaux bilateral talipes equinovarus (commonly known as (pseudo-Morquio type 2) (OMIM 184095) shows the clubfoot), and flexion contractures of the knees and described pattern but manifestations are limited to the hips. Sometimes, lordosis and slight scoliosis with musculoskeletal system (Nishimura et al., 2010). restricted joint movement are also observed. Congenital Another dysplasia is the spondylometaphyseal dyspla- distal spinal muscle atrophy is a pure motor neuron sia Kozlowski type (OMIM 184252): these patients disease and shows no sensory defects (Zimon et al., exhibit short stature, mostly due to shortening of the 2010). Scapuloperoneal spinal muscle atrophy (OMIM trunk. Defects are observed in the distal metaphysis of 181405) is a syndrome characterized by scapuloperoneal the femur, the femoral neck and trochanteric area. atrophy, laryngeal palsy, scapular winging, muscle Again, platyspondyly is a striking feature (Krakow wasting in the lower limbs, vocal cord paralysis, absence et al., 2009). The discovery that the pseudo-Morquio of tendon reflexes, and sometimes scoliosis and light type 2 syndrome is genetically a new channelop- sensory defects (DeLong and Siddique, 1992; Auer- athy triggered the identification of the genetic cause of Grumbach et al., 2010; Deng et al., 2010). metatropic dysplasia (OMIM 156530), which is the Another TRPV4 channelopathy is the autosomal most severe skeletal TRPV4 channelopathy. Some- dominant hereditary motor sensory neuropathy type times metatropic dysplasia is combined with lethal IIc (Charcot-Marie-Tooth neuropathy type 2; OMIM fetal akinesia. The nonlethal forms are characterized 606071), which is characterized by a variable degree of by short limbs, shortening of all long , enlarge- muscle weakness in the limbs, vocal cords, and in- ment of joints, severe kyphoscoliosis, severe platyspon- tercostal muscles, and by asymptomatic sensory loss. It dyly, severe metaphyseal enlargement, and defects in starts in infancy or childhood. The life expectancy of ossification (Krakow et al., 2009; Camacho et al., 2010; these patients is shortened because of respiratory Unger et al., 2011). failure. Facial asymmetry, tongue fasciculations, and Parastremmatic dysplasia (OMIM 168400) is char- third and sixth cranial nerve palsies have also been acterized by severe dwarfism, thoracic kyphosis, dis- reported (Auer-Grumbach et al., 2010; Deng et al., tortion and twisting of the limbs, contractures of the 2010; Landouré et al., 2010; Rossor et al., 2012). Many large joints, malformations of the vertebrae and pelvis, patients with Charcot-Marie-Tooth neuropathy type 2 708 Nilius and Szallasi

(but also scapuloperoneal spinal muscle atrophy) have is also implicated in Gitelman syndrome (Loffing skeletal symptoms such as pes cavus, hammertoes, et al., 2004), which is characterized by salt-losing lumbar hyperlordosis, and scoliosis (Fig. 16), as well as hypotension, hypomagnesemia, and hypokalemic me- bladder urgency, incontinence, tremor, and dysphagia. tabolic alkalosis due to mutations in the gene of the The TRPV4 mutations in these patients are gain of thiazide-sensitive sodium/chloride cotransporter (NCC) function, associated with neurotoxicity (Fig. 16). There SLC12A3. is clearly some overlap between neuronal/axonal Upregulation of both channels causes hypocalciuria defects and skeletal symptoms. Thus far, 21 mutations (Yang et al., 2010). Interestingly, an ancestral Trpv6 in TRPV4 have been described that can cause neu- haplotype that consist of three nonsynonymous poly- ropathies (Fig. 15). These mutations are mainly local- morphisms results in three mutations in the protein ized in the convex surface of the ARD of the TRPV4 with a gain-of-function phenotype. The frequency of the N terminus (Fig. 15). Mutations causing skeletal dys- ancestral Trpv6 haplotype (carriers are still in the plasia, although scattering through the whole length modern population) is significantly higher in Ca2+ of the channel protein, seem to be more frequently stone formers compared with nonstone-forming indi- located in the concave surface of the ARD. There are viduals. Thus, TRPV6 might play a role in calcium three hot spots for disease-causing mutations (Fig. 15): 1) stone formation in certain forms of absorptive hyper- the ARD domain; 2) the transmembrane region calciuria (Hughes et al., 2008; Suzuki et al., 2008). S3–S5; and 3) a C-terminal region were the channel associates with several members of the cytoskeleton, C. Melastatin Transient Receptor such as tubulin, actin, and microtubule-associated Potential Channelopathies protein 7 (Inada et al., 2012). TRPM1 was previously considered to be a tumor Indeed, the discovery that a large number of mu- suppressor protein in melanoma cells. The Trpm1 gene tations in the same gene (that are often located in the codes two transcripts: TRPM1 channel protein in its same domain of the channel) can cause nine different exons and miR-211 in one of its introns. The loss of diseases creates a challenging puzzle (see Nilius and TRPM1 channel protein is an excellent marker of Voets, 2013). The reasons underlying the phenotypic melanoma aggressiveness, whereas the expression variability (i.e., why these mutant channels result in of miR-211 is linked to the tumor suppressor function these different diseases) remain elusive (Nilius and of TRPM1 (Mazar et al., 2010; Guo et al., 2012; Margue Voets, 2013). Mutations causing neuropathies probably et al., 2013). have a lower penetrance than mutations causing TRPM1 is linked to the autosomal recessive congen- skeletal dysplasias; this may explain why skeletal ital stationary night blindness type 1C (OMIM diseases often show no neurologic symptoms. Because 613216), a clinically and genetically heterogeneous of the high expression of TRPV4 in the inner ear and group of retinal disorders characterized by nonpro- the urothelium, it is not surprising that some patients gressive impaired night vision and decreased visual have also hearing problems and/or bladder symptoms acuity. It is caused by ON bipolar cell dysfunction: such as overactive bladder and incontinence (reviewed TRPM1 channels, gated by the mGluR6 signaling in Verma et al., 2010). cascade, are necessary for the depolarizing light The TRPV4 variant P19S was suggested to predispose response of these cells (Audo et al., 2009; Nakamura the carriers to acquired chronic obstructive pulmonary et al., 2010). In Appaloosa horses, the coat spotting disease (COPD) because of a reduced airway clearance pattern is caused by a loss-of-function mutation in due to decreased cilia activity, which is supposed to be TRPM1. In these horses, TRPM1 dysfunction is due a TRPV4-dependent mechanism (Li et al., 2011b). The a retrovirus insertion that disrupts the transcription same mutation/polymorphism also causes hyponatremia of the TRPM1 gene by premature polyadenylation (Tian et al., 2009). (Bellone et al., 2013). Congenital stationary night TRPV5 and TRPV6 function as Ca2+ (re)absorption blindness type 1C results from the loss-of-function of channels. No TRPV5 or TRPV6 channelopathy has yet rod and cone ON bipolar cells in the mammalian been described. The TRPV5 gene exhibits an unusually retina. Patients also display myopia, reduced central high frequency of four nonsynonymous SNPs among vision, and nystagmus. Unlike the Appaloosa horses, African Americans, all of which increase Ca2+ reab- none of the patients shows abnormal skin pigmenta- sorption and cause hypercalciuria, predisposing the tion (Li et al., 2009). carriers for kidney stone formation (Na et al., 2009). TRPM2 and TRPM7 (Fig. 13), two chanzymes, have Interestingly, in Pendred syndrome, an inherited long been thought to cause Guamanian amyotrophic disorder that causes hereditary deafness, TRPV5 and lateral sclerosis (ALS-G) and parkinsonism-dementia TRPV6 in the inner ear are inhibited because of complex of Guam (PD-G), or parkinsonism-dementia endolymph acidification due to a malfunction of the complex, two related neurodegenerative disorders that 2 2 Cl /HCO3 exchanger SLC26A4, pendrin (Wangemann are endemic in the Western Pacific, including Guam et al., 2007). An indirect involvement of both channels (Plato et al., 2002). ALS-G and PD-G have a multifactorial TRP Channels as Drug Targets 709

Fig. 13. The topographical structure of TRPM7 channels. Reprinted with permission from Gaudet (2009). etiology, including soil and drinking water that is low in TRPM2 might be involved in bipolar disorder type I Ca2+ and Mg2+ buthighinFe2+,Mn2+,andAl3+,aswell (BD-I), characterized by one or more manic (or mixed) as the presence of the putative , L-b-N- episodes, usually followed by major depressive epi- methylamino-L-alanine, derived from the cycad plant sodes. One of the putative susceptibility loci of BD-I is (traditionally, bats that feast on cycad are consid- associated with the chromosomal region that encodes eredadelicacybythenatives). TRPM2 (Liu et al., 2001; Xu et al., 2006, 2009a). SNPs Mutations in the Trpm2 and Trpm7 genes have been in the promoter region of Trpm2 are also significantly implicated in the pathogenesis of these diseases. associated with BD-I. It was postulated that the TRPM2 and TRPM7 are thought to initiate neuronal TRPM2 polymorphism may contribute to the risk for cell death by sensing oxidative stress (TRPM2 is DB (Xu et al., 2009a), especially if it coexists with a redox sensor); indeed, TRPM2 and TRPM7 are a SNP within the iPLA2b gene (Xu et al., 2013). (The crucial for cell viability in neurodegenerative diseases iPLA2b gene encodes an enzyme that is involved in (for a review, see Benarroch, 2008; Szydlowska and oxidative stress.) Tymianski, 2010). A subset of patients with ALS-G and TRPM3 has been recently discussed as a part of the PD-G are heterozygotes for a missense mutation in the genetic background that may contribute to the comor- Trpm2 and Trpm7 genes, which cause fast inactivation bidity between autism and Duchenne muscular dys- and increased sensitivity to inhibitory Mg2+ (Hermosura trophy. Indeed, in some patients, a deletion involving et al., 2005, 2008; Hermosura and Garruto, 2007). It exons 1–9 of TRPM3 has been described (Pagnamenta seems that ion influx through both channels is physi- et al., 2011). TRPM3 might be also involved in the ologically important, and disruption of this influx may, pathogenesis of Kabuki syndrome (OMIM 147920), under certain conditions, contribute to disease states a congenital mental retardation syndrome character- (Hermosura et al., 2008). Because the ALS-G and PD-G ized distinct facial appearance (the name comes from environments are deficient in Ca2+ and Mg2+,anin- the facial resemblance of the patients to the stage creased sensitivity of TRPM7 to inhibition by Mg2+ could makeup used in traditional Japanese theater), heart even worsen the Mg2+ homeostasis in a Mg2+-deficient defects, urinary tract anomalies, hearing loss, hypoto- environment, leading to a reduced intracellular Mg2+ nia, short stature, joint laxity, and unusual dermato- concentration (Schmitz et al., 2003). However, a recent glyphic patterns (Kuniba et al., 2009). linkage analysis did not reveal any evidence in support TRPM4 mutations cause progressive familial heart of the linkage to the Trpm7 , indicating that block type I (OMIM 113900), which is a progressive TRPM7 is probably not associated with ALS-G/PD-G cardiac bundle branch disease affecting the His-Purkinje (Hara et al., 2010). system that exhibits autosomal dominant inheritance. 710 Nilius and Szallasi

TRPM4 mutants show a gain-of-function phenotype convoluted tubule, the most distal part of the probably because of increased surface expression, leading nephron). to depolarization-induced defects in the conduction and The primary defect in HSH is impaired intestinal electrical gridlock (Kruse et al., 2009; Liu et al., 2010a). Mg2+ reabsorption that causes a decrease in serum Mutations were also found in patients with atrioventric- Mg2+ levels; this, in turn, seems to lower PTH output ular block. No mutations were found in other patients by the parathyroid gland. A decrease of both PTH and with sinus node dysfunction or long QT syndrome serum Ca2+ levels (secondary hypocalcemia) results in (Stallmeyer et al., 2012). CNS symptoms. Most Trpm6 mutations are loss of It is unclear whether TRPM4 plays a role in function due to truncations of the channel protein by alteration of the arterial myogenic response (Bayliss insertion of stop codons. Single point mutations, frame effect) associated with stroke and cerebral autosomal shifts, exon splicing, deletions, and mutations affecting dominant arteriopathy with subcortical infarcts and alternative splicing were also described (for reviews, leukoencephalopathy (Folgering et al., 2008). A subset see Woudenberg-Vrenken et al., 2009; Dimke et al., of patients with who had a bifascic- 2011, 2013). ular block and/or a complete right bundle branch block TRPM8 has not yet been associated with any distinct was recently reported. In these patients, four TRPM4 channelopathy. However, the familial amyloidotic mutants were described, resulting in either decreased polyneuropathies (or familial amyloidotic neuropa- expression (P779R and K914X) or increased expression thies, neuropathic heredofamilial amyloidosis, familial (T873I and L1075P) of TRPM4 channels (Liu et al., amyloid polyneuropathy) are a rare group of autosomal 2013c). dominant neuropathies of autonomic and peripheral TRPM6 is involved in Mg2+ homeostasis (see Montell, nerves that might have some genetic relation to 2003; Cao et al., 2008; Ferrè et al., 2011; Dimke et al., TRPM8 (Gasperini and Small, 2012). 2011, 2013). More than 35 mutations in the Trpm6 gene have been associated with the disease hypomagnesaemia D. Ankyrin Transient Receptor with secondary hypocalcaemia (HSH)-1, also known as Potential Channelopathies HOMG1 (OMIM 602014) (Schlingmann et al., 2005, TRPA1 is associated with a pain-causing channelop- 2007). This form of hypomagnesemia has to be separated athy, the autosomal dominant familial episodic pain from other forms, such as hypomagnesemia 2 (HOMG2) syndrome. Patients have episodes of debilitating upper caused by misrouting of the FXYD2 Na+,K+-ATPase g2 body pain, triggered by fasting and physical stress. subunit, renal hypomagnesemia-3 (HOMG3)associated Moreover, they show enhanced cutaneous flare re- with hypercalciuria and nephrocalcinosis and caused sponse and secondary hyperalgesia to punctate stimuli. by mutation in the claudin gene CLDN16 gene, renal One point mutation in S4 causes a shift in gating hypomagnesemia-4 (HOMG4) caused by mutation in the properties of the channel with dramatic increase in EGF gene (impaired of the basolateral sorting of pro- inward currents and activation at normal resting poten- EGF), renal hypomagnesemia-5 (HOMG5)(associated tials. Specific TRPA1 antagonists inhibit the abnormal in with hypercalciuria, nephrocalcinosis, and severe ocular vitro response of the mutant TRPA1 channel, implying involvement) caused by mutation in the claudin gene new therapy for this syndrome (Kremeyer et al., 2010; CLDN19 gene, and renal hypomagnesemia-6 (HOMG6) Wood, 2010). caused by mutation in the divalent metal cation trans- An SNP causing a point mutation in the TRPA1 porter CNNM2 gene (Dimke et al., 2013). N terminus was recently discovered in patients with HSH1 (HOMG1) is a familial hypomagnesemia with acute pain and paradoxical heat sensation (Binder secondary hypocalcemia, a rare autosomal recessive et al., 2011). In vitro, this mutant TRPA1 displays disorder characterized by very low serum Mg2+ levels. higher expression at both cold and heat. However, the Hypocalcemia is a consequence of parathyroid failure mutant is not activated by cold but by heat, probably and PTH resistance that results from severe Mg2+ de- because of a loss of interaction with associated proteins ficiency. The disease manifests during the first months (May et al., 2012). of life with generalized convulsions or signs of in- creased neuromuscular excitability such as muscle E. Mucolipin Transient Receptor spasms or tetany. Untreated, the disease may be fatal Potential Channelopathies or lead to severe neurologic damage and mental TRPML1 mutations cause ML-IV (OMIM 252650), retardation. Successful and causal treatment com- an autosomal recessive neurodegenerative lysosomal prises mainly administration of Mg2+, usually intrave- storage disorder characterized by psychomotor retar- nously, followed by life-long high-dose oral treatment. dation and ophthalmologic abnormalities, including This treatment is effective because most of Mg2+ corneal opacity, retinal degeneration, and strabismus. reabsorption in the nephron occurs via a paracellular In the brain, agenesis of the corpus callosum was pathway in the thick ascending limb of the loop of described. Blood Fe2+ deficiency and achlorhydria also Henle (by contrast, TRPM6 resides in the distal characterize the disease. Over 80% of patients with TRP Channels as Drug Targets 711

ML-IV are Ashkenazi Jews (Slaugenhaupt, 2002; Zeevi pancreas, seminal tract, and arachnoid membrane. In et al., 2009; Bach et al., 2010). Over 21, mostly loss-of- the kidney, cysts can be formed in any segment of function, mutations in the Trpml1 gene have been the nephron and are associated with an increase in the identified in patients with ML-IV. Channel defects number of cells in the circumference of already dilated result in impairment of organelle Ca2+ release, which renal tubules. These cysts are filled with fluid that is is required for the correct fusion/fission events and probably secreted by the cyst epithelium. Well de- causes malfunctions in late endosome–lysosome for- veloped cysts occupy much of the mass of the abnor- mation (Lloyd-Evans and Platt, 2011; for a recent mally enlarged kidneys, and thereby compress and review, see Wakabayashi et al., 2011). destroy normal renal tissue and impair kidney function. Lysosomal storage diseases are caused by the in- ADPKD also causes cardiovascular abnormalities, such ability of the cells to process the material captured as forming of coronary artery aneurysms and intracra- during endocytosis. In ML-IV, lysosomal hydrolases nial “berry” aneurysms, leading to vessel rupture, in- are normal, in contrast with most storage diseases. The ternal bleeding, chronic subdural hematomas, and disorder results from a defect in transport along the defects of the vessel wall (see Dietrich et al., 2006). endosomal/lysosomal pathway, affecting membrane TRPP1 mutations are also related to structural sorting and fusion of both endosomes and autophago- defects in the heart (e.g., defective septum formation) somes with lysosomes (for a review, see Dong et al., (Wu et al., 2000). PKD1, a glycoprotein with a large 2010). Defects in these late steps of endocytosis and N-terminal extracellular region, associates with TRPP1 autophagy cause intracellular accumulation of lyso- to form functional channels. Mutations in PKD1 (85% of somal substrates. Patients with ML-IV display accu- cases; TRPP1 is only responsible for the remaining 15%) mulation of large vacuolar intracellular organelles that are the major case of ADPKD. More than 400 mutations are built up because of the defective late endosome/ have been described in PKD1/TRPP1 in patients with lysosome/phagosome fusion/fission events. They con- ADPKD (Audrézet et al., 2012). As discussed above, tain amphiphilic lipids (phospholipids, sphingolipids, TRPP1 associates with PKD1, which is required for its gangliosides, mucopolysaccharides, lipofuscins, etc.) function as a putative flow sensor via the primary cilium and other materials from cell organelle debris. A (for a review Patel and Honoré, 2010). TRPP1 is involved defective function of lysosomes also impairs autopha- in governing cellular processes via the Janus kinase/ gosome functions (i.e., clearance of the cell interior signal transducers and activators of transcription, p53, from toxic proteins and damaged cell organelles) mTOR, NFAT/AP-1 (activator protein-1), cAMP/PKA, (Vergarajauregui and Puertollano, 2008; Micsenyi et al., cAMP-dependent ERK, CDK, or Wnt signaling path- 2009). Another pathophysiological mechanism is related ways, which all may be partially involved in the to the function of TRPML1 as a Fe2+ and Zn2+ channel. pathogenesis (for review, see Takiar and Caplan, 2011). Absence of the lysosomal Fe2+/Zn2+ leak would result in Other functional defects may arise from dissociation lysosomes overloaded with these heavy metals, leading to of mutant TRPP1 from its submembranous cytoskeletal the loss of lysosomal function (Kiselyov et al., 2011). interaction partners (Chen et al., 2008b). The PKD1/ TRPML1-mediated lysosomal Ca2+ release is also TRPP1 complex regulates the translocation of the helix- dramatically reduced in another lipid-storage disease, loop-helix protein Id2, a crucial regulator of cell pro- the Niemann–Pick type C disease (OMIM 257220), al- liferation and differentiation, into the nucleus. An though the main disease-causing effect is due to mu- enhanced nuclear localization of Id2 in renal epithelial tations in the lysosomal two-pore TPC1 channel. cellsfrompatientswithAKPKDconstitutesamechanism Sphingomyelins normally undergo sphingomyelinase- for the hyperproliferative phenotype causing cyst forma- mediated hydrolysis in the lysosomes of normal cells, tion (Benezra, 2005; Li et al., 2005a). In general, the but accumulate distinctively in lysosomes of Niemann– whole pathogenesis of ADPKD is not well understood (for Pick type C cells. TRPML1 channel activity is inhibited an excellent review, see Chapin and Caplan, 2010). by sphingomyelins. Thus, abnormal accumulation of lumi- Because ADPKD is a ciliopathy, an association of TRPP1 nal lipids causes secondary lysosome storage by blocking with other ciliopathies (e.g., Joubert syndrome, a rare TRPML1- and Ca2+-dependent lysosomal trafficking (Tang that affects the cerebellum, an area of the et al., 2010b; Shen et al., 2012). TRPML2 and TRPML3 brain that controls balance and coordination) was have not yet been reported to cause any human disease, postulated (Garcia-Gonzalo et al., 2011). Another example although a TRPML3 mutation in Va mice causes deafness of TRPP1 involvement is Meckel syndrome (also known and altered fur pigmentation (Di Palma et al., 2002). as Meckel–Gruber syndrome, or dysencephalia splanch- nocystica), which is a rare, lethal, ciliopathic genetic F. Polycystic Transient Receptor disorder characterized by renal cystic dysplasia, CNS Potential Channelopathies malformations, polydactyly, hepatic developmental de- TRPP1 mutations cause the ADPKD (OMIM 613095), fects, and pulmonary hypoplasia (Mason et al., 2011). a disease that manifests in progressive development TRPP1 mutations are also involved in the pathogenesis of of large epithelium-lined cysts in the kidney, liver, the situs inversus (i.e., deformation of the left-right body 712 Nilius and Szallasi axis), which is due to a primary ciliary dyskinesia phenotypic analysis of knockout mice) need to be (Bataille et al., 2011; Yoshibaetal.,2012).Nodiseases carefully and critically evaluated because they do not have been reported thus far for TRPP2 and TRPP3. always mirror human diseases, which tend to involve complicated environmental and genetic factors. In other words, the preclinical model may not properly III. Transient Receptor Potential Channels: represent the disease. For example, the TRPV1 Acquired Diseases antagonist ABT-102 [(R)-(5-tert-butyl-2,3-dihydro-1H- As discussed above, the link between germ-line TRP inden-1-yl)-3-(1H-indazol-4-yl)-urea; structure shown gene mutations and human hereditary diseases (the in Fig. 9] blocked pain behavior in a rodent model of so-called TRP channelopathies) is firmly established OA (Honore et al., 2009). However, in a randomized (Figs. 14, 15, and 16). Indeed, two TRP channel controlled trial, it did not relieve hip arthritic pain in subfamilies (polycystin and mucolipin) were named client-owned dogs (Malek et al., 2012). Of note, the after the human diseases (polycytic kidney disease and phase II with the AstraZeneca TRPV1 mucolipidosis, respectively) with which they are asso- antagonist AZD1386 (Fig. 9) in patients with OA was ciated. Reversing the altered functions of these mutant prematurely terminated at an interim analysis for lack TRP channels by therapeutic intervention is expected of analgesic efficacy (Svensson et al., 2010; Miller et al., to provide clinical benefits in these genetic disorders. 2014). From a drug developmental point of view, diseases Another confounding factor that complicates the caused by gain-of-function mutations in TRP channels extrapolation of animal studies to patients is species- could be more amenable to therapeutic targeting since related differences. For instance, neurogenic inflam- overactivation of channels could be inhibited by small mation triggered by the release of proinflammatory molecule antagonists. By contrast, diseases caused by neuropeptides from primary sensory neurons plays loss-of-function mutations, particularly truncation types, a pivotal role in the ovalbumin model of asthma (see are difficult to target with small molecules, and a less Lundberg, 1995). Indeed, antagonists blocking neuro- validated approach such as gene therapy may be re- kinin (NK) 1 and 2 receptors ameliorate the symptoms quired to restore the normal TRP channel function. of experimental asthma in the rat or guinea pig In contrast with TRP channelopathies, the role of (Barnes, 2001), and NK1 receptor (NK1R) blockade TRP channels in the development, maintenance, and even causes weight loss in asthmatic, ovalbumin-obese shaping of acquired disease states is only beginning mice (Ramalho et al., 2013). These antagonists, to be understood. Generally speaking, disease im- however, showed little (if any) clinical benefit in plications arising from animal studies (including patients with asthma (Butler and Heaney, 2007). This

Fig. 14. The spectrum of human diseases attributed to mutant TRP channels (TRP channelopathies). For more details, see Nilius and Owsianik (2010b). TRP Channels as Drug Targets 713

Fig. 15. TRPV4 mutations and associated diseases. For more details, see Nilius and Voets (2013). is in agreement with the lack of convincing evidence for and TRPA1 are on the list of potential analgesic targets a clinically meaningful neurogenic inflammatory re- (Figs. 17, 18, 19, 20, and 21), although, as discussed sponse in human airways distal to the nasal mucosa below, the level of validity differs among them. Re- (Barnes, 2001). spiratory diseases that entail various pathophysiological TRP channels themselves show striking (and, from alternations in airway smooth muscle, immune system, a drug developmental point of view, quite worrisome) airway vasculature, and vagal sensory nerve are also rich species-related differences in their activation mecha- with TRP targets (reviewed in Abbott-Banner et al., 2013). nisms. For example, cold activates rodent, but not In addition, multiple TRP channels are implicated in human or monkey, TRPA1 (Chen et al., 2013a). Further- skin (Valdes-Rodriguez et al., 2013) and bladder more, hTRPA1 acts as a sensor for tissue acidosis, (Charrua et al., 2010; Avelino et al., 2013) disorders whereas protons, conversely, inhibit rTRPA1 (de la as well as cancer (Prevarskaya et al., 2010; Liberati Roche et al., 2013). This might have given false negative et al., 2013). Taking these multifaceted and sometimes results when testing TRPA1 antagonists as potential overlapping roles of TRP channels in a disease into analgesic agents in rodent models of cancer-induced bone consideration, targeting a single TRP channel might pain, in which the acidic microenvironment is believed to provide an insufficient effect because it may address be an important contributor to pain (Lozano-Ondoua only limited aspects of the disease. Thus, one may et al., 2013). argue that a “dirty” compound that acts on multiple Alternative splice variants were reported to influ- TRP channels may be superior to a selective compound ence TRP channel activities (in some instances in a in exerting therapeutic effects. tissue-specific manner). For example, the coexpression of TRPA1a and TRPA1b increases current density in A. The Role of Transient Receptor Potential Channels response to agonists (Zhou et al., 2013). By contrast, in Nociception, Pain, and Itch the TRPV1 splice variant VR.59sv functions as a dom- 1. Basic Concepts. Nociceptors were first described inant negative modulator of TRPV1 activity (Eilers by Charles Scott Sherrington more than a century ago. et al., 2007). The expression of VR.59 is reduced during A nociceptor (from the Latin term nocere, to hurt) is experimental cystitis and it was speculated that this defined as a “pain cell” that is capable of sensing contributes to bladder hyperalgesia (Charrua et al., noxious physical (chemical, thermal, and mechanical) 2008). stimuli in the periphery and transmitting the pain Although increasing numbers of TRP channels are signal to the CNS. These stimuli produce a sensation of reported to be involved in pathophysiology of diseases, intense pain leading to a protective avoidance reaction some diseases are endowed with multiple TRP targets. and facilitating survival. In a simplistic manner, the Pain has been one of the mainstreams of TRP research nociceptive pathway can be subdivided into three (recently reviewed in Patapoutian et al., 2009; Brederson phases: transduction, propagation, and transmission et al., 2013; Julius, 2013) and multiple TRP channels of the signal to the spinal cord. Traditionally, drug including TRPV1, TRPV3, TRPM2, TRPM3, TRPM8, discovery efforts largely covered pain targets involved 714 Nilius and Szallasi

Fig. 16. Charcot-Marie-Tooth disease type 2C is a TRPV4 channelopathy. (A) The disease is of variable severity, ranging from mild, late-onset weakness in a mother to severe quadriparesis and respiratory failure in her daughter. (B) Sequencing of the TRPV4 gene in two affected families revealed changes in position 805 (C . T) and 806 (G . A), respectively. (C) Protein homology of TRPV4 in various species. The human mutation results in amino acid substitution at Arg269, a highly conserved residue. (D) When expressed in Xenopus laevis oocytes, the mutant channels exhibit a gain-of- function phenotype. (E) DRG neurons expressing the mutant TRPV4 channels are sensitive to neurotoxicity. WT, wild type. Reprinted with permission from Landouré et al. (2010). *P , 0.05, statistical significance. in the propagation and transmission of the signal and transduction phase (first step of nociception) in an inflammatory pathways. However, the discovery of the effort to block pain at its source (reviewed in TRPV1 receptor (Caterina et al., 1997), followed by the Patapoutian et al., 2009; Moran et al., 2011; Brederson cloning of temperature-sensitive TRPV1-related chan- et al., 2013; Julius, 2013). nels (thermoTRPs) and the elucidation of their func- In mammals, primary sensory (nociceptive) neu- tion, brought a great degree of interest in exploring the rons form an anatomic connection between potentially TRP Channels as Drug Targets 715

Fig. 17. Targeting pain at the source: TRP channels expressed on peripheral sensory neurons and their activation mechanisms. See sections I.B–D for details. Courtesy of Dr. Marcello Trevisani. harmful external and internal agents and the CNS. Of orchestrate sunburn pain [and mediate ultraviolet B note, many non-neuronal cells (e.g., urothelial cells and (UVB)–induced tissue damage], presumably by upregu- keratinocytes) also express pain-sensing TRP channels, lating the expression of the endogenous algogenic in particular TRPV1 (Denda et al., 2001; Birder et al., substance, endothelin-1 (Moore et al., 2013). TRPV1- 2002; Southall et al., 2003; Wilder-Smith et al., 2007), positive sensory afferents express endothelin-1 recep- TRPV3 (Facer et al., 2007), and TRPV4 (Chung et al., tors; indeed, endothelin-1 both stimulates nociceptors 2003; Gevaert et al., 2007). It has been also suggested and sensitizes them to painful stimuli (Plant et al., (reviewed in Moran et al., 2011) that these cells may 2007) presumably by activating TRPA1 channels (Liang also function as unconventional (non-neuronal) pain et al., 2010). Thus, it is easy to visualize a positive sensors and communicate with polymodal nociceptive feedback loop between keratinocytes and sensory nerve C fibers (Southall et al., 2003; Birder, 2005). Indeed, endings that promotes neuronal sensitization. keratinocytes respond to capsaicin with eicosanoid, Primary sensory neurons are bipolar cells with particularly LTB4 release (Jain et al., 2011), and mouse somata in DRG and trigeminal ganglia. The central DRG neurons coexpress TRPV1 with the axons of these neurons enter the CNS, where they form receptor 1 BLT1 (Andoh and Kuraishi, 2005). Moreover, synapses with second-order neurons in the dorsal horn keratinocyte-specific TRPV3 transgenic mice show of the spinal cord (DRG neurons) or the spinal nucleus augmented PGE2 release in response to heat (Huang of the trigeminal tract (trigeminal ganglion neurons). et al., 2008). Epidermal TRPV4 was recently reported to Many neurons innervating the viscera are located in the nodose ganglia. Their peripheral fibers travel with the vagus nerve, whereas their central axons project to the area postrema (reviewed in Holzer, 1991; Szallasi and Blumberg, 1999). Most primary sensory neurons possess unmyelinated axons (C fibers) and are capsa- icin sensitive (Holzer, 1991). A small subset of neurons with thin-myelinated axons (Ad fibers) also expresses TRPV1 receptors. Interestingly, it has been shown that Ad fibers that do not normally express TRPV1 do so under inflammatory conditions or after injury (Rashid et al., 2003). This abnormal, TRPV1-positive Ad fiber Fig. 18. Systemic RTX (100 mg/kg s.c.) treatment reduces thermal population has been suggested to contribute to neuro- hyperalgesia in a rat model of neuropathic pain. Thermal hyperalgesia pathic pain in patients with diabetic polyneuropathy was initiated by placing loose ligatures on the sciatic nerve of the rat (day 0), and was determined by measuring paw flick latency in the hot plate test. (Rashid et al., 2003; Bishnoi and Premkumar, 2013). RTX was given either before (prophylactic group, black squares) or after Indeed, desensitization by TRPV1 agonists (e.g., capsai- (therapeutic group, black triangles) surgery. Please note the complete reversal of the thermal hyperalgesia in both groups compared with vehicle cin) relieves chronic pain in these patients (reviewed in control subjects (open circles). Knotkova et al., 2008; Szallasi and Sheta, 2012) despite 716 Nilius and Szallasi

Fig. 19. (A) Intrathecal RTX alleviates pain behavior in client-owned dogs with osteosarcoma (N), severe osteoarthritis (A), or both (N+A). (B) Visual analog score of dogs with bone cancer pain at baseline and after intrathecal administration of RTX (1.2 mg/kg). Pre-RTX (pretreatment) represents the day before general anesthesia and intrathecal injection. N represents the number of dogs still alive at each time point. Figure courtesy of Michael Iadarola and Dottie Cimino-Brown. *P , 0.05, statistical significance. the degeneration of C fibers (Lauria et al., 2006). Most et al., 2009). The fractional Ca2+ current via TRPV1, recently, TRPV1-expressing Ad fibers have been impli- however, depends on the mode of channel activation. For cated in the pathogenesis of breakthrough pain and other example, it is significantly smaller for proton activation typesofpainexacerbationconditionssuchaspostsurgical compared with capsaicin (Samways et al., 2008). This is 2+ hyperalgesia (Mitchell et al., 2014). important because [Ca ]i is thought to play a pivotal role Given the important role of TRPV1 in pain (and the in the phenomenon of capsaicin desensitization (see side effects of small molecule TRPV1 antagonists), Szallasi and Blumberg, 1999). novel strategies to inhibit this channel are particularly As reviewed elsewhere, TRPV1 is a promising target to interesting. A novel analgesic strategy is to antagonize relieve inflammatory pain (see Szallasi et al., 2007; Lázár the interaction between TRPV1 and AKAP79, a scaf- et al., 2009; Szolcsányi and Sándor, 2012; Brederson folding protein essential for positioning of serine- et al., 2013; Szolcsányi and Pintér, 2013). Indeed, both threonine A kinases adjacent to target phosphorylation genetic deletion (Caterina et al., 2000; Davis et al., 2000) sites (Btesh et al., 2013). Small peptides that prevent and pharmacological blockade of TRPV1 ameliorate heat this interaction have been identified and shown to be hyperalgesia in rodent models of inflammatory pain analgesic in mouse models of inflammatory hyper- (reviewed in Gunthorpe and Szallasi, 2008; Gunthorpe algesia, highlighting the potential therapeutic value of and Chizh, 2009). Of relevance is the finding that TRPV1 interfering with the interaction between TRPV1 and expression is increased in reflux esophagitis [or gastro- AKAP79 (Fischer et al., 2013). esophageal reflux disease (GERD)], in which “heartburn” TRP channels play a central role in thermal isduetoexposuretoregurgitatedacidicgastriccontents nociception (Moran et al., 2011; Julius, 2013) and as (Matthews et al., 2004; Bhat and Bielefeldt, 2006). Yet, well as in the detection of noxious chemicals (Nilius disappointingly, in randomized clinical trials, the TRPV1 et al., 2007b; Kumar et al., 2013). This is interesting antagonist AZD1386 (see Fig. 9 for structure) provided no biology but, per se, it would not make these channels symptomaticpainreliefinpatientswithGERD(Krarup potential targets for analgesic drugs. Importantly, et al., 2011, 2013). TRPV1 is also activated and/or sensitized by agents TRPV1 is also elevated in inflammatory bowel disease in “inflammatory soup” (Figs. 12 and 21), ranging from (IBD) and irritable bowel syndrome (also known as colon tissue acidosis (protons) through cytokines, NGF, BK, 12- irritabile), a fairly common condition of unknown HPETE, and 15-HPETE and other arachidonic acid etiology characterized by frequent bowel movements metabolites (reviewed in Caterina and Julius, 2001; and tenesmus (painful straining at stool) (Yiangou et al., Pingle et al., 2007; Szallasi et al., 2007). These agents 2001; Chan et al., 2003). Because there is no effective act in concert to lower the heat activation threshold of medical therapy, irritable bowel syndrome is frustrating TRPV1 (Di Marzo et al., 2002; Szallasi et al., 2007; Lázár for both patients and their physicians. Therefore, it is an TRP Channels as Drug Targets 717

neurons, coexpresses TRPV1 with the related channels TRPV3 and TRPV4 and also with TRPA1 (Figs. 17, 20, and 21) (Kobayashi et al., 2005). TRPV1, TRPV3, and TRPV4 are heat-activated channels, so their presence on the same neurons is not unexpected. It is more difficult to explain why these heat-responsive receptors are coexpressed with the “cold receptor” TRPA1. Of note, TRPA1 was reported to be heat activated in some studies (Hoffmann et al., 2013). Adding to the com- plexity, TRPA1 seems to be present on both peptidergic and nonpeptidergic neurons (Hjerling-Leffler et al., 2007). A second major subset of primary sensory neurons, encompassing both A fiber and C fiber neurons, is characterized by their TRPM8 expression (Figs. 17 and 21) (Kobayashi et al., 2005). The minimal overlap be- tween TRPV1 and TRPM8 expression suggests that TRPV1-positive neurons and TRPM8-expressing neurons are fundamentally different, although TRPA1 appears to be present on both TRPV1- and TRPM8-expressing pop- ulations (Kobayashi et al., 2005). In keeping with this concept, TRPV1-like immunoreactivity is elevated, where- as TRPM8 is, by contrast, reduced in injured human brachial plexus nerves (Facer et al., 2007). On the basis of these findings, it has been suggested that TRPV1 may be a more relevant therapeutic target than other ther- moTRPs for pain related to posttraumatic neuropathy Fig. 20. Schematic diagram showing the involvement of TRPA1 channels in nociception, neurogenic inflammation, and mechanical hypersensitiv- (Facer et al., 2007). Intriguingly, and in contrast with ity/allodynia. The magnified insert on the top illustrates the hypothetical expression in the DRG, TRPM8 is coexpressed with role on TRPA1 in the development and maintenance of central sensitization. According to this model, endogenous TRPA1 agonists such TRPV1 in vagal sensory neurons innervating the mouse as A3 (HXA3), 5,6-epoxyeicosatrienoic acid (5,6-EET), and ROS lung (Nassenstein et al., 2008). facilitate NMDA responses and thus amplify pain signaling in the spinal As discussed above, TRPV1 can form functional cord. Spinal TRPA1 activation also facilitates a process in which innocuous mechanical stimuli (from piezo2-expressing low-threshold heteromultimers with other TRP channels. Therefore, afferents) are aberrantly transmitted to lamina 1 projection neurons antagonists that do not distinguish between thermoTRPs and thus become painful. This mechanism is believed to play a role in mechanical allodynia. Reproduced with permission from Koivisto et al. may have a therapeutic value by targeting TRP hetero- (2014). multimers. The shared TRP domain in these channels may represent a target for such inhibitors (García- Sanz et al., 2007). Of note, BCTC (Fig. 9), originally exciting possibility that per os TRPV1 antagonists may described as a TRPV1 antagonist, also functions as provide symptomatic relief. Indeed, there is anecdotal a potent inhibitor of TRPV4 and TRPM8 channels evidence that eating hot spicy food exacerbates symp- (Weil et al., 2005). Clearly, studies using BCTC as a toms in patients with irritable bowel syndrome (see selective TRPV1 blocker need to be carefully reeval- Szallasi and Blumberg, 1999). uated(thesameappliestocapsazepine,another a. Differential transient receptor potential channel “dirty” TRPV1 inhibitor, widely used in the past to expression defines functional sensory neuron subtypes: dissect TRPV1-mediated responses). Implications for drug development. Primary sensory Maybe because they are ubiquitously expressed, the neurons are heterogenous in several aspects, including presence of TRPC channels in sensory neurons has their anatomy, neurochemistry, and function. For attracted little attention (Elg et al., 2007). In adult example, these neurons differ in the myelin sheet that mice, all seven members of the TRPC family (TRPC1– protects their axons (myelinated Ab-, thin-myelinated TRPC7) were detected in DRG neurons with TRPC1, Ad-, and unmyelinated C fibers), they use different TRPC3, and TRPC6 being the most abundant. Of note, mediators (e.g., peptidergic and nonpeptidergic), and TRPC3 was exclusively expressed by nonpeptidergic, they convey different somatosensory information to the TRPV1-negative neurons. TRPC3 was recently identi- CNS (e.g., touch, pain, itch, and temperature). One way fied as a key molecular target for the excitatory effect to subclassify primary sensory neurons is by the TRP of the IgG immune complex in rat DRG neurons (Qu channels that they express. A major population of et al., 2012). As discussed later, TRPC3 (in concert with neurons with C fibers, as well as a minor subset of Ad TRPA1) may also be a target for nonhistaminergic itch 718 Nilius and Szallasi

Fig. 21. TRP channels as nociceptors. Sensory neurons express a multitude of heat-sensitive TRP channels (e.g., TRPV1–TRPV4, TRPA1, and TRPM8), some of which are also activated by acids. TRPA1 has emerged as the key chemoreceptor that responds to scores of irritant and potentially harmful chemicals. TRPM8 is thought to sense environmental cold but its role in cold hyperalgesia is unclear. Some TRP channels (e.g., TRPV1 and TRPA1) also expressed in the CNS where they have been suggested to play a role in central sensitization. See sections I.B–D for more details. Reprinted with permission from Moran et al. (2011). in sensory afferents (Than et al., 2013). Furthermore, it open TRPV1 pore and gains access to its binding site on was suggested that TRPC1 and TRPC6 may cooperate the (Binshtok et al., 2007). This elegant with TRPV4 in mediating mechanical hyperalgesia approach affords selective targeting of TRPV1- (Alessandri-Haber et al., 2009). Indeed, in rat DRG expressing sensory neurons (Binshtok et al., 2007, neurons, the expression of TRPC4 is increased after 2009). This approach was recently further refined nerve injury (Wu et al., 2008), possibly as a regenera- by identifying such TRPV1 agonists (e.g., Cap-ET) that tive reaction promoting neurite growth. Furthermore, recognize and selectively target already sensitized knockdown by short hairpin RNA (shRNA) of TRPC1 TRPV1 channels in inflamed tissues and augment the reduces mechanosensitivity of mouse DRG neurons uptake of QX-314 (Li et al., 2011a). It is hoped that this (Staaf et al., 2009). Finally, rats with transposon- innovative approach may tame hyperactive TRPV1 in mediated TRPC4-knockout mutation showed amelio- diseased tissues and spare normal nociception. Of note, rated visceral pain reaction in response to colonic in combination with bupivacaine, QX-314 can be also mustard oil exposure (Westlund et al., 2014). used to selectively block TRPA1-expressing neurons Of note, thermoTRPs are also colocalized with other (Brenneis et al., 2014). Indeed, as discussed later, QX- receptors involved in pain transmission. In an in- 314 was used to silence neuronal pathways conducting novative study, capsaicin has been used to deliver so- itch (Roberson et al., 2013). dium channel blockers into neurons expressing TRPV1 b. Disease-related changes in transient receptor poten- (Binshtok et al., 2007). QX-314 [N-(2,6-dimethylphenyl- tial channel expression. TRP channels not only show carbamoylmethyl)triethylammonium bromide] is quater- bidirectional changes during disease states (upregula- nary derivative of lidocaine that is ineffective when tion or downregulation), but can be also expressed in administered alone because it is not capable of crossing cells that do not normally express such channels (re- the membrane. However, when coadministered with viewed in Szallasi et al., 2007). These observations have capsaicin, QX-314 enters the sensory neuron through the important practical implications for drug development. TRP Channels as Drug Targets 719

For example, animal experiments suggest that TRPM8 hypoalgesia paralleled the loss of TRPV1-like immuno- may be a relevant target to ameliorate cold hyperalgesia reactivity (Pabbidi et al., 2008). The molecular drivers of that develops after nerve injury (Katsura et al., 2006; these changes are unknown but it has been hypothe- Xing et al., 2007; Ji et al., 2008). In support of this sized that the downregulation of TRPV1 expression in hypothesis, mRNA encoding TRPM8 is increased in the diabetic skin is related to the diminished NGF levels rat DRG after chronic constriction injury (CCI) (Frederick (Facer et al., 2007). As reviewed elsewhere (Knotkova et al., 2007). However, in humans (unlike in rodents), et al., 2008), diabetic neuropathy is a traditional in- TRPM8 appears to be downregulated after nerve injury dication for capsaicin-containing topical preparations. (Facer et al., 2007) and in painful dental pulp (Alvarado However, the clinical experience with capsaicin is et al., 2007). Indeed, no evidence for the involvement of conflicting, with some studies reporting significant pain TRPM8 in cold allodynia has been found in patients with relief, whereas others have been unable to replicate neuropathic pain (Namer et al.,2008).Thisisanother these results (see Knotkova et al., 2008). Of note, in worrisome example of the species-related differences in addition to its interaction with TRPV1 receptors, TRP channel biology that hinder extrapolation of animal capsaicin also exerts a direct effect on mitochondria experiments to patients. (reviewed in Szallasi and Blumberg, 1999), which may By contrast, TRPA1 appears to be upregulated in play an important role in the development and human DRG after nerve injury (Anand et al., 2008). In maintenance of local analgesia (desensitization or, more rats, antisense knockdown of TRPA1 alleviates cold correctly, defunctionalization) in the treated area (Fig. hyperalgesia after spinal nerve ligation (Katsura et al., 22). How capsaicin is applied to the skin may have 2006). However, the relevance of these observations is a significant impact on this mitochondrial effect. In the unclear since cold allodynia appears to be independent skin of patients with diabetic neuropathy, TRPV1- of TRPA1 in patients with neuropathic pain (Namer expressing epidermal nerve fibers are markedly reduced et al., 2008). (Bley, 2013), accompanied by decreased TRPV3 expres- In rodents, the expression of TRPV4 is increased at sion in keratinocytes (Facer et al., 2007). This is in both the mRNA and protein levels after mechanical agreement with the loss of TRPV1-like immunoreactiv- nerve injury, induced by long-term compression of ity during the late stage of experimental diabetic DRG (Zhang et al., 2008f). As detailed below, TRPV4 neuropathy (Pabbidi et al., 2008). If so, TRPV1 blockade has also been linked to chemotherapy (e.g., taxol or may have a therapeutic value in early diabetic poly- vincristine)–induced neuropathy (Alessandri-Haber neuropathy (when TRPV1 is enhanced) but not in et al., 2004, 2008). When given intrathecally, TRPV4 established diabetic polyneuropathy (when TRPV1 is oligodeoxynucleotide antisense reverses mechanical downregulated). allodynia induced by long-term compression of DRG Although long-term administration is (Zhang et al., 2008f) and ameliorates mechanical hy- strictly speaking not a disease, it should be mentioned peralgesia in animal models of neuropathy of various here that long-term morphine administration upregu- etiologies such as diabetes, alcoholism, and chemother- lates TRPV1 expression in the spinal cord in a mito- apy (Alessandri-Haber et al., 2008). TRPV4 appears to gen-activated protein kinase–dependent manner (Chen be also involved in inflammatory pain, as implied by the et al., 2008c). This is intriguing because morphine reduced response to inflammatory soup in Trpv4(2/2) tolerance is often associated with the development of mice (Chen et al., 2007). This finding is consistent with thermal hyperalgesia. In fact, intrathecal pretreat- the role of TRPV4 as an osmosensor and the hypotonic ment with the TRPV1 antagonist SB366791 [N-(3- nature of the inflammatory soup. The relevance of these methoxyphenyl)-4-chlorocinnamide] has been shown to observations is, however, questioned by the apparent attenuate morphine tolerance and to prevent thermal lack of painful phenotype within the broad spectrum of hyperalgesia (Chen et al., 2008c). It is hoped that disease-associated TRPV4 channelopathies, including TRPV1 antagonists will reduce the need for both gain-of-function and loss-of-function TRPV4 muta- and, as an added benefit, also prevent tolerance to tions (Nilius and Voets, 2013). opioids. Interestingly, acute morphine administration TRPV1 shows bidirectional expression changes in has the opposite effect since it negatively modulates various disease states (see Szallasi, 1996; Szallasi et al., TRPV1 via inhibition of adenylate cyclase (Vetter 2007). TRPV1 expression is elevated after spinal cord et al., 2006). Withdrawal symptoms in patients injury (Wu et al., 2013). During inflammation and in addicted to , by contrast, may include cold bone cancer, TRPV1 levels also substantially increase hyperalgesia, which appears to be mediated by TRPM8 (Niiyama et al., 2007). Conversely, TRPV1 expression is (Shapovalov et al., 2013). Indeed, activation of m-opioid downregulated in neuropathic pain secondary to injury receptors induces internalization of TRPM8 with a re- (Lauria et al., 2006) or after RTX treatment (Szallasi bound after naloxone treatment. These “cold turkey” et al., 1999). In a murine model of diabetic neuropathy, patients may be helped to overcome their cold sen- the early thermal hyperalgesia was associated with sitivity by TRPM8 antagonists. Of note, the painful increased TRPV1 expression, whereas the late thermal adverse effect of apomorphine (a nonnarcotic morphine 720 Nilius and Szallasi

Fig. 22. Simplified model of capsaicin actions on sensory neurons. Note that capsaicin impairs mitochondrial functions both directly and indirectly (by evoking Ca2+ influx through TRPV1). This mitochondrial dysfunction was suggested to play an important role in the development of long-lasting capsaicin “desensitization” (defunctionalization). Reprinted with permission from Bley (2013). derivative used in the pharmacotherapy of patients well localized nociception (Abd-Elmeguid and Yu, with Parkinson disease) involves TRPA1 activation 2009). (Schulze et al., 2013). To elucidate the role of thermoTRPs in dental 2. Transient Receptor Potential Channels in Dental nociception, retrograde labeling with a fluorescent dye Pain. The tooth is a unique tissue in that it is subject was used to identify dental primary afferent neurons. A to extreme changes in temperature from ice cold to combination of molecular [single-cell reverse-transcription burning hot, depending on the food or drink consumed. polymerase chain reaction (RT-PCR)], immunohistochem- In contrast with other tissues, noxious hot or cold ical, and functional (patch clamping) studies revealed that temperatures do not evoke nociception in the teeth most (up to 85%) trigeminal ganglion neurons innervating under normal circumstances due to the thermal insulating capacity of enamel, the outer protective shell of the tooth (reviewed in Chung and Oh, 2013; Chung et al., 2013). However, when the enamel is decayed and dentin is exposed (e.g., due to caries or trauma), small changes in temperature and/or light touch can evoke sudden and intense toothache. Three complementary, rather than mutually exclu- sive, hypotheses have been put forward to explain the generation of dental pain, all potentially involving TRP channels (summarized in Fig. 23; reviewed in Chung and Oh, 2013): these are the neuronal, hydrodynamic (Fig. 24A) and odontoblast transducer (Fig. 24B) theories of dental nociception. The neuronal theory postulates the transduction of noxious temperatures by nerves innervating the dentin and pulp that express thermoTRPs. Indeed, the tooth pulp is densely in- nervated by primary sensory neurons with somata in trigeminal ganglia. The majority (70%–90%) of intra- Fig. 23. Schematic illustration of the three prevailing hypotheses to pulpal axons are unmyelinated C fibers which may explain dental pain. (A) Neural theory: nerve endings in the dentinal conduct dull, drawling dental pain. The remaining tubule are directly activated by external stimuli. (B) Hydrodynamic theory: fluid movement within the dentinal tubules is detected by nerve myelinated intrapulpal fibers are mostly Ad fibers endings. (C) Odontoblast transducer theory: odontoblasts act as pain that are responsible for the rapid, sharp, lancinating, sensors. Reproduced with permission from Chung et al. (2013). TRP Channels as Drug Targets 721

the generation of proinflammatory cytokines in periodontitis-susceptible Fischer 344 rats, a rodent model of human periodontal disease (Breivik et al., 2011) TRPV2 is predominantly expressed by medium to large-size dental afferent neurons in culture (Ichikawa and Sugimoto, 2000). Interestingly, the expression level of TRPV2 is higher in labeled dental afferent neurons (37%) than in unlabeled trigeminal ganglion neurons (14%). Indeed, half of the neurons that innervate the periodontal ligament express TRPV2 (Gibbs et al., 2011). Molecular and immunocytochemical studies also revealed the presence of cold-responsive TRPA1 and TRPM8 channels in retrogradely labeled rat dental afferent neurons (Park et al., 2006). Decayed tooth is usually more sensitive to cold than heat; thus, the expression prevalence of both TRPM8 and TRPA1 in dental afferents was unexpectedly lower than that of TRPV1. However, TRPA1 expression is increased in trigeminal ganglion neurons after injury applied to rodent teeth (Haas et al., 2011). Some of the dental afferents appeared to coexpress TRPM8 and/or TRPA1 with TRPV1 (Park et al., 2006). If this controversial observation holds true, such neurons may respond to temperature changes in either direction (warm or cold) with nerve impulses. Indeed, TRPA1 was reported to respond to both heat and cold (Hoffmann et al., 2013). A sensory role for odontoblasts has also been postulated (see Chung and Oh, 2013). Odontoblasts form the outermost cell layer between the dentin and the tooth pulp. The main function of odontoblasts is to deposit a mineralized Ca2+ matrix, which, in turn, is converted into dentin during tooth development and repair. Whereas it is generally accepted that odonto- blasts may double as cellular sensors [in support of this hypothesis, they express both voltage-gated Na+ channels (Allard et al., 2006) and store-operated Ca2+ channels (Shibukawa and Suzuki, 2003)], the expres- sion of thermoTRP channels in odontoblasts (and their Fig. 24. (A) Molecular mechanisms of the hydrodynamic theory: fluid role in toothache, the so-called odontoblast transducer movements caused by diverse external stimuli activate mechanoreceptors theory of dental pain; Fig. 24B) remains controversial. in dental primary afferents, possibly including some TRP channels such For example, odontoblasts obtained by in vitro differ- as TRPV4. (B) Molecular mechanisms of the odontoblast transducer theory: after injury, odontoblasts acquire a sensory phenotype and start entiation of neonatal rat pulpal cells were reported to expressing nociceptive TRP channels. Reproduced with permission from express a variety of TRP channels including TRPV1, Chung et al. (2013). TRPV2, TRPV3, andTRPV4, along with TRPM3 (Son et al., 2009). Similarly, in human odontoblasts differ- the tooth pulp express TRPV1 (Park et al., 2006; Kim entiated in vitro from the pulp of extracted molar teeth, et al., 2011b). Importantly, these neurons respond to expression of TRPV1, TRPA1, and TRPM8 was de- noxious heat (and also capsaicin) with elevated intracel- scribed (El Karim et al., 2011). By contrast, odonto- lular calcium levels (Chaudhary et al., 2001). Of note, blasts isolated short term from adult rat incisors TRPV1 expression was reported to be upregulated in showed no evidence of TRPV1, TRPV2, TRPA1, and/ trigeminal ganglion neurons during LPS-induced experi- or TRPM8 expression (Yeon et al., 2009). To resolve mental pulpitis (Chung et al., 2011). This increased these discrepant results, one might argue that either TRPV1 expression might contribute to the thermal hy- the actual odontoblasts are different from those peralgesia in patients with chronic pulpitis. In support of obtained via in vitro differentiation or that the pro- this hypothesis, desensitization of TRPV1-expressing cedure of short-term isolation itself eliminates the sensory neurons to RTX reduced bone loss and suppressed odontoblasts that express TRPV1 or TRPV2. Finally, 722 Nilius and Szallasi the discrepancy may simply reflect developmental response to hydrodynamic pressure (Yu et al., 2009a). differences between adult and neonatal rats. For If so, mechanosensitive TRP channels expressed in instance, TRPV1 appears to be widely expressed in odontoblasts might play an important role in this neonatal (Ritter and Dinh, 1992), but not adult process. (Cavanaugh et al., 2011), mouse brain. Furthermore, In summary, the contribution of TRP channels to TRPC1, TRPC3, and TRPC6 are highly expressed in gum disease and dental pain is most likely complex adult, but not neonatal, rodent DRG neurons; by and still poorly understood. According to the US contrast, TRPC2 and TRPC5 show an age-dependent Centers for Disease Control and Prevention, more decline in expression levels (Elg et al., 2007). than half of the adult population in the United States Although thermoTRP channels expressed on dental suffers from gum disease (gingivitis, periodontitis). afferents may explain the heat sensitivity of decayed Desensitization to RTX suppresses inflammation and teeth, temperature transduction alone cannot account resultant bone loss in a rodent model of periodontal for the sudden and intense dental pain induced by disease (Breivik et al., 2011). Furthermore, the TRPV1 normally innocuous stimuli such as exposure to air antagonist AZD1386 relieves acute dental pain after puffs and water spray or consumption of sweets. molar extraction in humans (Quiding et al., 2013). Furthermore, patients with chronic pulpitis often These observations suggest that TRPV1 (and maybe describe a pulsating pain sensation in response to also related thermoTRPs) is a viable therapeutic target hydrostatic pressure (Heyeraas and Berggren, 1999). in patients with periodontal disease; for example, it To explain the mechanical allodynia in patients with can be exploited by topical RTX (applied to the gingiva) toothache, the hydrodynamic theory of dental pain or TRPV1 antagonist treatment. (Fig. 24A) was formulated (Brannstrom, 1986; Lin 3. Transient Receptor Potential Channels in Visceral et al., 2011). According to this hypothesis, the move- Pain. With up to 10% of the general population ment of dentinal fluid (which is restricted when both affected, chronic pain and discomfort arising from the ends of the dentinal tubule are closed by the pulp and viscera represent a large unmet medical need. A enamel layer) gets exaggerated when dentin is exposed hallmark of chronic visceral pain is mechanical hyper- by dental caries or tooth crack (see Fig. 24A). algesia and allodynia (perceived as colicky pain) in Of TRP channels that exhibit mechanosensitivity response to peristaltic movements and/or distension and thus may respond to dentinal fluid movement, the that may be secondary to (or at least exacerbated by) expression of TRPV1 (Chaudhary et al., 2001; Park inflammatory disorders. Thus, compounds that simul- et al., 2006), TRPV2 (Ichikawa and Sugimoto, 2000; taneously block mechanosensory transduction and Gibbs et al., 2011), and TRPA1 (Park et al., 2006) has inflammation may prove particularly useful in re- been demonstrated in retrogradely labeled dental lieving visceral pain. There is good evidence that TRP afferent nerves. Furthermore, the expression of TRPV4 channels are involved in both of these processes (Fig. (Wei et al., 2011b) and TRPM3 (Vriens et al., 2011) was 25; see Holzer, 2004; Blackshaw et al., 2010, 2013; reported in trigeminal ganglion neurons. With regard Holzer, 2011a,b). to toothache, TRPA1 is of particular interest because of a. Esophagus and gastrointestinal tract. Sensory the broad range of stimuli to which it responds afferent nerve endings in the mucosa of the gastrointes- (chemical, cold, and mechanical), including bacterial tinal tract respond to chemical or fine tactile stimulation of endotoxins (Meseguer et al., 2014). Of note, TRPA1 is the epithelium, whereas those innervating the muscularis found at the tip of the inner hear hair bundles, where it was suggested to play a crucial role in detecting fluid movements (Corey et al., 2004). Thus, it is a reasonable assumption that TRPA1 may play a similar role in detecting fluid movements within dentinal tubules. However, the role of TRPA1 in mechanotransduction remains controversial because TRPA1-null mice showed normal response to loud noise (Kwan et al., 2006); therefore, the involvement of TRPA1 in the generation of dental pain by mechanical stimuli needs to be verified. Dentin formed before the tooth eruption is called primary dentin. However, dentin formation continues throughout the lifetime with (secondary dentin) or without (tertiary dentin) periodontal disease. The mechanism underlying ongoing dentin formation is unclear. It was suggested that human dental pulp stem Fig. 25. Role of TRP channels in visceral pain. Courtesy of Peter cells may differentiate into odontoblastic cells in Holzer. TRP Channels as Drug Targets 723 and the serosa mostly function as mechanosensitive patients with GERD. It should be kept in mind, tension receptors (see Brierley et al., 2004; Holzer, however, that TRPV1 is a major, but by no means 2011a). The majority of sensory fibers (e.g., .80% of exclusive, acid sensor in the gastrointestinal tract. splanchnic afferents originating in thoracolumbar DRG) Indeed, proton-induced responses were reduced (by that project into the visceral mucosa possess TRPV1 approximately 50%), but not abolished, in Trpv1 (see Robinson and Gebhart, 2008) and convey sensation knockout animals (Kichko and Reeh, 2009). Thus, the of bloating, discomfort, and pain to the CNS (Chen et al., minimal benefit of TRPV1 antagonism in patients with 2013b). Indeed, capsaicin administered topically into GERD (ClinicalTrials.gov identifier D9127C00002) the colon evokes intense pain and referred hyperalgesia was hardly unexpected (Krarup et al., 2013). Paren- (Laird et al., 2001). thetically, the first-generation TRPV1 antagonist A major subset (40%–60%) of vagal afferents arising capsazepine was reported to paradoxically aggravate from nodose ganglia is TRPV1 positive and is typically acid-induced gastric ulcer formation in the rat (Horie associated with satiety and nausea (Patterson et al., et al., 2004). If this observation holds true for humans, 2003). In accord, desensitization of these nerves to RTX it may represent another problematic adverse effect exerts a marked effect in different species, for per os TRPV1 antagonists. including dogs and ferrets (Yamakuni et al., 2002). TRPV1-positive nerves appear to mediate visceral Furthermore, mice fed capsaicin-containing chow gain pain in response to noxious rectal distension (Spencer less weight compared with littermates on a regular diet et al., 2008). This is somewhat surprising since TRPV1 (Zhang et al., 2007b; Leung, 2008). In addition, there is is not supposed to have mechanosensitive properties. anecdotal evidence that human capsaicin consumption Indeed, genetic deletion of TRPV1 has no significant leads to early satiety and weight loss (see Whiting effect on the mechanosensory function of somatic et al., 2012). On the basis of these observations, the use nociceptors (Caterina et al., 2000). For unclear reasons, of dietary capsaicin to boost weight loss as part of visceral TRPV1-expressing afferents seem to function a comprehensive weight management program was differently. For example, TRPV1-null animals show advocated. The phenotype of the Tpv1 knockout mouse deficits in visceral mechanoreceptors, and their behav- is, however, controversial. Early reports indicated no ioral responses to colorectal distension are markedly difference in body weight between Trpv1 knockout and reduced (Jones et al., 2005a). Of note, Trpv1(2/2) wild-type mice (Caterina et al., 1997). However, when mice exhibit decreased mechanical hyper-reactivity of kept on a high-fat diet, the TRPV1-null animals stayed the bladder during cystitis (Wang et al., 2008c), lean compared with control subjects and showed less implicating a role for TRPV1 in visceral pain associ- fatty change in the liver (Motter and Ahern, 2008). By ated with inflammatory disorders. Silencing by RNA contrast, another study found increased obesity (pre- interference of TRPV1 has been reported to ameliorate sumably secondary to less physical activity) in aged visceral pain in rats (Christoph et al., 2006). Moreover, Trpv1 knockout mice (Wanner et al., 2011). Appar- the first-generation TRPV1 antagonist, capsazepine, ently, both age and diet influence the phenotype of the diminished discomfort to colorectal distension in mice TRPV1-null mouse. (Sugiura et al., 2007), similar to the decrease seen in The finding that TRPV1 is directly activated by low TRPV1-null animals (Jones et al., 2005a). Interest- pH (Fig. 4) makes this channel a prime candidate for ingly, TRPV1 and TRPA1 antagonists synergistically sensing heartburn, caused by the reflux of acidic attenuate caerulein-induced pain behavior and pan- stomach contents into the gastroesophageal junction creatic inflammation (Schwartz et al., 2011; Terada (reflux esophagitis, also known as GERD). Indeed, et al., 2013) and prevent the transition from acute to TRPV1 is present on afferents innervating the mucosa chronic pain during experimental pancreatitis (Schwartz of the human esophagus (Matthews et al., 2004; Bhat et al., 2013); after this transition, TRP antagonism is and Bielefeldt, 2006), where its expression is increased ineffective. abuse is a well known cause of acute in mucosal biopsies taken from patients with GERD. pancreatitis. In this context, it is worth mentioning that Mice whose TRPV1 has been deleted by genetic is capable of activating TRPV1 (Trevisani et al., manipulation develop less esophagitis after acid 2002) and ethanol-induced acute pancreatitis is attenu- exposure compared with wild-type control subjects ated in TRPV1-null mice (Vigna et al., 2014). These (Fujino et al., 2006). This is important because findings argue for early intervention by TRPV1 (and also persistent reflux esophagitis is believed to play a TRPA1) antagonists. pivotal role in the development of intestinal metaplasia Increased TRPV1-immunoreactivity was observed in (Barrett esophagus), a premalignant condition. Fur- colonic sensory afferents in patients with IBD, both thermore, desensitization to RTX of gastric mucosal Crohn disease and ulcerative colitis (Yiangou et al., sensory afferents was shown to protect against ulcer 2001), and in rectal sensory fibers with rectal hyper- formation in response to acids (Szolcsányi, 1990). sensitivity and fecal urgency (Chan et al., 2003). It is These observations formed the experimental founda- currently unclear whether these changes in TRPV1 tion of the clinical trials with TRPV1 antagonists in expression are pathogenic or adaptive. In a rat model 724 Nilius and Szallasi of irritable bowel syndrome, TRPV1 antagonists pre- analgesic activity of TRPV1 and TRPA1 antagonism vent the development of visceral hypersensitivity was synergistic (Vermeulen et al., 2013), implying initiated by acetic acid treatment during the neonatal a therapeutic potential for a dual TRPV1/TRPA1 period (Winston et al., 2007). These findings imply antagonist in the pharmacotherapy of IBD. In further a pathogenic role for the dysfunction of TRPV1-positive support of this concept, TRPV1 blockade ameliorated colonic fibers in irritable bowel syndrome (Keszthelyi early, inflammatory pain, whereas TRPA1 blockade et al., 2013). In accord, one study reported a positive suppressed sensitization leading to chronic visceral pain correlation between the number of TRPV1-immunoreactive in a rat model of ulcerative colitis (Chen et al., 2013b). fibers in the rectosigmoid colon and the abdominal Analysis of TRPA1 mutants revealed that TNBS pain score in patients with irritable bowel syndrome covalently binds to cystine residues in the TRPA1 (Akbar et al., 2008). A second study also noted protein in the cytoplasmic N terminus (Engel et al., increased pain perception to rectal capsaicin applica- 2011a). Of note, peripheral blood mononuclear cell tion in patients with irritable bowel syndrome but no supernatants obtained from patients with diarrhea- evidence of TRPV1 upregulation (van Wanrooij et al., predominant IBD, but not constipation-predominant 2014). Taken together, these observations suggest patients or healthy individuals, caused mechanical that TRPV1 is a relevant therapeutic target for hypersensitivity of mouse colonic afferents that was treatment of visceral pain. TRPV1 may even mediate partially inhibited by TRPA1 antagonists (Hughes (at least in part) the well documented beneficial effect et al., 2013). of cannabinoids on visceral pain (De Petrocellis et al., In the rat, TRPA1 (via activation of p38 mitogen- 2012). activated protein kinase) seems to play an important In a rat model of IBD, topical capsaicin treatment role in gastric distension-induced visceral pain (Kondo reduces bowel ulceration in response to trinitrobenzene et al., 2013), raising the possibility that TRPA1 may be sulfonic acid (TNBS) (Goso et al., 1993a). In this model, involved in the development of epigastric pain in the small molecule TRPV1 antagonist JYL1421 [N-(4- patients with functional dyspepsia (FD). This hypoth- tertbutylbenzyl)-N-[3-fluoro-4-(methylsulfonylamino) esis was recently tested in a rat model of FD. Adult benzyl]-thiourea] suppressed microscopic colitis and rats subjected to inflammatory insult by intracolonic significantly reduced (but did not completely abolish) TNBS administration as neonates were reported to visceromotor response to colorectal distension (Miranda develop FD-like gastric hypersensitivity with no et al., 2007). TRPV1 also appears to be involved in the apparent involvement of TRPA1 (Winston and Sarna, postinflammatory hyperalgesia that occurs after reso- 2013). In this context, it is worth mentioning that lution of dextran sodium sulfate–induced experimental a combination of caraway and peppermint oil attenu- colitis (Eijkelkamp et al., 2007). Nonetheless, it may be ates postinflammatory visceral hyperalgesia in rats a premature conclusion that TRPV1 is exclusively (Harrington et al., 2011). Caraway oil is used as responsible for the beneficial effect of capsaicin de- a dietary supplement for digestive problems, including sensitization in preclinical models of colitis. Indeed, in a bloating and mild spasms of the stomach and intes- murine model of visceral pain TRPV1-null mice showed tines. In a multicenter, double-blind clinical study, the only partial (approximately 60%) reduction in pain combination of peppermint and caraway oil was response magnitude compared with wild-type control sub- reported to provide symptomatic relief in patients with jects (Jones et al., 2005a). FD (Madisch et al., 1999). Peppermint oil (and its What is responsible for the remaining 40% of the active ingredient, menthol) is a known TRPM8 agonist. pain behavior? The amiloride-sensitive acid-sensing Indeed, TRPM8 is expressed on a distinct subset of ion channels may be a major contributor (Sugiura colonic afferents with a possible (and rather controver- et al., 2007). Indeed, ASIC(2/2) mice display a re- sial) coexpression of TRPV1 (Harrington et al., 2011). duction in pain behavior that is similar in magnitude This is interesting because dietary capsaicin was to that observed in the TRPV1 knockouts (Jones et al., reported to cause visceral pain in patients with FD. 2005a). Furthermore, is has been shown that TRPA1 It was postulated that TRPM8 activation may interfere (presumably present on TRPV1-positive fibers) is with nociceptive transduction via TRPV1 and TRPA1 markedly upregulated during TNBS-evoked colitis (Blackshaw et al., 2010). Taken together, these ob- (Yang et al., 2008). Consistent with a pathogenic role servations imply a role for TRPV1, TRPA1, and TRPM8 of the increased TRPA1 expression, both intrathecal in the pathomechanism of FD; however, as yet, no administration of TRPA1 antisense oligodeoxynucleo- unequivocal evidence has been presented to support this tide (Yang et al., 2008) and intraperitoneal blockade of concept. TRPA1 by the small molecule antagonist TCS-5861528 H2S is an irritant colorless gas with offensive odor, (Vermeulen et al., 2013) was reported to reverse which is overproduced in the feces of patients with hyperalgesia to colonic distension. Furthermore, colitis ulcerative colitis presumably as a metabolic aberration induced by TNBS is markedly attenuated in TRPA1- due to altered colonic bacterial flora. H2S was recently null mice (Engel et al., 2011a,b). Importantly, the shown to function as a gasotransmitter acting on TRP Channels as Drug Targets 725 various channels, including TRPA1 (Miyamoto et al., sensitizes TRPV1 (Amadesi et al., 2004, 2006) and 2011). Intraluminal colonic H2S evokes visceral noci- TRPA1 (Tarada et al., 2013). Thus, PAR-2 appears to ceptive behavior in the mouse (Matsunami et al., function as a regulator of TRP channels (Surprenant, 2009). TRPA1 is a well established target for the 2007). Since gut bacteria produce high amounts of somatic pronociceptive actions of H2S. Indeed, the H2S- PAR-2, TRPV4 (along with TRPV1 and TRPA1) may be donor NaHS stimulates DRG neurons obtained from an attractive pharmacological target to relieve visceral wild-type, but not Trpa1(2/2), mice (Andersson et al., pain. This hypothesis has gained good experimental 2012), and NaHS-induced mechanical hyperalgesia support by the demonstration of increased TRPV4 was significantly suppressed (although not completely mRNA expression in patients with IBD and the efficacy eliminated) by both pharmacological blockade with of pharmacological TRPV4 antagonism in a mouse model AP18 [4-(4-chlorophenyl)-3-methyl-3-buten-2-one ox- (induced by TNBS) of colitis (Fichna et al., 2012). Of note, ime; Fig. 10] and genetic silencing of TRPA1 (Okubo Src inhibitor-1 suppressed PAR-2–induced activation of et al., 2012). The role of TRPA1 in H2S-induced visceral TRPV4, indicating the importanceoftyrosinephosphor- pain, however, remains controversial, with one group ylation (Poole et al., 2013). finding similar pain behavior in wild-type and Trpa1 The literature on the neuroimmune link between knockout mice (Andersson et al., 2012) and another sensory neurons and the aberrant immune response in group reporting the involvement of both TRPA1 and colitis (see Engel et al., 2011b) is growing (but still Cav3.2 channels in H2S-induced colonic pain and highly speculative). As discussed above, capsaicin- referred hyperalgesia (Tsubota-Matsunami et al., sensitive C fibers coexpress SP with CGRP (see Holzer, 2012). 1991). Unexpectedly, these neuropeptides seem to Another gasotransmitter in the gastrointestinal exert opposite effects in colitis. Indeed, SP-null mice tract is NO. In behavioral assays, peripheral NO- are protected against experimental (oxazolone- evoked nociception is absent when both TRPV1 and induced) colitis, whereas, conversely, the CGRP knock- TRPA1 are ablated such as in TRPV1/TRPA1 double- out animals show increased susceptibility (Engel et al., knockout mice (Miyamoto et al., 2009). As was the case 2012). Chemical ablation of SP-positive nerves by for H2S, the participation of TRPA1 in NO-induced capsaicin pretreatment, or pharmacological blockade visceral pain is unclear. Of note, in the bladder, the of SP receptors (NK1R) by CAM 4092, abolished the TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6- T cell– mediated transfer colitis in severe combined dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropyl- immunodeficiency (SCID) mice (Gad et al., 2009). phenyl)acetamide; Fig. 10] failed to inhibit the pain Unfortunately, the clinical data are equivocal and reaction induced by ifosfamide (an NO donor) (Pereira confusing since patients with IBD were reported to et al., 2013). have decreased (Jarcho et al., 2013), increased (Goode An intriguing, newly identified target for NO in the et al., 2000), or similar (ter Beek et al., 2007) NK1R gastrointestinal tract is TRPV2. In the mouse in- expression compared with healthy subjects. Further- testine, TRPV2 is expressed in both sensory afferents more, SP-like immunoreactivity was markedly reduced and inhibitory motor neurons (Mihara et al., 2010). In in gastrointestinal biopsies taken from patients with dissociated myenteric neurons, TRPV2-like currents ulcerative colitis (Kimura et al., 1994). were detected in association with increased intestinal Most recently, TRPC4 has been added to the list of motility. In the stomach of the mouse, approximately potential therapeutic targets in visceral pain (Westlund 84% of neuronal NOS-expressing myenteric neurons et al., 2014). In rats, genetic inactivation of TRPC4 by coexpressed TRPV2, and gastric emptying was accel- transposon-mediated knockout mutation, or pharmaco- erated by the TRPV2 activator probenecid (Mihara logical blockade by the TRPC4 antagonist ML-204 [4- et al., 2013). methyl-2-(1-piperidinyl)quinoline], induced tolerance TRPV4 has emerged as a molecular osmotransducer to visceral pain evoked by intracolonic mustard oil and mechanotransducer candidate on visceral affer- exposure. ents (Brierley et al., 2008). Compared with TRPV1, the b. Genitourinary tract. Multiple TRP channels are anatomic distribution of TRPV4 in the gastrointestinal expressed in the bladder (in urothelium, nerve endings, tract is more discrete: TRPV4 appears to be preferen- fibroblasts, and detrusor muscle; Fig. 26), where they tially expressed in colonic sensory neurons with are thought to function as sensors of stretch and a virtual absence on vagal afferents (Brierley et al., chemical irritation (see Charrua et al., 2010; Skryma 2008). Behavioral responses to painful colonic disten- et al., 2011; Avelino et al., 2013). There is a dense sion are significantly reduced in TRPV4(2/2) mice network of nerve fibers that express TRPV1 in the (Brierley et al., 2008), as is the mechanical hyper- suburothelium and muscular layer of both rat and algesia that occurs in response to PAR-2 (Grant et al., human renal pelvis, ureter, bladder, and urethra (see 2007). Indeed, TRPV4 has been implicated as a primary Avelino et al., 2013) but the existence of functional target in PAR-2–mediated sustained inflammatory TRPV1 in urothelial cells and detrusor smooth muscle signaling (Poole et al., 2013). Of note, PAR-2 also remains controversial (recently reviewed in Boudes 726 Nilius and Szallasi

Fig. 26. Roles of TRP channels in bladder function. The micturition reflex is mediated by TRPV1-positive afferents and probably, albeit to a lesser degree, also by TRPM8-expressing nerves. The same neurons convey nociceptive information (e.g., bladder pain secondary to cystitis) to the CNS. TRPV4 is thought to be a key player in bladder functions because it is present in both the urothelium and the detrusor muscle where is activated by stretch (bladder distension) and hypo-osmolar urine. TRPV1 may be activated by protons and the inflammatory milieu during cystitis (although the existence of functional TRPV1 in the urothelium remains controversial). The micturition reflex is under the control of a descending CNS pathway. When this pathway is disrupted (e.g., as a result of spinal cord injury or MS), the micturition reflex becomes autonomous and partly driven by TRPV1- expressing nerves. Collectively, these findings imply a therapeutic potential for TRPV1 and TRPV4 blockade and TRPM8 activation in the management of painful and/or overactive bladder disorders. Reprinted with permission from Moran et al. (2011). and De Ridder, 2012). As discussed elsewhere in this biochemical marker of nociceptive pain) and amelio- article, the involvement of neuronal TRPV1 (and rates mechanical hyperalgesia in experimental cystitis nonneuronal TRPV4) in the micturition reflex is well models (Wang et al., 2008c). Furthermore, the TRPV1 established; here, we focus on the role of TRPs in antagonist GRC-6211 (structure shown in Fig. 9) nociception. ameliorates nociceptive behavior and micturition re- The pain of interstitial cystitis is thought to be flex activity in chronically inflamed rat bladder amenable to TRPV1 antagonist therapy. In a feline (Charrua et al., 2009a). Combined, these findings model of interstitial cystitis, abnormally enhanced imply a therapeutic value for TRPV1 antagonists in capsaicin responses were detected (Sculptoreanu et al., the symptomatic treatment of interstitial cystitis. Yet 2005a) secondary to TRPV1 phosphorylation by PKC a large clinical trial with intravesical RTX to de- (Sculptoreanu et al., 2005b). In addition, increased sensitize TRPV1-expressing afferents provided no TRPV1 expression was reported in bladder afferents of clear benefit over placebo in patients with interstitial patients with interstitial cystitis (Mukerji et al., cystitis (Payne et al., 2005), although a subsequent 2006b). An interesting, alternative molecular pathway (and much smaller) study reported some improvement to increase TRPV1 activity during cystitis is by down- in a small subset of patients with interstitial cystitis regulation of TRPV1b, a TRPV1 splice variant that (Peng and Kuo, 2007). In summary, TRPV1 as a acts as a dominant negative modulator (Charrua et al., therapeutic target in interstitial cystitis remains 2008). In mice, genetic manipulation of the TRPV1 questionable (Mourtzoukou et al., 2008). One cannot gene prevents spinal c-fos overexpression (a surrogate help but wonder whether this is another example of the TRP Channels as Drug Targets 727 worrisome disconnect between preclinical models (ro- is present in lumbar DRG neurons, where its expres- bust effect for TRP targeting) and patients (no clinical sion is increased in transgenic animals overexpressing benefit for the same approach). NGF (Girard et al., 2013), but not in bladder nerve A complex change in mRNA levels encoding TRP endings. TRPV4(2/2) mice show an altered micturi- channels was recently reported in bladder biopsies tion pattern that is characterized by increased inter- taken from 22 patients with classic interstitial cystitis micturition intervals and spotting behavior due to and 17 patients with nonclassic interstitial cystitis spontaneous (not reflex-driven) detrusor muscle con- (Homma et al., 2013). In patients with nonclassic tractions (Gevaert et al., 2007). In addition, TRPV4 (i.e., nonulcerated) interstitial cystitis, TRPV2 was the knockout mice show reduced urinary frequency and only TRP channel to show increased expression. By increased void volume after bladder damage caused contrast, classic interstitial cystitis tissues showed in- by intravesical administration of creased levels of TRPA1, TRPM2, TRPM8, TRPV1, and (Everaerts et al., 2010b). On the basis of these findings, TRPV2 mRNAs and a concomitant decrease in TRPV4. it was postulated that TRPV4 plays a crucial role in the This supports the notion that interstitial cystitis is mechanosensory pathway in the bladder by detecting a heterogenous disease (Peeker and Fall, 2000). changes in intravesical pressure. Indeed, mechanical As discussed elsewhere in this review, in the rat, stretch activates urothelial TRPV4 in vitro (Mochizuki TRPA1 is highly coexpressed with TRPV1 on bladder et al., 2009). Of note, TRPV4 is increased in the afferents and is especially abundant in the outflow urothelium and detrusor muscle of rats with experi- region, implying a role in micturition (Streng et al., mental bladder outlet obstruction (Cho et al., 2013). 2008). Although it is tempting to speculate that TRPA1 Furthermore, TRPV4 is osmosensitive and the osmo- (a shared target for a broad range of chemical irritants) larity of urine changes during cystitis. However, there may contribute to bladder pain under inflammatory is no evidence that TRPV4 contributes to bladder pain conditions, the experimental data to support this in humans (as discussed above, some “TRPV4pathy” hypothesis are weak. In fact, even visceral pain evoked patients show abnormal micturition but none complain by mustard oil (a well established activator of TRPA1) of painful bladder). appears to be mediated via TRPV1 and not TRPA1 4. Contribution of Transient Receptor Potential Chan- (Everaerts et al., 2011). Parenthetically, allyl- nels to Neuropathic and Cancer Pain. Chronic pain isothiocyanate, the active ingredient in mustard oil, both affects a large segment of the population, an estimated directly activates TRPV1 (Gees et al., 2013) and sensitizes 50 million Americans, and costs the United States it to heat stimulation (Alpizar et al., 2014). Although billions of dollars in health care expenditures and lost TRPA1 was implicated in the hyperalgesia and urinary productivity. Although the Decade of Pain Control and bladder overactivity that accompany cyclophosphamide- Research has given new impetus to pain research, the induced cystitis (Meotti et al., 2013), these animals also translation of preclinical research into clinical practice show elevated TRPV1 expression (Dang et al., 2013) and is slow to occur. This is evident by the limited number of diminished nociceptive behavior in the presence of mechanistically novel therapeutic agents that have TRPV1 blockade (Charrua et al., 2009a). entered into the clinic for the treatment of pain in TRPM8 is expressed both in urothelium and on thin recent years. Consequently, agents that have been bladder afferents (Fig. 26; Stein et al., 2004). TRPM8 is around for decades, such as opiates and nonsteroidal an interesting target to explore for bladder pain in that anti-inflammatory drugs (NSAIDs), still represent a 1) it is increased on the bladder afferents, but not the mainstay in current pain therapy. However, NSAIDs urothelial cells, of patients with idiopathic detrusor have only modest effects on moderate to severe pain and overactivity, and 2) the increase in TRPM8 correlates their use is limited by a combination of gastrointestinal with pain severity (Mukerji et al., 2006b). Indeed, these and cardiovascular side effects. Opiates are effective patients show higher pain scores during cold water pain killers but their use is complicated by sedation, instillation (Mukerji et al., 2006c). The TRPM8 agonist constipation, and itch, as well as concerns regarding menthol evokes the micturition reflex in humans. tolerance and abuse. Clearly, new potent analgesic Conversely, the TRPM8 antagonist AMTB (structure drugs with an acceptable safety profile are needed. shown in Fig. 11) decreases the frequency of volume- a. Targeting vanilloid transient receptor potential 1 induced bladder contractions in a rat model of painful for pain relief: A brief overview of clinical studies. bladder syndrome (Lashinger et al., 2008). These Preclinical research has identified an array of new findings imply a therapeutic potential for TRPM8 molecular mechanisms that are involved in the de- antagonists in the management of bladder disorders velopment and maintenance of chronic pain and may that are characterized by pain and/or overactivity of represent attractive targets for pharmacological in- the detrusor muscle (see Andersson et al., 2010). tervention. Since the molecular cloning of the vanilloid In the bladder, TRPV4 is the most abundant TRP receptor TRPV1 (Caterina et al., 1997), overwhelming channel, especially in the urothelium and detrusor experimental evidence has been accumulated that sen- muscle (see Avelino et al., 2013). Perplexingly, TRPV4 sory neurons expressing TRP channels, in particular 728 Nilius and Szallasi

TRPV1, are important mediators of pathologic pain 2000), in particular PKCj (Numazaki et al., 2002; (Patapoutian et al., 2009; Brederson et al., 2013). For Mandadi et al., 2006), that requires the interaction of instance, rats desensitized to systemic RTX (300 mg/kg TRPV1 with the scaffolding protein AKAP79/150 (Jeske s.c.) are devoid of both the ongoing spontaneous pain et al., 2009). Indeed, small inhibitory peptides that (manifested in the guarding behavior of the injured interfere with this interaction exhibit analgesic poten- paw) and the evoked pain (quantified as thermal tial in rodent models of neuropathic pain (Fischer et al., hyperalgesia in the hot plate test) that develop after 2013). Small molecule antagonists that selectively block mechanical damage of the sciatic nerve (Bennett sensitized (i.e., phosphorylated by PKC) TRPV1 chan- model) (Fig. 18) (unpublished data). Strikingly, RTX nels were also reported (Sugimoto et al., 2013). also abolishes pain behavior when given to rats in As detailed above, in the periphery, TRPV1 functions a therapeutic fashion (i.e., in animals already in as a polymodal receptor with complex regulation (Fig. discomfort after the operation) (Fig. 18). 12; see Szallasi et al., 2007; Lázár et al., 2009; Despite 25 years of intensive research, the mecha- Szolcsányi and Sándor, 2012). Importantly, there is nism of action of RTX is still poorly understood. In the emerging evidence that TRPV1 may also play an rat, systemic RTX (up to 300 mg/kg s.c.) induces important role in the modulation of synaptic trans- a peculiar constellation of molecular changes. For mission in the spinal cord (first sensory synapse in the example, the expression of endogenous compounds dorsal horn where TRPV1 is coexpressed with m-opioid that suppress pain (e.g., galanin) is increased; by receptors), as well as in supraspinal nuclei (Maione contrast, the expression of proinflammatory and et al., 2009b; McGaraughty et al., 2009). Indeed, algogenic agents (e.g., SP and CGRP) is downregulated a study comparing the analgesic effects of TRPV1 (Farkas-Szallasi et al., 1995, 1996; Szallasi et al., antagonists with and without access to the CNS 1999). These changes, collectively referred to as provided compelling evidence that a dual (both periph- “vanilloid-induced messenger plasticity,” appear to be eral and central) action is required for full analgesic fully reversible and parallel the loss and restoration of action (Cui et al., 2006). It was speculated that TRPV1 the pain behavior (see Szallasi, 1996). Indeed, in in the dorsal horn of the spinal cord contributes to patients with overactive bladder (similar to rats), central sensitization and is subject to regulation by intravesical RTX induces a long-lasting (several endogenous agents, the so-called “endovanilloids” months) but fully reversible improvement in bladder (Palazzo et al., 2013). functions (Cruz et al., 1997; Silva et al., 2000, 2001, Two independent gene-targeting studies deleting 2005). This reversible desensitization (defunctionaliza- TRPV1 alleles conclusively showed that TRPV1 is tion) contrasts with the irreversible loss (RTX as a critical channel for mediating thermal hyperalgesia a “molecular scalpel”) of nociceptive behavior that under inflammatory pain conditions in mice (Caterina follows intrathecal RTX administration (Jeffry et al., et al., 2000; Davis et al., 2000). In addition, one study 2009; Iadarola and Mannes, 2011). showed that TRPV1-null mice are significantly less TRPV1 can be both upregulated and sensitized sensitive to acute noxious heat stimulation (Bölcskei during inflammation and injury (Bishnoi and Premkumar, et al., 2010). Rats with nonfunctioning TRPV1 (desen- 2013). Indeed, TRPV1 was suggested to play a central sitized to RTX) also show profound hypoalgesia in the role both in peripheral (Immke and Gavva, 2006) and hot plate test (Broberger et al., 2000). Indeed, in- central sensitization (Palazzo et al., 2012). In keeping creased cutaneous heat pain perception is a biomarker with this concept, the “wind-up” phenomenon that to quantitate analgesic capsaicin activity in men (Bley, develops after repeated C fiber stimulation (believed to 2013). In light of these findings, it was not completely be a marker of central sensitization) is abolished in unexpected that TRPV1 antagonists increased the rats desensitized to RTX (Xu et al., 1997). In a tri- noxious heat pain threshold in humans (discussed geminal neuropathic pain model, extensive TRPV1 later in the clinical trials section). hyperactivity was detected in central terminals in- The endogenous fatty acid OEA, which is synthe- nervating the dorsal horn (Kim et al., 2014b). This sized and released from the intestine upon feeding, hyperactivity was maintained by descending seroto- evokes visceral pain-related behavior in wild-type nergic input from the brainstem. This is in accord with mice. This effect was prevented by TRPV1 antagonism the report that TRPV1 antagonists that enter the CNS and was absent in TRPV1 knockout mice (Wang and [e.g., A-784168 (3,6-dihydro-39-(trifluoromethyl)-N-[4- Wang, 2005). Other loss-of-function studies, such as [(trifluoromethyl)sulfonyl]phenyl]-[1(2H),29-bipyridine]- transgenic mice expressing TRPV1 shRNA, have 4-carboxamide)] are superior in their analgesic activity conclusively shown that silencing the gene encoding compared with those that are peripherally restricted for TRPV1 by RNA interference significantly attenu- [e.g., A-795614 (1-(1H-indazol-4-yl)-3-[(1R)-5-piperidin- ates capsaicin-induced pain behavior and sensitivity 1-yl-2,3-dihydro-1H-inden-1-yl]urea)] (Cui et al., 2006). toward noxious heat (Kasama et al., 2007). A key mechanism of peripheral TRPV1 sensitization In a rat model of partial-thickness cutaneous is phosphorylation by PKC (Premkumar and Ahern, thermal injury, pharmacological blockade of TRPV1 TRP Channels as Drug Targets 729 almost completely (by 98%) blocked thermal allodynia et al., 2005). TRPV1 is increased in synovial samples (Green et al., 2013), indicating that pain evoked by taken from patients with OA (Engler et al., 2007; a second-degree burn is predominantly TRPV1 medi- Kelly et al., 2013), and the loss-of-function TRPV1 ated. This observation implies a clinical benefit for variant I585V has been linked to a decreased risk for TRPV1 antagonist-containing lotions in the pain painful OA (Valdes et al., 2011). Taken together, these control of burn patients. Authors speculated that findings identify TRPV1 as a promising target to oxidized LA metabolites generated during burn injury relieveOApain.However,theTRPV1antagonist are capable of activating TRPV1 as endogenous algogenic ABT-116 [urea, N-((2-(3,3-dimethylbutyl)-4-(trifluoro- agents. Of note, sunburn pain also involves epidermal methyl)phenyl)methyl)-N9-(1-methyl-1H-indazol-4-yl)] did TRPV4 (Moore et al., 2013). Activation of TRPV4 by UVB not provide any significant pain relief in dogs with in keratinocytes upregulates endothelin-1 expression. experimental synovitis (Cathcart et al., 2012) or naturally TRPV1-expressing neurons express endothelin-1 recep- occurring, age-related hip OA (Malek et al., 2012), and tors and endothelin-1 potentiates capsaicin-evoked cur- AZD1386 (Fig. 9) was withdrawn from clinical trials in rents in sensory neurons (Plant et al., 2007). Although not patients with OA after a periodic review of the data failed directly related to pain, it is worth mentioning here that to show any clinical benefit (Svensson et al., 2010; Miller sunburn activates TRPA1 in human melanocytes and et al., 2014). thereby increases pigmentation (suntan) (Bellono and It is not easy to reconcile these discrepant findings. Oancea, 2013; Bellono et al., 2013). One might speculate Clearly, they question the relevance of the rodent that creams containing TRPA1 agonists may help prevent (MIA) model of human OA pain (Zhang et al., 2013c). It sunburn, an important cause of skin cancers (if so, one is worth recalling here that in other preclinical pain can also easily visualize an interest by tanning salons in models, the analgesic efficacy of TRPV1 blockade was this approach). dependent on the duration (stage) of the disease. For An entire book was recently devoted to the analgesic instance, TRPV1 is increased in the early stage of activity of TRPV1 antagonists in preclinical chronic experimental diabetic neuropathy but is reduced (and (inflammatory and neuropathic) pain models (Gomtsyan eventually lost) with disease progression (Pabbidi and Faltynek, 2009). For an update on this topic, et al., 2008). Moreover, insulin (which is initially interested readers are referred to a number of compre- increased in the circulation of patients with T2DM) hensive reviews (Kort and Kym, 2012; Szallasi and increases TRPV1 expression in the plasma membrane Sheta, 2012; Brederson et al., 2013; Szolcsányi and (Van Buren et al., 2005). In skin biopsies taken from Pintér, 2013). As a representative example of the patients with long-standing diabetic neuropathy, problems that plague this field, here we briefly review TRPV1-like immunoreactivity is, however, markedly the OA data from rodents to clinical trials. reduced (Lauria et al., 2006). Of note, TRPV1 antag- In a rat model of OA (induced by intra-articular onists block acute pancreatitis pain (as well as the monoiodoacetate [MIA] injection), DRG neurons back- transition from acute to chronic pain) in rats, but labeled from the affected joints showed increased become ineffective when chronic pancreatitis is estab- TRPV1-like immunoreactivity (Fernihough et al., lished (Schwartz et al., 2013). On the basis of these 2005). Compared with naïve rats, these animals dis- findings, one might argue that TRPV1 is involved in played enhanced TRPV1-mediated CGRP release in the development, but not maintenance, of chronic response to capsaicin (Puttfarcken et al., 2010). Three neuropathic pain. One may also make an argument weeks after MIA injection, spinal neurons obtained that TRPV1 antagonists, if given early, might prevent from these rats showed increased spontaneous firing (or at least) delay the development of chronic pain. activity (supposedly corresponding to ongoing pain), This is consistent with the observations that capsaicin which was inhibited by the TRPV1 antagonist A-889425 reduces hyperalgesia and bone damage when given [1-(3-methylpyridin-2-yl)-N-(4-(trifluoromethylsulfonyl) prophylactically (i.e., before MIA injection) in the rat phenyl)-1,2,3,6-tetrahydropyridine-4-carboxamide] (Kalff et al., 2010); however, in multiple clinical trials it (Chu et al., 2011). Intra-articular administration of the showed no proven clinical benefit in patients with OA TRPV1 antagonist JNJ17203212 [4-[3-(trifluoromethyl)- (reviewed in Remadevi and Szallasi, 2008). Clearly, OA 2-pyridinyl]-N-[5-(trifluoromethyl)-2-pyridinyl]-1- is a complex disease with redundancy in pain path- piperazinecarboxamide; Fig. 9] almost completely ways, and TRPV1 is only one of many pain targets in abolished the weight-bearing asymmetry in mice with patients with OA (Killock, 2013; Zhang et al., 2013c). MIA-induced OA 2 hours after treatment (Kelly et al., TRPV1-expressing sensory afferents remain a prom- 2013). Furthermore, desensitization to capsaicin re- ising therapeutic target in neuropathic pain. Indeed, duced both pain and bone damage induced by MIA targeted silencing by QX-314 and capsaicin of TRPV1- (Kalff et al., 2010). In mice, genetic deletion of TRPV1 positive axons abolishes thermal, mechanical, and reduced (but did not eliminate) tissue damage and (somewhat surprisingly) cold hyperalgesia in the rat mechanical hyperalgesia in the complete Freund’s (Brenneis et al., 2013), and perineural (Kissin and adjuvant (CFA) model of chronic arthritis (Szabó Szallasi, 2011) or epidural RTX (a molecular scalpel 730 Nilius and Szallasi that selectively abolishes TRPV1-expressing nerves) TRPA1 was silenced via siRNA knockdown. Taken produces lasting analgesia in rats with neuropathic together, these findings suggest that the peripheral pain secondary to spinal nerve ligation (Lee et al., antinociceptive actions of cannabinoids are mediated by 2012). Importantly, intra-articular RTX injections TRP channels, in particular TRPA1/TRPV1 heteromers ameliorate pain behavior and restore ambulation in (see Akopian et al., 2008). In other words, TRPA1/ dogs with severe OA pain (Fig. 19A; D. Cimino-Brown TRPV1 heteromers may represent novel “ionotropic and M. Iadarola, personal communication). As dis- cannabinoid receptors” (Akopian et al., 2009). cussed below, intrathecal RTX is undergoing clinical The clinical value of capsaicin (and its less pungent trials in patients with chronic, intractable cancer pain. synthetic congener, olvanil; Fig. 5) was extensively Although epidural RTX is well tolerated at doses at discussed elsewhere by us (Knotkova et al., 2008; which it induces analgesia (ED50 = 0.265 mg in the rat), Szallasi and Sheta, 2012) and others (Wong and it can cause sedation and hyperventilation at approx- Gavva, 2009; McCormack, 2010; O’Neill et al., 2012; imately 10-fold higher doses (.2 mg) and the treatment Bley, 2013), including in a recent systematic review can be even fatal if the dose exceeds 10 mg (Lee et al., of the Cochrane Database by Derry et al., (2013). 2012). Localized injection (into the affected painful Although ALGRX-4975 (patent owner, AlgoRx; Adlea), joints) should minimize the risk for these adverse an injectable capsaicin preparation, showed promise in effects. Finally, it is worth mentioning that RTX does early clinical studies to relieve OA and postoperative not influence the level of TRPV1 mRNA and protein in pain (e.g., after buniectomy or knee replacement non-neural cells (Kun et al., 2012). surgery; see Remadevi and Szallasi, 2008), Anesiva Cancer pain is another promising indication for (the company developing Adlea) ran out of funds to TRPV1 blockade. Here it suffices to mention that complete the clinical trials and filed for bankruptcy in TRPV1 expression is enhanced in DRG neurons 2010 (www.fiercebiotech.com/story/anesiva-ends-2009- ipsilateral to bone cancer (osteosarcoma) in the mouse bankrupcy-petition/2010-01-04). (Niiyama et al., 2007). In mice and dogs, treatment Another major setback for capsaicin therapy was with RTX to desensitize TRPV1-containing neurons the recent unanimous decision by a US Food and ameliorates bone cancer pain (Fig. 19; Brown et al., Drug Administration (FDA) panel not to recommend 2005; Menendez et al., 2006). In the mouse, this effect Qutenza, a high-dose (8%) capsaicin patch, for HIV- was mimicked by both genetic disruption of the TRPV1 related pain for lack of proof for clinical efficacy (www. gene and pharmacological TRPV1 blockade by the bloomberg.com/news/2012-03-08/neurogesx-fails-to-win- selective antagonist JNJ17203212 (Fig. 9; Ghilardi u-s-approval-to-sell-patch-for-hiv-related-pain.html). et al., 2005). It is noteworthy that intrathecal RTX Qutenza is still available for postherpetic neuralgia injection was reported to provide long-lasting (several (PNH) in the United States (Acorda, Ardsley, NY), and weeks) pain relief in dogs with advanced osteosarcoma, for nondiabetic peripheral neuropathic pain in Europe although it had no effect on the natural progression of (Astellas Europe, Chertsey, UK). Each 280 cm2 patch the disease (Fig. 19B; reviewed in Iadarola and contains a total of 179 mg capsaicin (i.e., 640 mg/cm2; Mannes, 2011; Iadarola and Gonnella, 2013). The www.qutenza.com). In the United States, a kit of two treatment was well tolerated. These findings paved patches retails for $1430.87. Clinical trials with the way for the clinical trials at the National Cancer Qutenza in patients with PNH and other forms of Institute with intrathecal RTX in patients with in- neuropathic pain were detailed elsewhere (Noto et al., tractable cancer pain (ClinicalTrials.gov identifier 2009). In brief, Qutenza induced a reversible loss of NCT00804154). TRPV1-positive dermal afferents (Fig. 27). It was well Of note, based on coimmunoprecipitation and fluores- tolerated (with a few exceptions, all subjects were able cence resonance energy transfer interaction experiments, to complete 30- to 60-minute treatment duration the existence of functional TRPA1/TRPV1 heteromers without asking for early removal of the patches), and was postulated in sensory neurons (Staruschenko et al., it gave a modest analgesic effect over placebo (Peppin 2010). The cannabinoid agonists WIN 55,212-2 [(R)- et al., 2011; Bley, 2013). Although herpes zoster is not (+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo uncommon (it affects about 800,000 adults in the [1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] United States annually) (Schmader, 2002), the proba- and AM1241 [(2-iodo-5-nitrophenyl)-[1-[(1-methylpiperidin- bility of long-standing pain of clinical importance is low 2-yl)methyl]indol-3-yl]methanone] were shown to di- (Helgason et al., 2000); therefore, the market for rectly activate TRPA1 (Akopian et al., 2008). Un- Qutenza is limited. Indeed, Qutenza is projected to expectedly, TRPA1 activation by these compounds also garner peak sales of just $50 million across the seven blocked the capsaicin-induced nocifensive behavior, major markets. NGX-1998 (Neuroges-X) is a liquid implying heterologous desensitization between TRPA1 formulation of high-concentration capsaicin (20% w/w). and TRPV1. Indeed, WIN 55,212-2 caused profound It has a shorter application time (5 minutes compared changes in the phosphorylation status of the TRPV1 with Qutenza, which needs to be left on the painful protein (Jeske et al., 2006). This effect was absent if area for at least 30 minutes); therefore, it is more TRP Channels as Drug Targets 731

general anesthesia. The RTX treatment was well tolerated and the animals were discharged to home the day after treatment. Intrathecal RTX administra- tion resulted in a profound analgesic action (Fig. 19B): The animals became ambulatory, walking on four legs. RTX administration, however, had no influence on disease progression. By 14 weeks, only four dogs stayed alive (Iadarola and Gonnella, 2013). The first human patient with intractable cancer pain was enrolled into the phase I RTX trials at the National Cancer Institute in October 2009 (ClinicalTrials.gov identifier NCT00804154). With more than 50 pharmaceutical companies filing over 1000 patents (for survey of the patent literature, see Voight and Kort, 2010), the preclinical literature on small molecule TRPV1 antagonists is vast and exten- sively reviewed both by us (Appendino and Szallasi, 2006; Gharat and Szallasi, 2007; Khairatkar-Joshi and Fig. 27. The high-dose capsaicin patch Qutenza induces a reversible loss of TRPV1-positive dermal afferents in the treated human skin area. Szallasi, 2009; Szallasi and Sheta, 2012; Brederson Reprinted with permission from Bley (2013). et al., 2013) and others (Gunthorpe and Chizh, 2009; Kort and Kym, 2012; Palazzo et al., 2012). Although convenient for the patients. NGX-1998 was reported to a number of small molecule TRPV1 antagonists have provide modest pain relief up to 12 weeks in 165 been advanced to clinical trials, thus far none have patients with PHN (www.empr.com/liquid-capsaicin- progressed beyond phase II. GlaxoSmithKline was ngx-1998-provides-pain-relief-after-five-minutes/article/ among the first to disclose its phase I results obtained 241907/#). with SB705498 [1-(2-bromophenyl)-3-[(3R)-1-[5- Civamide (synthetic zu-capsaicin) is being developed (trifluoromethyl)pyridin-2-yl]pyrrolidin-3-yl]urea] (Chizh by Winston Pharmaceuticals (Vernon Hills, IL; www. et al., 2007). In a proof-of-concept study, a single oral winstonlabs.com) for indications such as cluster head- dose of 400 mg of SB705498 (structure shown in Fig. 9) ache (ClinicalTrials.gov identifier NCT01341548), mi- substantially reduced pain and flare evoked by cutane- graine (Diamond et al., 2000), and OA (Schnitzer et al., ous capsaicin challenge (0.075% capsaicin cream applied 2012). Civanex (a cream containing 0.075% civamide) to the forearm) compared with placebo. SB705498 did is intended to be used in combination with COX-2 not show any serious adverse effects in the study. inhibitors in patients with moderate to severe OA pain. Subsequently, the effect of per os SB705498 on dermal In 2010, Health Canada issued a Notice of Compliance heat sensitivity after capsaicin or UVB challenge was for Civanex (as of today, the FDA approval is still evaluated. Somewhat unexpectedly, SB705498 caused pending). a modest, but significant (up to 1.3°C), increase in the There is a recent revival of interest in RTX as noxious heat perception threshold both in rodents and a molecular scalpel to achieve permanent analgesia in humans. In December 2005, an active-controlled, placebo- patients with intractable cancer pain, fueled by the controlled, randomized, single-blind, phase II trial was ability of the compound to relieve pain and restore initiated in subjects with dental pain after third molar ambulation in dogs with osteosarcoma (Fig. 19). tooth extraction. The subjects were to receive a single oral Canine osteosarcoma is a naturally occurring bone dose of SB705498, placebo, or co-codamol. The study was cancer pain that usually affects the long bones in one completed in February 2008, and results have not yet been limb. In the dog, RTX injections (1–3 mg/kg) can be revealed (ClinicalTrials.gov identifier NCT00281684). made into either the lumbar cistern (for hind limb In 2008, Amgen announced the early termination of tumors) or the cisterna magna (for forelimb tumors). At a phase Ib dental pain (molar extraction) study with this dose, intracisternal RTX administration caused their clinical candidate molecule, AMG517 [N-[4-({6-[4- nearly complete loss of sensitivity to noxious thermal (trifluoromethyl)phenyl]pyrimidin-4-yl}oxy)-1,3-benzothiazol- stimulation when tested 2 days after treatment (Brown 2-yl]acetamide;Fig.9],becauseitcausedalasting(1–4days) et al., 2005). Dogs with intractable osteosarcoma pain marked hyperthermia response (up to 40.2°C) in human (that had become unresponsive or intolerant to con- volunteers (Gavva et al., 2008). ventional pain management) were enrolled into the AstraZeneca was developing AZD1386 (Fig. 9) for the intrathecal RTX administration studies. Most of the potential oral treatment of chronic nociceptive pain animals were candidates for euthanasia because of and GERD. In April 2008, an active-controlled, placebo- poor pain control. Because intrathecal RTX causes an controlled, randomized, double-blind phase II trial was acute pain reaction, the injection is performed under initiated in subjects with pain due to third molar 732 Nilius and Szallasi extraction. AZD1386 (55 mg p.o.) caused significant According to the company website, XEN-D0501 is pain relief (Krarup et al., 2011). The drug was well about to enter phase II development for the indica- tolerated although a modest increase in body tempera- tions of overactive bladder and chronic cough (www. ture (approximately 0.4°C in average) was noticed in provesica.com). In healthy subjects, XEN-D0501 was most patients, exceeding 38°C in one individual (Krarup rapidly absorbed (tmax between 0.5 and 4 hours et al., 2011). Disappointingly, AZD1386 did not provide postdose), well tolerated, andcausedonlymildhyper- any clinically meaningful pain relief in patients with OA thermia (0.74°C) at the highest dose (5 mg) tested (Svensson et al., 2010; Miller et al., 2014) or GERD (Round et al., 2011). Finally, Amorepacific, a skincare (Krarup et al., 2013). Of note, in 2012 AstraZeneca company based in South Korea, is developing PAC-14028 announced the closing of its neuroscience division, in- [(E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)- clusive of the TRPV1 antagonist program (www.fierce- ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)- biotech.com/story/astrazeneca-cutting-2200-rd-jobs- acrylamide; Fig. 9] for atopic dermatitis (Lim and Park, slashing-neuroscience-restructuring). 2012). A phase II trial with GRC-6211 (Glenmark-Eli Lilly) There are two schools of thought with regard to the for OA pain was suspended due to undisclosed reasons major adverse effects (febrile reaction and impaired (see Fig. 9 for structure) (Kitagawa et al., 2012). No body heat pain sensation) of TRPV1 antagonists. The first temperature or thermosensation data were reported for considers these on-target side effects. Many proponents this compound (www.glenmarkpharma.com/GLN_NWS/ of this theory believe that side effects of TRPV1 news.aspx?res=P_GLN_NWS_RLS). blockade can be adequately managed by sensible Merck/Neurogen was developing MK-2295 (Fig. 9) precaution. Indeed, the hyperthermia response shows for the potential treatment of pain and cough. MK- attenuation after repeated dosing, and the initial 2295 markedly increased the noxious heat pain increase in body temperature can be adequately threshold in humans (unlike the hyperthermia re- managed by common antipyretic drugs such as sponse, this did not show any attenuation with acetaminophen (Gavva et al., 2007). Moreover, burns repeated dosing), placing the study participants at (that are usually mild) can be prevented by simply the risk of scalding injury (reviewed in Eid, 2011). warning the patients to be careful. In 2011, Abbott reported that its clinical candidate A more attractive approach is to eliminate the ABT-102 (structure shown in Fig. 9) caused the undesirable side effect of TRPV1 antagonists by elevation of mean core body temperature by 0.6°C in chemical modification of the pharmacophore. In the healthy volunteers after short-term administration of rat, it was feasible to eliminate hyperthermia while a 4-mg dose (Rowbotham et al., 2011). ABT-102 was preserving antihyperalgesia by differential modulation well tolerated with repeated dosing in humans; by day of distinct modes of TRPV1 activation (Lehto et al., 7, temperature increases were no longer significant for 2008). Subsequently, several companies (Astellas, any dose tested. Abbott also reported that ABT-102 Muchida, Grünenthal, etc) described so-called “modal- caused an increase in cutaneous and oral heat pain ity-specific” TRPV1 antagonists that do not raise body thresholds measured by quantitative sensory testing, temperature in the rat. Representative examples are and that the deficit in noxious heat perception did not AS1928370 [(R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro- attenuate with the 7-day twice-daily dosing regimen 7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl- (Rowbotham et al., 2011). 4-carboxamide; Fig. 9]) (Watabiki et al., 2011) and Compound PHE377 has completed a phase I clinical A-1165442 [(R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman- trial (www.pharmeste.com; PharmEste). PHE377 4-yl)-3-(3-methylisoquinolin-5-yl)urea; Fig. 9] (Reilly (structure not revealed) is interesting in that it was et al., 2012). reported not to cause any detectable hyperthermia in Modality specificity is an attractive concept. TRPV1 rats or dogs (see Trevisani and Szallasi, 2010). JTS-653 is unique in that it functions as a “multisteric by Japan (Fig. 9) was reported to ameliorate nocisensor” (Szolcsányi and Sándor, 2012). Side- PNH pain in rodent models (Kitagawa et al., 2013a). In directed mutagenesis experiments furnished strong the rat bladder, JTS-653 suppressed overactivity with- evidence that point mutations can selectively eliminate out affecting normal micturition (Kitagawa et al., TRPV1 activation mechanisms (Fig. 4). For example, 2013b). According to the company website, this com- the Y511A mutant (affecting the TM3 region) responds pound is now in phase II clinical trials in Japanese to heat and protons but not capsaicin (Jordt et al., patients with painful overactive bladder (www.jt.com/ 2000). Conversely, the T633A mutant (that changes investors/results/S_information/pharmacauticals/pdf/ the pore) shows normal capsaicin activation but does P.L.20220207_E.pdf). not respond to heat or protons (Ryu et al., 2007). These Although it is not intended to be a pain , findings imply that such modality-selective antagonists for the sake of completeness, it should be mentioned may be synthesized that leave heat sensing intact. For herethatProvesica(asubsidiary of Xention) successfully example, the R114E mutant (in the N terminus) shows completed its phase I clinical trials with XEN-D0501. normal heat responses but is insensitive to both protons TRP Channels as Drug Targets 733 and capsaicin (Jung et al., 2002a). Indeed, a new medium diameter peripheral nerve axons in the brachial methodology was developed to screen for TRPV1 nerve plexus of patients with traumatic nerve injury antagonists that do not inhibit capsaicin activation (Facer et al., 2007). TRPV3 is also increased in the spinal (Zicha et al., 2013). Added to this complexity are reports nerve ligation model of neuropathic pain in the rat (see of allosteric TRPV1 modulators (Kaszas et al., 2012; Bevan and Andersson, 2009). Lebovitz et al., 2012) and partial agonists/antagonists No robust phenotype has been reported for Trpv3 (Blumberg et al., 2011). The Amgen group postulated knockout mice in inflammatory pain; however, several that hyperthermia is a function of proton activation lines of evidence support a role for TRPV3 in pain (Lehto et al., 2008): Antagonists that do not interfere processing. Trpv3 knockout mice showed deficits in with the proton activation of TRPV1 are devoid of response to acute noxious thermal stimuli in water hyperthermia. This concept appears to be true for some immersion (.48°C) and hot plate (55°C) assays and compounds (AS1928370 and A-1165442) but not others exhibited deficits in responses to innocuous and (PHE377 and JTS-653) (compare structures in Fig. 9). noxious heat (within the first hour) in a thermal place In summary, we still do not understand why some preference assay (Moqrich et al., 2005). Knockdown of TRPV1 antagonists induce a marked febrile reaction, TRPV3 with shRNA reduced nocifensive behavior whereas others do not. It may be of relevance that induced by intraplanar injection of farnesyl pyrophos- TRPV1 is expressed in arteriolar smooth muscle phate (structure shown in Fig. 8), a TRPV3 agonist, in (Cavanaugh et al., 2011). The TRPV1 agonist RTX inflamed rats and also blocked farnesyl pyrophosphate– evokes “reddening” (hyperemia) when applied to the induced thermal hyperalgesia (Bang et al., 2010). Hydra rodent ear (Szallasi and Blumberg., 1989). Although Biosciences has reported efficacy of TRPV3 receptor this effect is traditionally attributed to neurogenic antagonists in models of inflammatory pain (CFA), inflammation, a direct action on TRPV1 expressed on formalin-induced flinching, thermal injury, and spinal vascular smooth muscle cells cannot be ruled out sensitization (see Reilly and Kym, 2011). Glenmark (vasodilatation/vasoconstriction in the skin is an Pharmaceuticals has reported efficacy of TRPV3 antag- important mechanism of body temperature regulation). onists in inflammatory and nerve injury models (see The literature is, however, confusing. Functional Khairatkar-Joshi et al., 2010). TRPV1 expression was reported in murine skeletal Pharmaceutical interest in the role for TRPV3 in muscle (Lotteau et al., 2013) where capsaicin was pain has lead to the discovery of small molecule TRPV3 noted to induce hypertrophy (Ito et al., 2013). In the antagonists and their characterization in preclinical perfused rat hind limb, RTX causes vasoconstriction pain models (see Huang and Chung, 2013). Hydra and increased O2 consumption (Eldershaw et al., Biosciences reported a selective (.40-fold versus other 1994). Taken together, these observations imply that TRP channels) and moderately potent tetrahydroqui- TRPV1 agonism (and possibly antagonism) may in- noline amide TRPV3 antagonist (IC50 of 0.2–1 mM) fluence body temperature regulation via non-neuronal with in vivo efficacy (200 mg/kg i.p.) in decreasing TRPV1 receptors. Neuronal and non-neuronal TRPV1 thermal hyperalgesia in inflammatory or burn models may have distinct structure-activity relations. If this of pain (see Reilly and Kym, 2011). No effect was hypothesis holds true, such antagonists can be observed in the contralateral paw, suggesting that the synthesized that block TRPV1 in sensory neurons compound did not have a systemic effect on noxious (important for pain relief) but do not affect non- thermosensation. Hydra Biosciences disclosed a quina- neuronal TRPV1 involved in body temperature zolinone as a second TRPV3 chemotype with moderate regulation. potency (IC50 of 0.2–1 mM) and good selectivity against b. Vanilloid transient receptor potential 3. Although other thermoTRP channels (10-fold versus TRPA1 and TRPV3 is most abundantly expressed in the skin TRPM8; 20-fold versus TRPV1). This chemotype was keratinocytes, expression has been also reported in also efficacious in reducing thermal hyperalgesia in the mouse peripheral and central nervous systems (Smith carrageenan model of inflammatory pain. et al., 2002; Xu et al., 2002; Facer et al., 2007). Glenmark Pharmaceuticals has disclosed TRPV3 However, the functional significance of central TRPV3 antagonists with antinociceptive efficacy in preclinical receptors is largely unknown. In humans, no clear inflammatory and neuropathic pain models (see picture has yet emerged with regard to non-neuronal Khairatkar-Joshi et al., 2010; Preti et al., 2012). (keratinocyte) TRPV3 expression in painful condi- GRC15300 (structure not disclosed) was described as 45 2+ tions. For example, TRPV3 is increased in keratino- a potent (IC50 = 79.5 nM in 2-APB–induced Ca cytes of patients with mastalgia (painful breast uptake in hTRPV3/Chinese hamster ovary (CHO) cells, tissue) (Gopinath et al., 2005), but is decreased in and 137 nM in human keratinocytes) and selective the skin of patients with diabetic neuropathy (Facer (.100Â versus TRPV1, TRPV4, TRPA1, and TRPM8) et al., 2007). More consistent observations were re- TRPV3 antagonist (Khairatkar-Joshi et al., 2010). ported with regard to neuronal TRPV3 expression GRC15300 demonstrated a dose-dependent (ED50 = after nerve injury: TRPV3 is increased in small and 1.66 mg/kg) reversal of mechanical hyperalgesia in the 734 Nilius and Szallasi

CFA model of inflammatory pain with efficacy superior et al., 2011). Importantly, the development of cold to at 2 and 4 hours after oral administration. allodynia paralleled the increase in TRPM8 staining In the in the CCI model of neuropathic pain, GRC15300 after CCI (Rossi et al., 2012). decreased mechanical hyperalgesia with short-term and TRPM8 is a cold-sensitive nociceptor (McKemy et al., repeated dosing (ED50 = 8.25 mg/kg p.o. after 8 days of 2002). Indeed, conditional ablation of TRPM8 in adult dosing). Short-term efficacy was comparable to prega- mice renders the animals unable to distinguish warm balin in this model. No tolerance was observed with from cool or to avoid noxious cold (Knowlton et al., subchronic administration. GRC15300 also demon- 2013). It is noteworthy that cold allodynia is markedly strated antinociceptive efficacy in postoperative paw diminished in these animals. By contrast, both Pirt incision pain (ED50 = 0.92 mg/kg), as well as in the MIA- (Tang et al., 2013) and artemin (Lippoldt et al., 2013) induced OA (ED50 = 11.9 mg/kg) models of pain. The enhance TRPM8-dependent cold pain. Indeed, Pirt- safety profile of GRC15300 was described with no null mice exhibit decreased behavioral responses to significant effects at hERG (human ether-a-go-go-re- cold temperatures (Tang et al., 2013). [Somewhat lated gene) K+ channel current, no alteration in re- confusingly, Pirt also potentiates the heat-responsive sponse to noxious heat in naïve rat tail flick assays, and TRPV1 channel (Kim et al., 2008a).] Cooling (e.g., ice no locomotor deficits in the rotarod assay (up to 30 mg/ packs) is a time-honored analgesic approach to sooth kg). In 2010, Glenmark Pharmaceuticals announced a acute pain secondary to skin burn or muscle strains. partnership with Sanofi-Aventis to advance GRC15300/ Although cooling can clearly activate TRPM8, the SAR 292833 into clinical trials for neuropathic pain beneficial effects of ice packs on pain are probably not (www.glenmarkpharma.com/GLN_NWS/pdf/Sanofi- TRPM8 mediated. Menthol (Fig. 6), a naturally occur- Aventis&Glenmark_sign_license_agreement.pdf). One ring TRPM8 agonist isolated from peppermint leaves, year later, the phase I trial was successfully completed has been used for centuries in folk medicine as an (it was well tolerated with good pharmacokinetic analgesic and agent. Today, menthol profile) and the molecule was advanced into proof-of- (similar to capsaicin) is used in over-the-counter (OTC) concept studies. pain without a clinically proven benefit. Of c. Melastatin transient receptor potential 8. note, menthol is also broadly used in lozenges, nasal Although TRPM8 is a well established molecular pain sprays, inhalers, and cough syrups for the relief of nasal target (see McKemy, 2010), its medicinal chemistry is congestion associated with rhinitis and upper respira- rudimentary compared with TRPV1 or TRPA1 (see tory tract irritation, and it is added to certain cigarette DeFalco et al., 2011). It is often debated in the brands to reduce the respiratory irritation that the literature whether TRPM8 activation is proalgesic smoke causes (see Preti et al., 2012). Menthol has, and analgesic. In other words, should we focus our therefore, been claimed as an adjuvant agent in efforts on developing specific TRPM8 agonists or formulations addressing rhinitis and upper tract airway antagonists? In our opinion, this is not an either/or inflammation (Preti et al., 2012). However, there is good question. It is not unlikely that (as is the case for evidence that menthol has no measurable effect on TRPV1) both TRPM8 agonists and antagonists have nasal airflow (Lindemann et al., 2008). therapeutic potential. In animal experiments, the behavioral responses to TRPM8 is expressed in a subpopulation of primary topical menthol are complex and often conflicting. For afferent sensory neurons originating from both DRG example, a recent study found that low (0.01%–1%) and and trigeminal ganglia (Dhaka et al., 2008). Most high (10%–40%) concentrations of menthol had oppo- studies agree that TRPM8-positive neurons are dis- site effects on thermal preference in rats, with de- tinct from those expressing TRPV1 and/or TRPA1 (Fig. creased cold avoidance at high concentrations (Klein 17; Takashima et al., 2007). A number of studies have et al., 2010). Proudfoot et al. (2006) showed that investigated the expression of TRPM8 in experimental TRPM8 agonists, including menthol, relieved pain in models of chronic pain with conflicting findings. For certain neuropathic models; however, a second study example, TRPM8 levels (measured by in situ hybrid- was unable to replicate this finding (Caspani and ization) decreased in the injured L5 DRG but remained Heppenstall, 2009; Caspani et al., 2009). The interpre- unchanged in the neighboring (uninjured) L4 DRG in tation of these studies is further complicated by the the spinal nerve ligation model of neuropathic pain findings that menthol is not selective for TRPM8. In (Obata et al., 2006). By contrast, in the CCI model, fact, menthol was shown to inhibit voltage-gated Na+ TRPM8 RNA levels showed a delayed increase (reach- (Haeseler et al., 2002) and Ca2+ channels (Swandulla ing statistical significance only at 14 days after CCI) in et al., 1987), which could reduce nociceptive trans- affected DRG neurons (Frederick et al., 2007). In the mission. This may explain the observation that re- same model, immunostaining revealed a rapid (already sponses to menthol were markedly reduced, but not noticeable 4 days after surgery) increase in the per- abolished, in TRPM8-null mice (Colburn et al., 2007). centage of TRPM8-immunoreactive neurons compared To complicate the picture even further, cold hypersen- with the sham-operated group (Xing et al., 2007; Su sitivity to menthol was abolished in TRPA1-deficient TRP Channels as Drug Targets 735 mice but developed normally in TRPM8-null animals neurons, TRPA1 is expressed by TRPV1-positive, (Gentry et al., 2010). peptidergic, small diameter neurons in DRG (Figs. 17, The pharmacology and medicinal chemistry of TRPM8 20, and 21; Story et al., 2003; Kobayashi et al., 2005). modulators were recently reviewed in detail (DeFalco Ablation of TRPV1+ neurons by RTX in mice results in et al., 2011; Fernández-Pena and Viana, 2013). Here we an almost complete loss of sensitivity to allyl- highlight only a few interesting findings related to isothiocyanate, a TRPA1 agonist (Pecze et al., 2009). analgesic potential of these agents. In 2006, Bayer Health- These data support a high degree of colocalization care reported a series of 2-benzyloxy-benzoic acid amide between TRPV1 and TRPA1 in sensory neurons. In derivatives such as AMTB (Alonso-Alija et al., 2006) for the human DRG, TRPA1 is localized primarily to small and treatment of airway disorders (reviewed in Preti et al., medium DRG neurons (Fig. 17), although some expres- 2012). Relevant to our review, AMTB (structure shown in sion in large neurons was also reported (Anand et al., Fig. 11) was reported to attenuate the bladder nociceptive 2008). Neuropathic injury increases neuronal expres- reflex responses in the rat (Lashinger et al., 2008), po- sion of TRPA1 (Anand et al., 2008; Ji et al., 2008). tentially opening a new therapeutic window in the TRPA1 localizes to central terminals of primary afferent treatment of painful bladder syndrome. neurons in the dorsal spinal cord of rats and humans In 2007, Janssen Pharmaceuticals described a series (Anand et al., 2008; Kim et al., 2010), where it is of benzothiophene phosphonate TRPM8 antagonists. believed to play an important role in central sensitiza- In vitro, phosphonate 25 inhibited TRPM8 receptors in tion and the development of mechanical allodynia (Fig. various species (rats, dogs, and humans) with similar 20; see McGaraughty et al., 2010; Koivisto et al., 2014). IC50 values of 29, 53, and 54 nM, respectively Non-neuronal localization of TRPA1 mRNA and protein (Matthews et al., 2012). In vivo, this compound was also reported in human skin keratinocytes (Anand inhibited icilin-induced “wet-dog shakes” in rats with et al., 2008; Atoyan et al., 2009) and melanocytes an ED50 value of 24 mg/kg p.o., and also suppressed (Bellono et al., 2013). neuropathic pain (ED50 = 14.8 mg/kg) in the CCI TRPA1 as a target for analgesic drugs has been model. The substitution of the benzothiophene core extensively reviewed elsewhere (Garrison and Stucky, with a benzimidazole ring increased potency: a repre- 2011; Strassmaier and Bakthavatchalam, 2011; sentative compound in this series blocked canine Andrade et al., 2012; Bautista et al., 2013; Brederson TRPM8 with an IC50 value of 6 nM (Parks et al., et al., 2013; Radresa et al., 2013; Koivisto et al., 2014). 2011). Vinylcycloalkyl-substituted benzimidazole TRPM8 The potential role of TRPA1 in chemotherapy-induced antagonists were reported to suppress both icilin-induced neuropathic pain is described below. An emerging wet-dog shakes and CCI-induced neuropathic pain indication for TRPA1 blockade is pain secondary to behavior by approximately 80% at a dose of 10 mg/kg diabetic neuropathy. , a toxic offshoot of p.o. (Calvo et al., 2012). The selective TRPM8 blocker glycolysis, is held responsible by some for the de- PBMC [1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2- velopment of long-term diabetic complications, includ- aminoethyl)carbamate; structure shown in Fig. 11] ing diabetic neuropathy (Vander Jagt, 2008). Indeed, in inhibited cold hypersensitivity in the CCI model of mice, methylglyoxal activates Nav1.8 channels and chronic pain but, disappointingly, was ineffective against evokes pain that is absent in the Nav1.8-null Scn(2/2) chemotherapeutic agent (oxaliplatin)–induced neuro- animals; in patients, a plasma level of 600 nM pathic cold hypersensitivity (Knowlton et al., 2011). methylglyoxal is required for the development of pain Additional notable TRPM8 antagonists in the preclinical (Bierhaus et al., 2012). Methylglyoxal is a potent stage of drug development include JNJ41876666 [3-[7- activator of murine TRPA1 (Andersson et al., 2013). trifluoromethyl-5-(2-trifluoromethyl-phenyl)-1H-benzimi- Importantly, methylglyoxal also activates human dazol-2-yl]-1-oxa-2-aza-spiro[4.5]dec-2-eneHydrochloride] TRPA1 (Ohkawara et al., 2012) and this interaction and AMG9678 [(R)-1-(4-(trifluoromethyl)phenyl)-N-((S)- was implicated in the pathomechanism of metabolic 1,1,1-trifluoropropan-2-yl)-3,4-dihydroisoquinoline-2(1H)- neuropathies (Eberhardt et al., 2012). Another interest- carboxamide] (structures shown in Fig. 11). ing aspect of the TRPA1-diabetes link is the ability of Similar to TRPV1, TRPM8 is involved in thermoregu- the antidiabetic drug glibenclamide to activate TRPA1 lation. Menthol increases body temperature, whereas on sensory neurons (Babes et al., 2013). Glibenclamide pharmacological TRPM8 blockade decreases body tem- is known to cause abdominal pain as a side effect in perature (Knowlton et al., 2011; Gavva et al., 2012). The some patients, and it was suggested that its direct effect hypothermic response is, however, mild (,1°C) and dis- on TRPA1 is responsible. appears upon repeated dosing; therefore, it may not pose TRPA1 antagonists ameliorate mechanical hyper- a problem for developing TRPM8 antagonists as thera- algesia and (even more important) prevent cutaneous peutics (unlike the febrile response to TRPV1 antago- nerve fiber loss in diabetic rodents (Wei et al., 2009b, nists) (Gavva et al., 2012). 2010, 2011a, 2013; Koivisto et al., 2012). , d. Ankyrin transient receptor potential 1. TRPA1 is a common cause of chemotherapy-induced peripheral expressed both in neuronal and non-neuronal tissues. In neuropathy (CIPN), worsens cold hyperalgesia in 736 Nilius and Szallasi diabetic rats via mitochondrial ROS generation and in dose-escalating studies in healthy volunteers (phase Ia). resultant TRPA1 activation (Barrière et al., 2012). Reportedly, the compound was well tolerated and Hydra/ Parenthetically, a similar mechanism (i.e., sensory Cubist now plan to test the compounds in patients with nerve terminal mitochondrial dysfunction causing acute, postsurgical pain (www.painresearchforum. TRPA1 activation) was recently linked to asthma org/news/22419-filling-pain-drug-pipeline). Of note, in re- (Nesuashvili et al., 2013). cent animal studies, TRPV1, but not TRPA1, blockade Another promising indication for pharmacological inhibited cutaneous incision–mediated hypersensitivity TRPA1 blockade is OA pain. The selective TRPA1 (Barabas and Stucky, 2013). The second TRPA1 antag- antagonist A-967079 [(1E,3E)-1-(4-fluorophenyl)-2- onist to enter clinical trials was GRC15736 by Glenmark methyl-1-pentene-3-one oxime; see Fig. 10 for struc- Pharmaceuticals. In phase I studies, the compound was ture] blocks OA pain in rats with an ED50 of 23.2 mg/kg well tolerated at plasma levels comparable to those that p.o. without altering physiologic noxious cold sensation were analgesic in the preclinical models (www.rttnews. (Chen et al., 2011). com/1819388/glenmark-s-novel-molecule-grc-17536- Several companies (e.g., Hydra Biosciences, Abbott, completes-phase-i-clinical-trials-in-europe). Glenmark Glenmark Pharmaceuticals, Janssen Pharmaceuticals, Pharmaceuticals plans to initiate phase II proof-of- and Merck) have reported TRPA1-selective antagonists concept studies with this compound for pain and (representative structures for HC-030031, CHEM58611528, respiratory indications. AP18, A-967079, and JNJ41477670 are shown in Fig. An intriguing alternative approach to silence 10). The medicinal chemistry of TRPA1 antagonists was TRPA1-expressing neurons is by a combination of reviewed elsewhere (Rech et al., 2010; Strassmeier and cinnamaldehyde and QX-314. Cinnamaldehyde (a Bakthavatchalam, 2011). In brief, the Hydra Bioscien- TRPA1 agonist) activates TRPA1 and renders it perme- ces antagonists contain a xanthine alkaloid core that is able to QX-314 (Lennertz et al., 2012). This approach is also present in caffeine. TRPA1 potency was enhanced very similar to (although probably less effective than) through modifications of the purine core and manipu- that previously described to silence TRPV1+ afferents by lation of the aryl group from phenyl to thiazole. Further a combination of capsaicin and QX-314 (Binshtok et al., modification of a second aryl functionality attached to 2007; Ries et al., 2009) since TRPA1 is less permeable to aliphatic pyrrolidine capping group led to exceptionally QX-314 than TRPV1 (Nakagawa and Hiura, 2013). potent TRPA1 antagonists such as HC-03001 (struc- Of note, TRPA1 was suggested to be a target for the ture shown in Fig. 10; reviewed in Strassmeier and analgesic action of acetaminophen (Andersson et al., Bakthavatchalam, 2011; De Petrocellis and Schiano 2011) and (Wang et al., 2013c), two time- Moriello, 2013). honored NSAIDs. Glenmark Pharmaceuticals also described TRPA1 5. The Contribution of Transient Receptor Potentials antagonists that were based on the modifications of to Chemotherapy-Induced Neuropathic Pain. CIPN the xanthine alkaloid core. Chemists at Glenmark represents a major dose-limiting side effect for many Pharmaceuticalsmodifiedthisringbyreplacingthe commonly used antineoplastic agents, both platinum- five-membered imidazole fragment of the purine core based (e.g., cisplatin and oxiplatin) and other (e.g., with different heterocycles, including isothiazole, pyr- vincristine, paclitaxel and thalidomide) agents. CIPN role, thiophene, pyridine, and imidazole (reviewed in can be especially severe in patients with coexisting Preti et al., 2012). Abbott reported TRPA1 antagonists independent risk factors for neuropathic pain such as based on an oxime lead series as exemplified by diabetes and/or alcohol abuse (see Nassini et al., 2013). A-967079. In vitro, A-967079 is more potent for human Cisplatin combination chemotherapy is a cornerstone TRPA1 (IC50 = 67 nM) than rat TRPA1 (IC50 = 290 nM) of the treatment of many cancers. Cisplatin is known to and demonstrates good oral bioavailability (Chen et al., cause a chronic peripheral sensory neuropathy that is 2011). In preclinical studies, A-967079 inhibited spon- manifested as neuropathic pain and sensory ataxia and taneous and mechanically evoked firing of spinal wide may develop off-therapy with a delayed onset, even dynamic range and nociceptive-specific neurons in OA several months after the completion of treatment (Park rats (McGaraughty et al., 2010). Janssen Pharmaceut- et al., 2008). This form of cisplatin-induced neuropathy icals described two lead compound series: 1) highly is often irreversible, is progressive, and shows signs of potent biaryl pyrimidines that contain a pyrrolidine spinal demyelinating disease. The initial response rate carboxamide side chain, and 2) compounds based on to cisplatin is high, but many patients will eventually the thioxodihydroindenopyrimidinone core (see relapse with cisplatin-resistant cancer. The third- Strassmeier and Bakthavatchalam, 2011). Finally, Merck generation platinum drug oxaliplatin is typically ad- described TRPA1 antagonists based on a hydroxy- ministered in a combination known as FOLFOX (with substituted aminodecalin core. fluorouracil and leucovorin) for the treatment of ad- To date, two TRPA1 antagonists have been advanced vanced colorectal cancer. Oxaliplatin has less ototoxic- into clinical trials. Cubist Pharmaceuticals and Hydra ity and nephrotoxicity than cisplatin but it can cause Biosciences were jointly testing CB-625 in the Netherlands an acute painful neuropathy soon after administration TRP Channels as Drug Targets 737

(see Hoff et al., 2012), leading to paresthesias (which in mitochondrial toxicity is Ca2+ overload. Indeed, Ca2 usually start periorally and then spread to the +-chelating agents were shown to reverse paclitaxel- extremities) and severe cold hypersensitivity. Indeed, evoked pain (Siau and Bennett, 2006). Of note, mito- exposure to cold can trigger an acute, transient chondrial dysfunction has been linked to neuropathic syndrome characterized by cramps, paresthesia, and pain (Botez and Herrmann, 2010; Reichling and Levine, dysesthesia in patients taking oxaliplatin, presumably 2011; Vincent et al., 2011) and migraine (Stuart and by activating TRPA1 (Zhao et al., 2012a). Furthermore, Griffiths, 2012), pointing to TRP channels as down- oxaliplatin may cause chronic peripheral neuropathy stream targets for the oxidative stress. resembling that linked to cisplatin (see Nassini et al., Finally, significant changes in the expression of 2013). various genes, including those controlling mitochon- Paclitaxel (originally derived from the bark of the drial dysfunction due to vincristine- and bortezomib- Pacific yew, Taxus brevofolia) is a mitotic inhibitor that associated peripheral neuropathy, have been demon- is also effective against cisplatin-resistant tumors. strated in humans (Broyl et al., 2010). Given their role Paclitaxel acts by promoting the assembly of abnormal in both generating neuropathic pain and regulating bundles of microtubules in the mitotic machinery. mitochondrial calcium homeostasis, TRP channels are Microtubules are, however, also important for the attractive targets to explore in CIPN (see Nassini development and maintenance of neurons. In the rat, et al., 2013). paclitaxel was shown to accumulate both in Schwann Patients on platinum-based chemotherapy regimens cells and DRG neurons, causing the formation of often complain about heat hypersensitivity (Carozzi unusual microtubule aggregates with resultant de- et al., 2010). There is a growing body of evidence that myelination and loss of axoplasmic transport. These this adverse effect is predominantly mediated by findings suggest that paclitaxel causes a combination TRPV1. In the mouse, cisplatin was reported to of sensory axonopathy and ganglioneuropathy with an upregulate TRPV1 mRNA both in vivo and in cultured end result of severe sensory peripheral neuropathy DRG neurons (Ta et al., 2010). In cisplatin-treated (Dougherty et al., 2004). Signs and symptoms of pe- mice, the increase in TRPV1 mRNA was associated ripheral neuropathy may begin as early as 24 hours with enhanced nociceptor responsiveness, and these after administration of a single, high dose of paclitaxel. animals (similar to the patients) developed thermal, Affected patients commonly complain of numbness, but not mechanical, hyperalgesia. Likewise, enhanced tingling, and burning pain, which typically occurs in capsaicin responses were seen in rats treated with a “glove and stocking” distribution (Dougherty et al., oxaliplatin (Anand et al., 2010). 2004). Sensory symptoms usually start symmetrically Patients with CIPN also exhibit mechanical allody- in the feet but can also appear simultaneously in both nia. This has been attributed to TRPV4 activation, hands and feet. Although mild cases have been mostly based on the observation that TRPV4(2/2) reported to resolve after discontinuation of therapy, mice exhibit reduced mechanical hyperalgesia com- the sensory abnormalities can persist in patients who pared with wild-type animals in response to treatment develop severe neuropathy. Despite attempts to min- with paclitaxel (Alessandri-Haber et al., 2004) and imize paclitaxel-induced neuropathy by changes in vincristine (Alessandri-Haber et al., 2008). This is in dosing and formulation, this adverse effect remains a keeping with the reports that in various models of major barrier to achieving the desired clinical response painful peripheral neuropathy, including those that in many patients. develop in diabetic animals, the mechanical hyper- Although antineoplastic agents linked to CIPN vary algesia is markedly reduced by intrathecal adminis- in both their chemical structure and mechanism of tration of oligodeoxynucleotides that are antisense to action, they seem to share a common target, mitochon- TRPV4 (Alessandri-Haber et al., 2008). Paclitaxel and dria (Jaggi and Singh, 2013). For example, paclitaxel- vincristine do not activate TRPV4 directly, nor do they induced painful peripheral neuropathy is associated enhance TRPV4 mRNA levels (Alessandri-Haber et al., with the presence of swollen and vacuolated axonal 2004). So how do these agents sensitize TRPV4? Accord- mitochondria (Flatters et al., 2006). Likewise, bortezo- ing to a recent theory, a unique signaling cascade is mib was shown to cause intracytoplasmatic vacuolization activated by integrins during CIPN that, in turn, leads to in DRG satellite cells, probably due to mitochondrial and membrane insertion and/or activation of the TRPV4 endoplasmic reticulum enlargement (Cavaletti et al., channel in sensory neurons via Src tyrosine kinase (Odell 2007). The molecular targets that mediate paclitaxel- et al., 2005). An alternative model focuses on PAR-2 (Chen and bortezomib-induced mitochondrial toxicity are, how- et al., 2011). This model holds that paclitaxel releases ever, different. Whereas paclitaxel appears to gate the mast cell tryptase and thereby activates PAR-2 in DRG mitochondrial permeability transition pore (Flatters neurons. There is good evidence that PAR-2 activation et al., 2006), bortezomib activates mitochondrial-based mayindirectlysensitize(viaPKA,PKC«,andPLC) apoptotic pathways, including activation of caspases a number of TRP channels, including TRPV1, TRPA1, (Cavaletti et al., 2007; Broyl et al., 2010). A major player and TRPV4, thereby contributing to the molecular 738 Nilius and Szallasi pathomechanism of mechanical allodynia and thermal mRNA correlated to the development of cold hyper- hyperalgesia. The extrapolation of these observations sensitivity. These findings were interpreted to imply to humans is, however, hindered by the apparent lack that oxaliplatin-induced cold hypersensitivity is, at of painful phenotype in patients carrying gain-of- least in part, mediated by TRPM8. If so, TRPM8 function TRPV4 mutations. activators such as menthol and icilin should exacerbate In summary, the participation of TRPV4 in the CIPN symptoms. Indeed, wet-dog shake and jumping development and maintenance of mechanical allodynia behaviors elicited by icilin were enhanced in mice is controversial. Furthermore, neither TRPV4 nor treated with oxaliplatin. Icilin is, however, not selec- TRPV1 can account for the cold hyperalgesia that tive for TRPM8. Icilin also activates TRPA1 and this patients with CIPN often display (see Nassini et al., response seems to be potentiated by oxaliplatin (Anand 2013). et al., 2010). Furthermore, topical menthol application Geppetti and co-workers recently explored the role of was reported to show a paradoxical analgesic effect in TRPA1 in rodent CIPN models (Nassini et al., 2011; CIPN induced by bortezomib (Colvin et al., 2008). In Materazzi et al., 2012; Trevisan et al., 2013). They addition, menthol was able to significantly reverse showed that TRPA1 entirely mediates both the CIPN induced by carboplatin, and its prolonged mechanical and cold hypersensitivity that develops in application during chemotherapy appeared to prevent mice and rats in response to oxaliplatin and cisplatin the worsening of CIPN. Clearly, more basic and clinical administration. Moreover, in paclitaxel-treated ani- investigation is needed to clarify the role of TRPM8 in mals, the TRPV4-resistant component of the mechan- CIPN. ical hyperalgesia was also mediated by TRPA1 6. Transient Receptor Potential Channels in (Nassini et al., 2011). Oxaliplatin and paclitaxel do Migraine. not directly gate TRPA1 because they do not cause any a. The role of vanilloid transient receptor potential 1 Ca2+ response in mouse or rat DRG neurons. However, in migraine and cluster headache. Migraine is a com- in CHO cells transfected with the mouse TRPA1 mon, debilitating episodic disorder characterized by channel, oxaliplatin evokes intracellular Ca2+ mobili- unilateral throbbing headache, phonophobia, photo- zation in a glutathione-sensitive manner that is absent phobia, and nausea, which is sometimes preceded by in untransfected CHO cells. To explain these seemingly premonitory symptoms (so-called “aura”) (reviewed in contradictory results, the authors have hypothesized Nassini et al., 2010a; Benemei et al., 2013). For unclear that the Ca2+ response to oxaliplatin challenge re- reasons (that may include the hormonal milieu), quires two conditions: 1) the presence of TRPA1, and 2) migraine is more common in women (18%) than in cells that generate sufficient levels of oxidative stress men (6%). The initiation of migraine attacks has been in response to oxaliplatin to activate TRPA1 (see attributed to a number of trigger mechanisms (re- Nassini et al., 2013). It is possible that DRG neurons viewed in Kelman, 2007), including both environmen- do not produce oxidative stress products in amounts tal (e.g., air pollution, odors, alcohol consumption, as sufficient to activate TRPA1, whereas CHO cells well as changes in temperature or weather) and possess the metabolic and enzymatic repertoire to physiologic factors (e.g., hormonal milieu or stress). produce high enough ROS levels (Nassini et al., 2011). Migraine headache is believed (at least in part) to Cells that are in the proximity of TRPA1-expressing reflect the activation of the trigeminovascular system, nerve terminals may release oxidative stress by- resulting in neurogenic inflammation of the meninges products generated by paclitaxel and thus activate and the release of CGRP, a potent vasodilator TRPA1. In support of this hypothesis, paclitaxel was (Geppetti et al., 2005; Raddant and Russo, 2011; reported to worsen cold hyperalgesia in diabetic rats Messlinger et al., 2012). This concept has gained compared with normoglycemic animals and this effect strong experimental support by the efficacy of CGRP was prevented both by the ROS scavenger N-acetyl- antagonists in clinical trials (Olesen and Ashina, 2011; cysteine and the selective TRPA1 antagonist, HC- Salvatore and Kane, 2011). TRP channels are promis- 030031 (Nassini et al., 2011). Paclitaxel treatment was ing targets in migraine (see Nassini et al., 2010a, 2013; associated with an accumulation of atypical mitochon- Dux et al., 2012; Oxford and Hurley, 2013), both in dria and an increase in mitochondrial ROS production. preventing attacks (e.g., TRPA1 is a well established Interestingly, platinum-based drugs also increase target for air pollutants and odors) and in relieving TRPA1 expression in DRG neurons. In rats, the novel headache (TRPV1 is thought to play a central role in TRPA1 antagonist ADM-09 ameliorates oxaliplatin- neurogenic inflammation including CGRP release). induced neuropathic pain (Nativi et al., 2013). Retrograde labeling studies demonstrated that a dis- In sensory neurons, TRPM8 receptor expression is tinct subset (approximately 25%) of the trigeminal increased after nerve injury (Frederick et al., 2007). A ganglion cells projecting to the dura express TRPV1, similar increase in TRPM8 mRNA was noted in DRG and 80% of these neurons are positive for CGRP neurons of mice treated with oxaliplatin (Gauchan (Shimizu et al., 2007; Chatchaisak et al., 2013). This et al., 2009). Importantly, this increase in TRPM8 high level of colocalization is consistent with the TRP Channels as Drug Targets 739 concept that TRPV1 activation in the dura stimulates stimuli; these responses were reversed by the TRPV1 CGRP release and subsequent vasodilatation (Dux antagonist SB705498 (Lambert et al., 2009). Finally, in et al., 2003). In addition to its presence in presynaptic humans, desensitization to intranasal civamide appli- terminals of trigeminal afferents, TRPV1 has also been cation was reported to ameliorate cluster headache detected, albeit at a much lower abundance, in (Saper et al., 2002), a pain condition that shares some arteriolar endothelium and smooth muscle (Kark pathophysiological characteristics of migraine, includ- et al., 2008; Cavanaugh et al., 2011; Czikora et al., ing involvement of the trigeminovascular system. 2012). Indeed, TRPV1 expressed in cochlear arterioles Combined, these findings imply an important role for has been proposed to mediate the inner ear disturban- TRPV1 in the initiation and propagation of migraine, ces in migraine (Vass et al., 2004) as well as in the development and maintenance of It is noteworthy that migraine is comorbid with hyperalgesia and allodynia that often accompany a number of CNS disease states such as epilepsy, migraine attacks. It was speculated that TRPV1 is an which implies that central sites might also be involved important mediator of CGRP release, responsible for in the triggering of migraine attacks. In a murine vasodilatation and plasma extravasation in the me- model of temporal lobe epilepsy, increased TRPV1 ninges (see Szallasi et al., 2006). In turn, mediators expression was noted in the dentate gyrus compared generated during neurogenic inflammation sensitize with control brain (Bhaskaran and Smith, 2010). This trigeminal nociceptors presumably by potentiating is in keeping with the report of increased TRPV1 TRPV1 channel function via phosphorylation and expression in the cortex and hippocampus of patients increased membrane trafficking (Meents et al., 2010). with mesial temporal lobe epilepsy (Sun et al., 2013). A In support of this theory, NGF (a component of the similarly increased TRPV1 expression was described in inflammatory soup and a proven TRPV1 modulator) cortical lesions removed from tuberous sclerosis was found to be elevated in the cerebral spinal fluid patients to ameliorate epileptic seizures (Shu et al., (Sarchielli et al., 2007), plasma and saliva (Jang et al., 2013). Taken together, these findings imply that 2011) of migraineurs. However, the TRPV1 antagonist TRPV1 may represent a novel antiepileptogenic target, A-993610 [(S)-1-(3-chloropyridin-2-yl)-3-methyl-N-(4- also relevant for preventing migraine attacks. How- (trifluoromethyl)phenyl)-1,2,3,6-tetrahydropyridine-4- ever, conflicting reports have made it difficult, if not carboxamide] failed to prevent cortical spreading impossible, to assign any pathophysiologic role for depression in a rat model of migraine, arguing against TRPV1 in the CNS. Recent experimental data a therapeutic potential for TRPV1 antagonism in the (obtained by TRPV1 reporter mice, a sensitive genetic management of acute migraine (Summ et al., 2011). approach for lineage tracing) have even questioned the Of note, a clinical trial with the TRPV1 antagonist very existence of brain TRPV1, except for a few small SB705498 in patients with migraine was completed regions in the CNS, most notably the hypothalamus but the outcome has not yet been made public. (Cavanaugh et al., 2011). Other studies reported Chronic migraine (defined as 15 or more headache a dramatic (almost complete) loss of TRPV1 in the days a month with headache episodes lasting 4 hours rat brain by postnatal day 26 (Köles et al., 2013). These or longer for at least 3 consecutive months) is a de- discrepant reports must be reconciled before brain bilitating condition that affects approximately 3% of TRPV1 can be validated as a potential target to patients with migraine with. As of today, it is unclear prevent migraine attacks. why migraine becomes chronic but functional magnetic In preclinical studies, a large body of evidence resonance imaging studies suggest a role for the implicates TRPV1 in migraine pain (reviewed in dysfunction of the descending antinociceptive systems. Szallasi et al., 2006; Goadsby, 2007; Meents et al., It was speculated that TRPV1 activation in the 2010). In the mouse, TRPV1 is highly expressed in trigeminal nucleus caudalis might contribute to the trigeminal dural afferents, where it mediates neuro- chronification of migraine by altering microglia (Shi- transmitter release (Huang et al., 2012). This provides bata, 2012). If this hypothesis holds true, TRPV1 the anatomic basis for understanding the functional antagonists may prove useful in preventing the de- significance of TRPV1 activation in headache patho- velopment of chronic migraine in at-risk patients even physiology. Capsaicin evokes CGRP release from dural if these antagonists provide no benefit during the acute tissue via TRPV1 activation, which in turn leads to attacks. CGRP-dependent vasodilatation (Eltorp et al., 2000). Finally, capsaicin-sensitive trigeminal afferents in- Furthermore, in trigeminal nucleus caudalis slices, nervating the nasal mucosa were implicated in the capsaicin produces repetitive forma- pathomechanism of sinus headache and hemicrania. tion and subsequent CGRP release that is inhibited by There is anecdotal evidence that capsaicin-containing the antimigraine drug (Evans et al., nasal sprays may relieve sinus headache (indeed, 2012). Importantly, in the cat, application of the a number of OTC capsaicin preparations are commer- inflammatory soup to the dura increased blood cially available for this indication). Clinical trials with flow and led to neuronal sensitization to mechanical intranasal civamide (a synthetic capsaicin congener) 740 Nilius and Szallasi are now complete, but the results have not yet been ganglion than in DRG neurons (Kobayashi et al., 2005). disclosed (ClinicalTrials.gov identifiers NCT00069082 TRPM8 knockout mice exhibit significant deficiencies in and NCT00033839). Preliminary studies suggest a link behavioral responses to a range of cold temperatures, but between migraine and TRPV1 variants (Carreño et al., the importance of TRPM8 in the development of cold 2012; Rainero et al., 2013). hyperalgesia and allodynia is still hotly debated. Al- b. Ankyrin transient receptor potential 1 as a candi- though OTC menthol patches are available for migraine, date sensor for environmental irritants that provoke the evidence for their clinical efficacy is at best anecdotal. migraine attacks. TRPA1 is highly coexpressed with d. Migraine as a transient receptor potential TRPV1 in a subpopulation of dural afferents. TRPA1 channelopathy? The observation that more than half detects environmental irritants such as acrolein (found of patients have at least one first-degree relative with in cigarette smoke, a known inducer of headache) migraine implies a strong genetic predisposition (Shyti (reviewed in Bessac and Jordt, 2008; Facchinetti and et al., 2011). Indeed, some rare familial forms of Patacchini, 2010; Nilius et al., 2011, 2012) and migraine are due to SNPs in genes encoding ion umbellulone (structure shown in Fig. 7), the active channels, including the Cav2.1 voltage-gated calcium agent in the Californian “headache tree” Umbellularia channel, the Nav1.1 voltage-gated sodium channel, and californica (Zhong et al., 2011; Edelmayer et al., 2012; the TWIK-related spinal cord K+ channel (TRESK) Nassini et al., 2012a). Moreover, TRPA1 is activated by (reviewed in Van den Maagdenberg et al., 2010; endogenous products of inflammation and oxidative Silberstein and Dodick, 2013). These mutations are stress (see Bautista et al., 2013). Importantly, these all predicted to be gain of function, leading to an TRPA1 agonists induce CGRP release from dural increase in neuronal excitability. For example, mice tissue and stimulate meningeal vasodilatation (Kun- bearing familial hemiplegic migraine mutations in the kler et al., 2011). There is good evidence that activation Cav2.1 protein exhibit enhanced susceptibility to corti- by irritants of TRPA1 receptors at trigeminal nerve cal spreading depression, presumably due to increased terminals in the nasal mucosa and subsequent activa- release of (Van den Maagdenberg tion of the trigeminovascular system play an important et al., 2010). On the basis of these observations, it is role in air pollution–induced headache (see Nassini a reasonable assumption that gain-of-function TRP et al., 2013; Oxford and Hurley, 2013). Studies on the channel gene mutations may also contribute to potential role of TRPA1 in migraine are, however, still migraine. in their infancy (reviewed in Benemei et al., 2013). Thus far, a genetic polymorphism in two TRP genes, c. Possible Involvement of other transient receptor TRPV1 (Carreño et al., 2012) and TRPM8 (Chasman potential channels in migraine. In addition to TRPV1, et al., 2011), has been implicated in the pathomechan- three other members of the vanilloid subfamily of TRP ism of migraine, although the available evidence is channels (TRPV2, TRPV3, and TRPV4) are expressed rather weak and indirect. Cortical hyperexcitability is on trigeminal neurons innervating the dura, but no associated with both migraine and epilepsy. Indeed, specific evidence has yet appeared that either TRPV2 antiepilepsy drugs that were useful in migraine pro- or TRPV3 is important in migraine (Rainero et al., phylaxis reduced susceptibility to cortical spreading 2013). By contrast, TRPV4 is strongly implicated in depression in animal studies. A recent study describes some migraine symptoms. TRPV4 has been identified a SNP allele in the TRPV1 channel that is associated as a probable mechanosensor and osmosensor on dural with enhanced synaptic transmission in the cerebral afferents (Wei et al., 2011b), and it was hypothesized cortex and produces larger currents in heterologous that activation of TRPV4 may be the source of the expression systems (Mori et al., 2012). When subjected throbbing head pain seen in migraine after movement to transcranial magnetic stimulation using a paired or coughing. Indeed, activation of TRPV4 on dural pulse stimulus in the motor cortex, individuals homo- afferents was shown to produce headache-related zygous for this allele exhibited larger short-interval behavior in a rat model of migraine (Wei et al., intracortical facilitation. Again, this report is difficult 2011b). In this study, approximately 50% of dural to reconcile with the apparent absence of TRPV1 in the afferents identified by retrograde labeling showed mouse brain cortex (Cavanaugh et al., 2011). sensitivity to hypotonic solutions and/or 4a-PDD (Fig. 7. Transient Receptor Potential Channels and 8), both known chemical activators of TRPV4. Topical Pruritus. Itch (pruritus) is an unpleasant sensation application of these substances to the dura produced that elicits the desire (or reflex) to scratch. Itch has allodynia that could be blocked by a TRPV4 antagonist many similarities to pain; indeed, it was referred to as in vivo. Unfortunately, adverse effects will probably the “skin equivalent of chronic pain” (Kini et al., 2011). preclude systemic TRPV4 antagonist therapy, but Chronic itch is a large, unmet medical need. According topical administration (e.g., nasal spray) may be of to a questionnaire-based German study, the lifetime therapeutic value in patients with migraine. prevalence of chronic pruritus exceeds 20% (Matterne TRPM8 might be interesting with regard to migraine in et al., 2009), although the real prevalence is unknown that it appears to be more highly expressed in trigeminal because many patients never seek medical attention. TRP Channels as Drug Targets 741

The similarities and differences between itch and C-afferent neurons that respond to both chemical and pain sensation (Fig. 28) are only beginning to be thermal, but not mechanical, stimuli and exhibit understood (reviewed in Goutos, 2013). We scratch a combination of low conduction velocity and high ourselves until it hurts to relieve itch, suggesting transcutaneous electrical threshold (Schmelz et al., a common neuronal pathway for pain and itch 2003; Ständer et al., 2003, 2011). These neurons express (Akiyama et al., 2012). Indeed, scratching blocks TRPV1 (Fig. 29). Indeed, in the itch pathway, the H1 responses of spinal second-order neurons to pruritic, histamine receptor is indirectly coupled downstream to but not algesic, stimuli (Nishida et al., 2013). The TRPV1 via PLA2 (Kim et al., 2004; Shim and Oh, 2008; intensity theory states that the very same primary Imamachi et al., 2009). Histamine plays a pivotal role in afferents are capable of triggering of both pruritus and pruritic, urticarial skin diseases in which antihist- pain (reviewed in Ross, 2011; Tóth and Bíró, 2013; Tóth amines provide almost instantaneous symptomatic et al., 2014). This model is supported by the observa- relief (Kiss and Keserü, 2012). However, antihistamines tions that 1) intradermal capsaicin injection can cause are without any clinical benefit in many disorders both itch and pain (Shimada and LaMotte, 2008; Klein characterized by chronic pruritus (including atopic et al., 2011; Sikand et al., 2011) in a dose-dependent dermatitis/eczema, and allergic as well as dry skin manner, and 2) desensitization to topical capsaicin itch), implying the existence of a second, histamine- creams alleviates both pain and pruritus (see Szallasi insensitive pruriceptive pathway (Handwerker, 2010). and Blumberg, 1999). However, modern research Intradermal insertion of spicules from the pods of the points to the existence of an autonomous pruriceptive cowhage plant Mucuna pruriens (broadly used in system (Fig. 28), organized more or less independently Ayurvedic medicine) was shown to activate mechanosen- from nociception (labeled-line theory) (reviewed in Ma, sitive, but not mechanoinsensitive, C fibers, as well as 2012; Tóth et al., 2014). This pruriceptive system is a subset of nociceptive, myelinated A fibers (Ringkamp thought to have two functionally and anatomically et al., 2011). Although both evoke a similar itchy distinct subdivisions, distinguished by a characteristic sensation, the skin reaction to histamine and cowhage response (or the lack of it) to histamine (see Tóth and is different in that histamine produces a wheal reaction, Bíró, 2013). whereas cowhage does not (Johanek et al., 2007). The Histamine released from activated mast cells and active ingredient in cowhage was identified as mucunain basophils plays a central role in allergic responses. (Shelley and Arthur, 1955), a novel cysteine protease that Histamine was shown to directly activate sensory activates PAR-2 and PAR-4 (Reddy et al., 2008). neurons by interacting at H1 and H4 receptors to PAR-2 is a major itch mediator: PAR-2 agonists evoke itch in humans (see Shim and Oh, 2008). enhance, whereas an anti-PAR-2 antibody attenuates, Histamine-sensitive neurons are unmyelinated sensory scratching behavior (Akiyama et al., 2010). When

Fig. 28. Primary afferents may selectively convey pain and itch, even if the noxious chemical lacks specificity. (A) Note that almost all noxious chemicals can (depending on how they were applied) elicit either itch or pain. (B) Discrimination between itch and pain is achieved by stimulating distinct subsets of sensory afferents. Reprinted with permission from Ross (2011). 742 Nilius and Szallasi

Fig. 29. The involvement of TRPV1 and TRPA1 in pain and itch. See text for more details. VSMC, vascular smooth muscle cell. Reprinted with permission from Ross (2011). applied to the human skin, mucunain clearly evokes intact (Han et al., 2013). Furthermore, only a minority a histamine-independent itch reaction. However, the (43%) of chloroquine-sensitive DRG neurons was found possibility could not be excluded that both mucunain to express TRPA1; the majority (57%) seemed to and histamine target TRPV1 via different second respond to chloroquine in a TRPC3-mediated fashion messengers, PAR-2 and PLA2, respectively. Indeed, it (Than et al., 2013). If confirmed, this observation will was postulated that endogenous pruritogenic substan- furnish TRPC3 with a previously unsuspected function ces act on TRPV1 indirectly via PAR-2 activation, in itch pathogenesis. based a rat model of hepatogenic pruritus induced by There is increasing experimental support for TRPA1 bile duct ligation (Belghiti et al., 2013). as a novel target for antipruritic drugs. Urushiol, the A recent breakthrough in pruritus research was the contact allergen in poison ivy, evokes pruritogenic observation that the antimalarial drug chloroquine responses in wild-type, but not in TRPA1-null, mice evokes scratching behavior in wild-type, but not (Liu et al., 2013a). Chronic itch evoked by impaired TRPA1-null, mice via the Mas-related G protein– skin barrier is also attenuated in the TRPA1-deficient coupled receptor A3 (MrgprA3) (Fig. 29; Wilson et al., animals (Wilson et al., 2013a). TRPV1 as an itch target 2011). Similarly, SLIGRL (an itch-producing peptide is more problematic. Topical capsaicin relieves pruri- that, such as cowhage, activates PAR-2) causes itch by tus (see Papoiu and Yosipovitch, 2010); however, activating TRPA1 receptors via MrgprC11 (Fig. 29; somewhat paradoxically, chemical ablation of sensory Wilson et al., 2011; Liu et al., 2013a). Thus, in a much afferents by systemic capsaicin administration results simplified manner, it can be postulated that the in chronic pruritic dermatitis in mice with skin pruriceptive system has two major subdivisions: ulceration due to extensive scratching behavior (www. a histamine-sensitive/TRPV1-positive subdivision, in abstractstosubmit.com/IBRO2011/abstracts/main.php?do). which the histamine receptor H1 is coupled to TRPV1 The TRPV1 antagonist PAC-14028 (Fig. 9) reduces, via PLA2; and a histamine-insensitive/TRPA1- but does not eliminate, scratching behavior in a murine expressing subdivision, in which TRPA1 is the down- model of atopic dermatitis (Yun et al., 2011a,b). By stream target of MrgrpA3 and MrgrpC11 (see Fig. 29). contrast, SB705498 (compare structures in Fig. 9) did In support of this model, activity-dependent silencing not provide any symptomatic relief in patients with of TRPV1- and TRPA1-positive afferents selectively seasonal allergic rhinitis (Bareille et al., 2013). Finally, blocks histamine- and chloroquine-induced scratching, comparable attenuation of the intradermal LTB4- respectively (Roberson et al., 2013). The real situation induced scratching behavior by TRPV1 [SB366791, is, however, probably more complex because genetic 97% inhibition] and TRPA1 [TCS-5861528, 2-(1,3- inactivation of Pirt abolishes both histaminergic and dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N- nonhistaminergic itch (Patel et al., 2011). Indeed, [4-(1-methylpropyl)phenyl]acetamide; 82% inhibition] TRPV1 appears to be coexpressed with TRPA1 in both antagonism in mice was recently reported (Fernandes nociceptive and pruriceptive afferents, both of which et al., 2013). are positive for CGRPa (McCoy et al., 2013b). Genetic IL-31 produced by TH2 cells evokes itch when ablation of these CGRPa-positive afferents reduce injected into the skin of mice (Dillon et al., 2004) or sensitivity to thermal and chemical (capsaicin) pain, dogs (Gonzales et al., 2013). Several lines of experi- as well as itch induced by histamine and chloroquine. mental evidence point to IL-31 as a critical neuro- By contrast, ablation of MgrpA3-positive neurons by immune link between pathogenic T cells and pruriceptive genetic manipulation leads to a substantial decrease in afferents. When overexpressed in transgenic mice, scratching behavior, whereas pain sensitivity remains IL-31 induces a pruritic skin condition that resembles TRP Channels as Drug Targets 743 human atopic dermatitis (Dillon et al., 2004). Impor- and Bíró, 2013; Nilius et al., 2014). Indeed, Olmsted tantly, TH2 cells overproduce IL-31 in both canine syndrome, a rare genetic disorder that is due to a gain- (Gonzales et al., 2013) and human (Sonkoly et al., of-function (G573S) mutation in TRPV3 (Lin et al., 2006) atopic dermatitis. A large subset of sensory 2012), is characterized by debilitating itch. This is in neurons that coexpress TRPV1 with TRPA1 carry keeping with the phenotype of transgenic mice over- a receptor for IL-31 (Cevikbas et al., 2013); indeed, expressing human TRPV3 G573S that includes loss of the IL-31–induced pruritus is ameliorated in both hair, dermatitis, and severe scratching behavior TRPV1- and TRPA1-deficient mice. (Huang et al., 2008). DS-Nh mice that carry a gain- It was suggested that pain- and itch-transmitting of-function TRPV3 isoform also display dermatitis and afferents are distinguished by the central mediators a hairless phenotype (Asakawa et al., 2006). Con- that they use. Indeed, TRPV1-expressing pruriceptive versely, TRPV3 knockout mice exhibit ameliorated afferents contain gastrin-releasing peptide (GRP) in scratching response in a model of dry skin–induced humans (Timmes et al., 2013). Mice whose GRP pruritus (Cheng et al., 2010b). Importantly, TRPV3 receptors have been deleted by genetic manipulation appears to be under the negative control of Mg2+ and it were reported to show normal pain response but no was postulated that dietary Mg2+ deficiency may scratching (itch) behavior (Sun and Chen, 2007). By liberate TRPV3 from this tonic inhibitory control, contrast, TRPV1-positive nociceptive afferents lack leading to a constitutively active TRPV3 channel GRP but carry the vesicular glutamate transporter-2. (Luo et al., 2012a). Indeed, a Mg2+-deficient diet Importantly, loss of vesicular glutamate transporter-2 causes dermatitis and scratching behavior in rats renders mice less sensitive to pain but, paradoxically, (Choi et al., 2008). Taken together, these findings more sensitive to itch (Lagerström et al., 2010; Liu imply a therapeutic potential for topically applied et al., 2010b). Mice that express a constitutively active TRPV3 antagonists in the management of pruritus form of the serine/threonine kinase BRAF (member B secondary to (atopic) dermatitis. of the Raf-kinase family of growth signal transduction The role of TRPV3 in the development of atopic proteins kinases) in their sensory neurons gated by dermatitis is, however, controversial. The pathogenesis Nav1.8 (so-called BRAFNav1.8 mice) show spontaneous of atopic dermatitis is thought to involve compromised scratching behavior indicative of chronic pruritus skin barrier formation and the skin barrier is clearly (Zhao et al., 2013b). A subset of TRPV1+ neurons defective in the Trpv3(2/2) animals (Cheng et al., contains the neuropeptide natriuretic polypeptide 2010b). To explain the phenotype of the Trpv3(2/2) b (Nppb). Nppb evokes potent scratching when injected mice, it was postulated that TRPV3 forms a complex intrathecally to mice (Mishra and Hoon, 2013); con- with ADAM17 (A Disintegrin And Metalloproteinase versely, Nppb(2/2) mice show markedly reduced 17), EGFR, and TGase1 that is required for normal responses to various itch-producing agents. terminal keratinocyte differentiation (Cheng et al., To resolve these somewhat conflicting findings, it 2010a). Dysregulation of this complex is believed to was postulated that specific subsets of interneurons impair barrier function and lead to dry skin. Thus, may help distinguish itch from pain in the spinal cord. a certain level of basal TRPV3 activity seems to be For example, mice that lack Bhlbh5-positive (a tran- essential for normal skin function, with both increased scription factor whose absence leads to abnormal and decreased activity leading to skin disease. In neuronal differentiation) interneurons show dramati- accord, keratinocyte migration and wound healing are cally enhanced scratching behavior in response to impaired in the Trpv3(2/2) mice. If so, one would not pruritogenic agents but no change in nociception (Ross want to apply a TRPV3 antagonist cream on pruritic et al., 2010). This model is in keeping with the but damaged (scratched/ulcerated) skin. On the other observation that morphine, which activates m-opioid hand, topical TRPV3 agonist administration might be receptors in the spinal cord, may relieve pain and useful in the management of nonhealing skin ulcers cause/enhance pruritus at the same time. (although it could cause pruritus as an on-target side The processing of itch in the CNS is complex and poorly effect). Recent data implicate TRPA1 as the primary understood. In primates, two distinct populations of itch mediator in atopic dermatitis (Oh et al., 2013; spinothalamic tract neurons were found to transmit Wilson et al., 2013a,b). Diseased (atopic dermatitis) histaminergic and nonhistaminergic (e.g., cowhage-evoked) keratinocytes release thymic stromal lymphopoietin itch. In accord, positron emission tomography studies (TSLP) via the Recent data implicate TRPA1 as the revealed different cerebral activity patterns during itch primary itch mediator in atopic dermatitis (Oh et al., evoked by histamine and cowhage. Generally speaking, in 2013; Wilson et al., 2013a). Diseased atopic dermatitis the , the activity in the posterior cingulate keratinocytes release thymic stromal lymphopoietin cortex correlates to itch, whereas the thalamus is active (TSLP) via the ORAI1/NFAT pathway; in turn, TSLP during pain only (Mochizuki et al., 2007). activates TRPA1-expressing cutaneous afferents to An intriguing and possibly underappreciated pruri- trigger itch (Wilson et al., 2013b). Importantly, TSLP togenic target is TRPV3 (recently reviewed in Nilius also regulates immune cells that are responsible for 744 Nilius and Szallasi the development of asthma in patients with atopic The pulmonary vessels are lined by endothelial cells. dermatitis, the so-called “atopic march.” It is notewor- When these cells become “leaky,” it will manifest as thy that keratinocytes isolated from DS-Nh mice pulmonary edema. The proliferation of vascular smooth carrying a gain-of-function TRPV3 mutation also show muscle (in particular, the occurrence of smooth muscle increased responses to TSLP (Yamamoto-Kasai et al., in small arterioles that do not normally have a muscular 2013). As a note of caution, the pathobiology of chronic layer) is a hallmark of pulmonary hypertension. itch is complex and still poorly understood (see Raap Last but not least, the mammalian respiratory tract et al., 2011); therefore, it is probably prudent to temper is lined with a dense plexus of sensory fibers (reviewed our expectations with regard to the therapeutic in Canning, 2006). Activation of this subset of nerve potential of blocking individual TRP channels. fibers by irritant and/or inflammatory stimuli triggers multiple reflexes such as sneezing, coughing, mucus B. Transient Receptor Potential Channels in secretion, bronchospasm, and apnea that limit venti- Airway Disorders lation and dilute/expel foreign materials. Analogous to 1. What Should We Target in Patients with Chronic pain, in which inflammatory mediators produce a hy- Cough: Ankyrin Transient Receptor Potential 1, Vanil- persensitivity to various stimuli, reflexes including loid Transient Receptor Potential 1, or Both? In cough are proposed to be sensitized by mediators of a somewhat simplified manner, the airways transport inflammation and oxidant stress such that they become gases to and from the pulmonary alveoli where the gas triggered by innocuous stimuli (see Undem and Carr, exchange with the circulating blood takes place. 2010; O’Neill et al., 2013; Spina and Page, 2013). This is Functionally, the lungs can be divided into the airways believed to be the underlying mechanism of sensory and the pulmonary vasculature. The airways are hyper-reactivity syndrome (see Millqvist, 2011). a gateway not only to gas exchange but also to airborne In summary, the airways are built of epithelium, pollutants, allergens, and infectious agents. The main smooth muscle, and connective tissue (fibroblasts), airways are lined by a ciliated columnar epithelium with an important contribution of sensory nerves and covered by mucus, the main function of which is to resident immune cells. The pulmonary vasculature filter out and remove harmful particulate matters. consists of endothelial cells, smooth muscle, and Motile cilia directly sense noxious substances entering connective tissue. TRP channels are expressed in all the airways and initiate defensive mechanisms (Shah of these tissues and cells (see Fig. 30) and there is good et al., 2009). Indeed, ciliary dysfunction (“ciliopathy”)is evidence that they play a pivotal role in the de- now recognized as an early sign of chronic inflamma- velopment and maintenance of chronic lung diseases, tory lung diseases (Horváth and Sorscher, 2008). The ranging from chronic cough through COPD and asthma possible connection between smoking, ciliopathy, and to idiopathic pulmonary fibrosis (IPF) and IPAH (see COPD is now subject to intensive research (weill.cornell. Nassini et al., 2010c; Abbott-Banner et al., 2013). edu/news/releases//wcmc/wcmc_2012/07_12_12b.shtml). Combined, these diseases already affect hundreds of The airway mucus is produced by goblet cells, and millions of patients worldwide and their prevalence is goblet cell hyperplasia is a hallmark of allergic asthma. on the rise. By the end of this decade, COPD alone is The columnar respiratory epithelium may undergo predicted to be the third leading cause of death and squamous metaplasia in response to long-term irrita- fourth commonest cause of disability. Unfortunately, tion such as cigarette smoke. This can not only available COPD treatment options (including inhaled compromise mucociliary airway clearance but can also , long-acting , and roflu- create an environment for the emergence of squamous milast, a phosphodiesterase-4 inhibitor) provide only cell carcinoma (SQCC). symptomatic relief and do not halt (or reverse) disease Large airways (bronchi) are held open by cartilage progression. With regard to IPF and IPAH, the rings, whereas smaller airways (bronchioli) are sup- therapeutic options are even less satisfactory. Clearly, ported by the surrounding lung tissue. Both contain there is an urgent need for novel therapeutics and, as circular smooth muscle that can regulate the airway discussed below, there is a cautious optimism in the diameter by constricting or dilating. Bronchioli termi- field that targeting certain TRP channels/functions nate in alveolar sacs in which a single layer of may have important therapeutic benefits in selected pa- pneumocytes line delicate fibrous septae containing tients with chronic respiratorydisease(seeTakemura a dense capillary network, the site of gas exchange et al., 2008; Moran et al., 2011; Preti et al., 2012; between the pulmonary circulation and the external Abbott-Banner et al., 2013). A number of tools (e.g., milieu. Destruction of these septae creates large air selective TRPV1 and TRPA1 antagonists and TRPV4 spaces called emphysema, whereas the proliferation of agonists) are already available for clinical studies and fibroblasts in the septae leads to pulmonary fibrosis. now the major challenge (similar to the pain field) is to Both of these pathologic processes will eventually identify the patients who are most likely to benefit compromise gas exchange, leading to hypoxemia and from these molecules. For example, asthma is a postu- hypercapnia. lated therapeutic target for TRPA1 (and maybe also TRP Channels as Drug Targets 745

Fig. 30. The diverse roles of TRP channels in the airways and their potential involvement in airway disorders. See text for details. Reprinted with permission from Moran et al. (2011).

TRPV1, TRPV4, TRPM4, TRPC1, and TRPC4) antag- activation profiles and neurochemistry. A third (and onists (see Facchinetti and Patacchini, 2010; Abbott- probably the least understood) population of C fibers in Banner et al., 2013). Asthma is, however, a heteroge- the respiratory tract originates from the DRG. Adding nous disease with at least six different forms and to this anatomic complexity is the fact that capsaicin- patients belonging to these different forms are likely to sensitive C fibers show striking species-related differ- respond differently to TRPA1 blockade. ences in the airways. For example, when exposed to a. Vanilloid transient receptor potential 1. The most inhaled capsaicin, rats do not cough or show broncho- common type of sensory nerves in the mammalian constriction; however, they develop a marked neuro- respiratory tract are C fibers (see Canning, 2006) that genic inflammatory (edema) response that is absent in express TRPV1 (see Geppetti et al., 2006). These fibers animals desensitized to capsaicin (Lundberg and Saria, are relatively unresponsive to mechanical changes that 1983). occur during respiration but can be selectively stimu- Guinea pigs are very sensitive to inhaled capsaicin: lated by BK (and other inflammatory mediators), acidic they severely cough and display both airway edema solutions, and capsaicin. Parenthetically, a recent and constriction (see Canning, 2006). Capsaicin-evoked study reported the presence of TRPV1 in bronchial cough occurs only in conscious (but not in anesthetized) epithelium with a marked upregulation in individuals animals (see Undem and Carr, 2010). Humans also with asthma (McGarvey et al., 2014). Moreover, respond with cough to inhaled capsaicin (Fig. 31A). TRPV1 expression was detected in airway smooth Study subjects describe an itchy, urge-to-cough sensa- muscle cells with a similarly increased expression in tion that slowly builds up in intensity and is relieved asthmatic rats (Zhao et al., 2013a). These are un- by voluntary coughing. This is very different from the expected and controversial findings that need to be explosive, involuntary cough response that occurs in confirmed. Below, we focus on neuronal TRPV1 recep- response to aspiration even in anesthetized animals. tors in the airways, the existence of which is firmly Humans also develop a neurogenic inflammatory established. response in the airways (see Joos et al., 1995). There There are at least two distinct types of vagal C fibers, is good evidence (as reviewed in Baraniuk, 2001; bronchial and pulmonary, that innervate different Lacroix and Landis, 2008) that in upper airway regions of the respiratory tract and also differ in their mucosa, neurogenic inflammation is involved in the 746 Nilius and Szallasi

therapy employing intranasal capsaicin desensitiza- tion provides symptomatic relief in these individuals (Marabini et al., 1991). This procedure is, however, poorly tolerated (painful) and thus has not been widely adopted. It is still hotly debated whether neurogenic inflammation in humans can play any significant role in lower airway diseases such as asthma and/or COPD (for different views, please see Barnes, 2001; Widdi- combe, 2003; Groneberg et al., 2004b; Butler and Heaney, 2007; Ramalho et al., 2011). According to the majority opinion, it does not. There are limited data on capsaicin effects in human airways. In vitro, capsaicin was shown to induce contractions in isolated human bronchi; however, this effect required high (10 mM) capsaicin concentrations and was very weak compared with the contractile response detected in the guinea pig (Lundberg et al., 1983). In human volunteers, inhaled capsaicin evokes cough and causes mild retrosternal discomfort, as well as a transient and mild increase in airway resistance (Fuller et al., 1985). This bronchoconstrictor response did not differ between patients with asthma and smokers or nonsmokers who were otherwise healthy (Fuller et al., 1985). Vagal Ad cough afferents and bronchopulmonary C fibers converge in the nucleus of the solitary tract (see Canning, 2006). This was suggested to form the anatomic basis by which increased C fiber drive may enhance cough reflex sensitivity. The control of cough reflex by descending neural pathways was reviewed elsewhere (Mazzone et al., 2011). Of note, the increased C fiber drive may also originate outside of the respiratory tract. GERD is a well recognized cause of chronic cough (see Pauwels et al., 2009), and antireflux surgery eliminates chronic cough in a subset of patients with GERD (Hoppo et al., 2013). Yet, intra- luminal esophageal challenge with capsaicin or acids causes only pain but no cough response in humans (Wu et al., 2002). The pH challenge in the esophagus, however, sensitizes the cough response to inhaled capsaicin (Ing and Ngu, 1999). In keeping with this model, capsaicin (or SP) microinjected into the com- missural subnucleus of the nucleus of the solitary tract was shown to facilitate the cough reflex (Canning and Mori, 2011). Of note, at least in rodents, Ad fibers that do not respond to capsaicin under physiologic con- ditions may acquire capsaicin sensitivity after ovalbu- min sensitization (Zhang et al., 2008b). Furthermore, + Fig. 31. Increased sensitivity to inhaled capsaicin identifies a specific the “silent majority” of TRPV1 C fibers (that are subset of patients with chronic cough (B) compared with healthy control inactive under physiologic conditions) start discharg- subjects (A). Bronchial biopsies reveal enhanced TRPV1 expression in these patients (C), implying the therapeutic potential for TRPV1 antagonists. (A) ing during disturbances (Andresen et al., 2012). and (B) are original experimental notes kindly contributed by K.F. Chung. Capsaicin is a prototypical respiratory irritant that (C) is reprinted with permission from Groneberg et al. (2004a). evokes protective reflexes such as cough, sneeze, and fluid secretion when applied to the human respiratory pathogenesis of rhinosinusitis. Indeed, TRPV1 expres- mucosa. Increased sensitivity to capsaicin aerosols sion is increased in the nasal mucosa of patients with occurs in a number of respiratory disorders of varying vasomotor rhinitis (Van Gerven et al., 2013) and severity and etiology. For instance, inhaled capsaicin TRP Channels as Drug Targets 747 provokes exaggerated cough response in patients with tobacco products) that cause pronounced respiratory cough variant, but not classic, asthma (Fig. 31B; irritation in humans (see Bessac and Jordt, 2008). Nakajima et al., 2006), as well as in sensory airway Since tobacco smoking is thought to be the single most hyper-reactivity syndrome (SHR) (Ternesten-Hassèus important factor responsible for the development of et al., 2013). Indeed, the capsaicin inhalation test is COPD, it was a breakthrough observation that ciga- broadly used as a diagnostic tool to identify patients rette smoke extract (CSE) activates DRG neurons with SHR (Johansson et al., 2002). Of note, a reduced obtained from wild-type, but not TRPA1 knockout, urge-to-cough threshold to inhaled capsaicin was mice (of note, deleting TRPV1 has no effect on CSE observed in otherwise healthy smokers (Dicpinigaitis, activation) (Andrè et al., 2008). Parenthetically, airway 2003). TRPA1 is also a target for wood smoke (Shapiro et al., Theoretically, the increase in capsaicin-induced 2013). Indeed, cigarette smoke activates pulmonary cough may be due to the following: 1) enhanced TRPV1 C fibers by interacting at TRPA1 (Lin et al., 2010). expression and/or activity in airway sensory neurons, Interestingly, nicotine itself activates both TRPA1 2) airway epithelial barrier permeability alterations (Talavera et al., 2009) and TRPV1, albeit at much that allow capsaicin to more readily access airway higher concentrations at which it activates nicotinic nociceptor terminals, and 3) heightened CNS respon- acetylcholine receptors (Kichko et al., 2013). Paren- siveness to afferent input. Bronchial biopsies obtained thetically, nicotine also appears to activate TRPC1 and from a subset of patients with chronic cough revealed TRPC6 channels (Wang et al., 2014a). The implications an increase in TRPV1-like immunoreactivity (Fig. 31C) of this finding to smoking-associated lung diseases will that correlated to the sensitivity of the patients to be discussed later. Activation of sensory neurons by capsaicin-evoked cough (Groneberg et al., 2004a; high-concentration nicotine was markedly reduced in Mitchell et al., 2005). Furthermore, in cultured sensory the TRPA1/TRPV1 double-null animals. One may neurons, increased TRPV1 expression was observed argue that TRPA1 activation in the airways might 2–4 hours after rhinovirus (Abdullah et al., mediate the irritant sensation (perceived as pleasur- 2014). Intriguingly, treatment with the nonselective able by smokers) that is (at least in part) responsible COX inhibitor indomethacin increased capsaicin- for the tobacco addiction. If so, inhaled TRPA1 induced cough thresholds in patients with asthma or antagonists (by masking this pleasant feeling) might chronic bronchitis but not in healthy subjects aid smokers in kicking the habit. In keeping with this (Fujimura et al., 1995). By contrast, at least in rodents, hypothesis, TRPA1 polymorphism has been linked to acetaminophen causes airway hyper-reactivity through menthol preference in heavy smokers (Uhl et al., 2011). TRPA1 (Nassini et al., 2010b). These results offer Of note, off-target TRPA1 activation seems to compelling evidence that inflammatory mediators pro- mediate the undesirable side effects of many broadly duced in diseased airways enhance airway reflex used drugs. For example, the antidiabetic drug sensitivity in a manner that can be at least partially glibenclamide was proposed to evoke abdominal pain reversed by pharmacological intervention. In a large via TRPA1 activation (Babes et al., 2013). Moreover, European study, six TRPV1 gene SNPs appeared to multiple classes of molecules (including confer higher risk for chronic cough (Smit et al., 2012). lidocaine and propofol) have been shown to cause pain In preclinical models, various TRPV1 antagonists were in a TRPA1-mediated fashion (Fischer et al., 2010). reported to potently inhibit the evoked (e.g., by in- Although these data raise the possibility that surgical haling aerosolized citric acid) tussive response (re- anesthesia may paradoxically increase postoperative viewed in Geppetti et al., 2006; McLeod et al., 2008; pain, their more immediate impact is to identify Preti et al., 2012). Moreover, TRPV1 antagonists TRPA1 as a possible mediator of the respiratory attenuated antigen-provoked cough in guinea pigs complications of gas anesthetics, which can include sensitized to ovalbumin (McLeod et al., 2006), and coughing and laryngospasm (Eilers et al., 2010). In reduced epithelial injury in a mouse model of asthma support of this hypothesis, the TRPA1 blocker HC- (Rehman et al., 2013). In a clinical study, the TRPV1 030031 prevents desflurane-induced increases in air- antagonist SB705498 (Fig. 9), however, did not block way resistance in guinea pigs (Mutoh et al., 2013). “spontaneous” cough in patients afflicted with chronic Additional hazardous irritants, including isocya- cough, indicating that the clinically important cough nates, ozone, and chlorine, activate TRPA1 in vitro may be mediated by a target on capsaicin-sensitive and cause respiratory irritation in vivo (reviewed in afferents, which is unrelated to TRPV1 (Smith et al., Bessac and Jordt, 2008). These molecules are broadly 2012; Khalid et al., 2014). This has refocused attention toxic and exposure to them also causes marked on TRPA1 as a promising therapeutic target in airway symptoms in human airways. Importantly, interrup- disorders. tion of TRPA1 function in rodents via gene disruption b. Ankyrin transient receptor potential 1. Many of or pharmacological blockade nearly abolishes sensory the irritants that activate TRPA1 are air pollutants neuron activation and/or respiratory reflexes, includ- produced by the combustion of materials (including ing cough produced by short-term exposure to these 748 Nilius and Szallasi irritants, pointing to TRPA1 as the sole (or at least antioxidant treatment (Geraghty et al., 2011). TRPA1 is most important) molecular target for a wide range of a major target for ROS in the respiratory tract. respiratory irritants. One noteworthy exception is Moreover, as discussed above, TRPA1 is a main me- nicotine, which (as we saw above) activates sensory diator of CSE-induced IL-8 production in respiratory neurons via both TRP (TRPA1 and TRPV1) and epithelium. MMP1 is increased in the lungs of patients nicotinic acetylcholine receptors. The complex inter- with emphysema. Indeed, transgenic mice expressing play between these two mechanisms in vivo, however, human MMP1 develop changes resembling human awaits further clarification because TRPA1 is neces- emphysema (D’Armiento et al., 1992; Foronjy et al., sary for increases in the Penh (enhanced pause) 2003). The promoter of the MMP1 gene has a CSE- respiratory measurement caused by intranasal nico- responsive element (Mercer et al., 2009) and a poly- tine; however, similar provocations produce robust morphism in the MMP1 gene (G1607GG) has been respiratory sensory irritation responses in TRPA1- linked to rapidly declining lung function in smokers deficient mice (reviewed in Moran et al., 2011). (Wallace et al., 2012). Interestingly, MMP1 also exhibits In preclinical models, increasing evidence supports a neurotropic function in that it sensitizes vagal C fibers a pivotal role for TRPA1 in airway allergic inflamma- to chronic cough. On the basis of these observations, one tion (reviewed in Nassini et al., 2010c; Abbott-Banner might visualize a complex feedback loop in which et al., 2013). Mice either lacking TRPA1 or pretreated smoking upregulates MMP1 expression via TRPA1 with the TRPA1 blocker HC-030031 were protected (leading to emphysema) and then MMP1 elevates from airway inflammation (both in bronchoalveolar C fiber drive (directly or indirectly involving TRPA1) lavage [BAL] and lung tissue), bronchial hyper- to cause chronic cough. reactivity to acetylcholine, and transcription of the Interestingly, increased IL-8 levels were also found in gene product for the gel-forming mucin, MUC5AC the BAL of patients with IPF (Willems et al., 2013), and (Caceres et al., 2009). Of note, knocking out TRPV1 did polymorphism in the IL-8 gene has been linked to IPF not alter any of these parameters, providing evidence (Ahn et al., 2011). It was recently speculated that COPD that TRPA1 has a distinct role in this model of allergic and IPF may be “distinct horns of the same devil” (Chilosi disease. The TRPA1 blocker HC-030031 (but not the et al., 2012). Given the proposed role of non-neuronal TRPV1 antagonist JNJ17203212) was also shown to TRPA1 (e.g., in lung fibroblasts) in IL-8 production, it attenuate the late asthmatic response in rodent models might be argued that the relentless progression of IPF of allergic asthma (Raemdonck et al., 2012). TRPA1 couldbesloweddownbyTRPA1antagonists. has also been implicated in airway neurogenic in- Although it is technically not a chronic airway flammation caused by the acetaminophen metabolite disorder (and is better suited to the section on cancer), N-acetyl-p-benzo-quinoneimine in multiple small ani- it is worth mentioning here that human small cell mal species (Nassini et al., 2010b). Neurogenic in- (poorly differentiated neuroendocrine) carcinoma cells flammation is clearly a key mechanism in experimental express TRPA1 (Schaefer et al., 2013). Small cell asthma, and the pivotal role of TRPA1 in the initiation carcinoma is a dreaded smoking-related lung tumor. and maintenance of the neurogenic inflammation in The TRPA1 agonist allyl-isothiocyanate evokes Ca2+ rodent and guinea pig airways is firmly established. currents in small cell carcinoma cells and promotes the Neurogenic inflammation, however, is unlikely to play survival of tumor cells via ERK phosphorylation. any significant role in the pathomechanism of allergic TRPA1 is expressed in airway epithelial cells, where disease or COPD in human airways (see Widdicombe, it serves as a receptor for toxic lung inhalants, many of 2003). Therefore, it was an important recent discovery which are carcinogenic (Büch et al., 2013). Thus, one that non-neuronal cells in human airways (both might speculate that TRPA1 is instrumental for both epithelial cells and fibroblasts) express functional the initiation and progression of lung cancer. TRPA1 and respond to CSE exposure with IL-8 Chronic cough is a large, unmet medical need. The production in a TRPA1-mediated fashion (Nassini exact prevalence of chronic cough has proven difficult et al., 2012b). This finding is not completely un- to determine, with reports ranging from 9% to 33% of expected since TRPA1 was originally cloned from the general population, depending on environmental human lung fibroblasts, and the presence of functional factors such as smoke exposure and air pollution TRPA1 in lung fibroblasts was also demonstrated (reviewed in Chung and Pavord, 2008). In heavy (Mukhopadhyay et al., 2011). Taken together, these smokers living in Delhi, a large metropolis with compelling data demonstrate that TRPA1 can contrib- a problem of air pollution that is fairly common in ute to neurogenic inflammation in laboratory animal the developing world, the prevalence of chronic cough models and non-neurogenic inflammation in human approaches 70% (Chhabra et al., 2001, 2008). Bende airways. and Millqvist (2012) recently reported an age- CSE increases both matrix metalloprotease-1 dependent increase in the prevalence of chronic cough (MMP1) and IL-8 production by human small air- in nonsmoking Swedish adults, with a peak (13%) way epithelial cells; these effects are prevented by occurring in middle-aged women (age 40– 49 years). TRP Channels as Drug Targets 749

The symptomatic management of chronic cough has receptor (Carr et al., 2003), whereas PGE2 was changed surprisingly little during the past 50 years. It demonstrated to activate TRPA1 to evoke coughs still heavily relies on and its derivatives such (Grace et al., 2012). However, somewhat unexpectedly, as dextromethorphan. This is a problem because there it turned out that the complete elimination of BK- and is little evidence that codeine derivates can provide any PGE2-evoked cough responses requires a simultaneous clinical benefit in cough relief over placebo in patients blockade of TRPV1 and TRPA1 (Brozmanova et al., with chronic cough (Ramsay et al., 2008), yet they 2012; Grace et al., 2012). Subsequently, it was reported have significant adverse effects (ranging from itch to that the blockade of cigarette smoke–induced activa- hallucinations) as well as a well documented abuse tion of the laryngeal nerve similarly required the potential (www.nhtsa.gov/people/injury/research/ simultaneous antagonism of TRPV1 and TRPA1 (Liu job185drugs/dextromethorphan.htm). et al., 2013b). These observations may be interpreted to The current American College of Chest Physicians imply that a dual TRPV1/TRPA1 inhibitor might be guidelines recommend that cough treatment follow superior as an antitussive agent to selective inhibitors. a thorough diagnostic work-up (Irwin, 2006). However, Theoretically, there are three strategies to relieve no underlying disease can be diagnosed in up to 46% of cough by targeting airway C fibers: 1) to prevent patients with chronic cough (effectivehealthcare.ahrq. C fiber activation by inflammatory stimuli (e.g., by gov/ehc/products/356/1370/CER_100_ChronicCough_ NSAIDs or antihistamines); 2) to block generator ExecutiveSummary_20130102.pdf). This recognition potential formation (in which TRPV1 and TRPA1 are has raised the provocative question of whether idio- believed to be the major players); and 3) to inhibit pathic chronic cough is a real disease or a failure of action potential formation (reviewed in Undem and diagnosis (Morice et al., 2011). As of today, most experts Carr, 2010; Muroi and Undem, 2011). Regarding option seem to agree that idiopathic chronic cough is a real 1, COX metabolites activate TRPA1 (Materazzi et al., disease, although a consensus on terminology (variably 2008), and the nonselective COX inhibitor indometh- referred to as idiopathic chronic cough, cough hypersen- acin was reported to ameliorate the cough response in sitivity syndrome, and SHR) is still lacking. This is most experimental animals, as well as in patients with likely a heterogenous group of patients whose unifying chronic bronchitis (Fujimura et al., 1995). Yet, in- feature is their unique sensitivity to inhaled capsaicin domethacin is also known to cause paradoxical cough (compare Fig. 31, A and B). as a side effect in a small subset (2.3%) of patients Guinea pigs cough in response to inhaled capsaicin (www.ehealthme.com/ds/indomethacin/cough). In the (or citric acid) and this response is exaggerated in guinea pig, Nav1.7 knockdown by shRNA abolishes ovalbumin-sensitized animals (reviewed in Canning citric acid–induced cough (Muroi et al., 2013); this and Mori, 2011). TRPV1 antagonists such as BCTC observation supports the feasibility of the third (McLeod et al., 2006) and V112220 (Leung et al., 2007) approach. This is in keeping with the antitussive block cough evoked by capsaicin or acid challenge with action of nebulized lidocaine in patients with intracta- an efficacy similar to that of codeine. Guinea pigs also ble cough (reviewed in Slaton et al., 2013). Arguing cough when exposed to the inhaled TRPA1 agonist against this approach is the lack of cough phenotype in allyl-isothiocyanate (Andrè et al., 2009); this cough patients with congenital lack of pain due to loss-of- response is ameliorated by the TRPA1 antagonists HC- function Nav1.7 mutations (Cox et al., 2006). 030031 (Andrè et al., 2009) and AP18 (Brozmanova A popular (and controversial) theory holds that et al., 2012). Inhaled allyl-isothiocyanate also provokes chronic cough is due to the airway remodeling cough in human volunteers (Birrell et al., 2009). (plasticity) that may occur in response to ongoing Perhaps surprisingly, TRPV1 activation is far more irritation (critically reviewed in Niimi, 2011). Certain effective than TRPA1 activation to evoke coughing in agents generated during inflammation (e.g., NGF) guinea pigs (Brozmanova et al., 2012). This is in were shown to upregulate TRPV1 (and also SP) keeping with the finding that capsaicin (1 mM) is more expression in nociceptive DRG neurons (Ji et al., active than allyl-isothiocyanate (100 mM) in causing 2002). Furthermore, it was reported that Ad fibers sustained action potential generation in isolated that do not express TRPV1 under normal conditions tracheal C fibers. become capsaicin sensitive in preclinical pain models Thus, TRPV1 antagonists block cough response (reviewed in Ueda, 2006). It is not unlikely that similar evoked by TRPV1 agonists, and TRPA1 antagonists changes in TRPV1 expression may also occur during inhibit cough evoked by TRPA agonists. Of course, it airway inflammation. Indeed, vagal Ad fibers (both was expected; however, what about “spontaneous” slowly and rapidly adapting) were reported to acquire (pathologic) cough? Spontaneous cough is difficult to capsaicin sensitivity after ovalbumin sensitization; study in experimental animals. A reasonably good this stems from newly expressed TRPV1 in NF+ model is cough evoked by inflammatory mediators such (i.e., myelinated) nodose ganglion neurons (Zhang as BK and PGE2. In the airways, BK was previously et al., 2008b). Importantly, these findings in experimen- shown to be coupled to TRPV1 downstream of the B2 talanimalscorrelatetotheclinical observations. SP-like 750 Nilius and Szallasi immunoreactivity is increased in bronchial biopsies pathobiology but no longer plays a significant role in taken from patients with cough variant, but not classic, the maintenance of the established disease. If so, asthma (Lee et al., 2003b). For TRPV1-like immunore- knockout animals are not good models to study chronic activity (Fig. 31C), a similar phenomenon was described cough and/or asthma because they do not reflect the in patients with chronic cough (Groneberg et al., 2004a; established disease. Indeed, a loss-of-function TRPV1 Mitchell et al., 2005). These increases in SP- and TRPV1- variant (I585V) was associated with a lower risk for like immunoreactivity are associated with greatly childhood asthma (Cantero-Recasens et al., 2010). Yet exaggerated cough in response to inhaled capsaicin there is no experimental evidence to imply a therapeu- (Cho et al., 2003; Ternesten-Hassèus et al., 2013). tic potential for TRPV1 antagonists in the pharmaco- Interestingly, elevated SP levels were also found in therapy of allergic airway diseases. For example, the the plasma (Otsuka et al., 2011) and nasal secretions TRPV1 antagonist SB705498 when applied to the (Cho et al., 2003) of patients with chronic cough. nasal mucosa reduced the capsaicin effect, proving Indeed, it was postulated that elevated SP in nasal target involvement (Holland et al., 2013); however, it secretions defines a distinct population of patients with did not provide any symptomatic relief in patients chronic cough, those with “upper airway cough syn- with seasonal allergic rhinitis (Alenmyr et al., 2012; drome” (Lim et al., 2011). The interpretation of these Bareille et al., 2013). A number of TRPV1 antagonists observations is, however, not straightforward since have entered clinical trials as potential antitussive inhaled SP does not provoke cough in the guinea pig drugs (the latest addition is XEN-D0105; www. (El-Hashim and Amine, 2005), nor do tachykinin ariopharma.com); unfortunately, the results are not NK1R (the receptor SP) antagonists relieve spontane- available. ous coughing in men (Joos et al., 1996). Thus far, 2. Transient Receptor Potential Channels Other than elevated SP appears to be marker of overactive sensory Vanilloid Transient Receptor Potential 1 and Ankyrin nerves in the respiratory tract, rather than a disease- Transient Receptor Potential 1 in Airway Structural causing substance. and Inflammatory Cells: Implications for Therapy. Bronchial biopsies reveal increased inducible nitric- In addition to TRPV1 and TRPA1, a number of TRP oxide synthase (iNOS) activity in the airway epithe- channels belonging to the TRPV (TRPV2 and TRPV4), lium of individuals with asthma (Lane et al., 2004). TRPM (TRPM4 and TRPM8), and TRPC (TRPC1, Increased iNOS was also found in the nasal mucosa of TRPC3, and TRPC6) subfamilies are expressed in the patients with allergic rhinitis (Kawamoto et al., 1999; airways (see Abbott-Banner et al., 2013). Of these Yuksel et al., 2008). Exhaled NO is a good biomarker of TRPs, TRPV4 appears to be the most intriguing as chronic cough (Chatkin et al., 1999). Indeed, patients a therapeutic target but it is also probably the most with chronic cough hypersensitivity syndrome show problematic. TRPV4 is highly expressed in bronchial significant nitrosative stress in their nasal mucosa smooth muscle, where it is thought to contribute to Ca2+ (Bae et al., 2012). It was recently recognized that OA- mobilization (Jia et al., 2004). TRPV4 was reported to be NO2 is a highly electrophilic TRPA1 activator; thus, activated (Chen et al., 2008a), or at least potentiated elevated NO may cause persistent airway inflamma- (Güler et al., 2002), by hypoosmolar solutions. Because tion by activating TRPA1 in the respiratory tract individuals with asthma produce copious amounts of (Taylor-Clark et al., 2009). In addition, the long-term airway secretions with low osmolarity, one attractive nitrative stress may exacerbate airway remodeling. hypothesis for further exploration is that activation of The importance of iNOS in the pathogenesis of allergic TRPV4 by hypo-osmolar secretions in bronchial smooth airway disorders was highlighted by the finding that muscle is a major cause of bronchoconstriction in these allergen (ovalbumin) exposure causes PARP-1 activa- patients (see Liedtke and Simon, 2004). If this model tion and oxidative damage in the airways of wild-type, holds true, inhaled TRPV4 blockers may represent but not iNOS knockout, mice (Naura et al., 2009). The a new class of drugs. The possible link v nonselective NOS inhibitor N -nitro-L-arginine methyl between TRPV4 and COPD is also gaining strength: ester inhibits cough both in naïve and ovalbumin- TRPV4 is expressed in murine and human airway sensitized guinea pigs whereas the selective iNOS epithelial cells, where it is believed to play a key role antagonist ONO1714 [(1S,5S,6R,7R)-7-chloro-3-imino- in mucociliary clearance by regulating ciliary beats 5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride] ex- (Lorenzo et al., 2008; Alenmyr et al., 2014). Impairment erts antitussive activity only in the sensitized animals of this airway protective function by toxic inhalants (Li (Hori et al., 2011). On the basis of these observations, et al., 2011b) and/or carrying a gain-of-function TRPV4 the selective iNOS inhibitor GW274150 was advanced variant (Zhu et al., 2009) is thought to increase the risk into clinical trials, where, disappointingly, it blocked for developing COPD. neither the early nor the late asthmatic response In addition to bronchial epithelium and smooth (Singh et al., 2007). To explain these seemingly muscle, TRPV4 is also expressed in vascular endothe- contradictory findings, one might hypothesize that lium (Watanabe et al., 2002). It is worth mentioning iNOS is required for an initiating event in asthma here that in endothelial cells, TRPV4 is coexpressed TRP Channels as Drug Targets 751 with a number of TRPs such as TRPC1–TRPC6 and these results imply that both TRPC1 and TRPC6 are TRPM4 (see Nilius et al., 2003). Shear stress-induced important players in the pathogenesis of pulmonary vasodilation was absent in the TRPV4-null mice hypertension. Indeed, chronic cigarette smoke expo- (Hartmannsgruber et al., 2007). However, the acute sure upregulates TRPC1 and TRPC6 in mice; in these biology of TRPV4 most readily manifests itself in the animals, the normal vascular tone is restored only alveolar septae of the lung (Alvarez et al., 2006). In when both TRPC1 and TRPC6 are knocked down by isolated and perfused mouse lungs, activation of siRNA (Wang et al., 2014a). TRPV4 by elevated vascular pressure or injurious Smooth muscle cells in diseased airways typically high-pressure mechanical ventilation causes extravas- display abnormalities in contraction and/or prolifera- cular leakage of fluid (i.e., pulmonary edema) reflected tion. Although bronchodilators are efficacious therapies by increases in the lung’s filtration coefficient capable of reversing airflow obstruction, treatments (Hamanaka et al., 2007; Huh et al., 2012). Consistent that reduce contractility and pathologic remodeling of with these findings, intravenous administration of the airway smooth muscle remain targets of active in- TRPV4 agonist GSK1016790A (Fig. 8) causes circula- vestigation. In human airway smooth muscle cells, tory collapse characterized by the failure of the TRPC1, TRPC3, TRPC4, and TRPC6 mRNA were alveolar septal barrier (Willette et al., 2008; Thorneloe detected (see Abbott-Banner et al., 2013). After in- et al., 2012). cubation with the inflammatory cytokine TNFa, The exact role of TRPV4 in lung injury is complex human airway smooth muscle cells exhibit marked and only partially understood. The TRPV4 agonist changes in Ca2+ homeostasis that are accompanied by GSK1016790A, which seems to act by recruiting increased expression of the TRPC3 protein (White inactive TRPV4 channels from intracellular depots et al., 2006). Moreover, enhanced acetylcholine- (Sullivan et al., 2012), can produce dramatic deforma- induced cytosolic Ca2+ elevations in human airway tion of cultured human endothelial cells (Willette et al., smooth muscle cells are blocked by RNA silencing of 2008), but it is only part of the story. It was recently TRPC3. These data are consistent with findings in shown that TRPV4 channels expressed on alveolar allergically inflamed mice in which TRPC3-directed macrophages can play a critical role in ventilator- antibodies inhibited nonselective cation conductances induced injury of mouse isolated lungs (Hamanaka and restored resting membrane potentials of airway et al., 2010). Although it might take some time to smooth muscle cells to more hyperpolarized values, dissect out the relative contribution of different similar to those seen in control subjects (reviewed in TRPV4-expressing cells to lung injury, TRPV4 antag- Wang and Zheng, 2011). Compensatory elevation of onism promises to be a novel and effective approach to TRPC3 expression in Trpc6(2/2) mice may also mitigate pulmonary edema. Indeed, TRPV4 appears to explain why Trpc6-deficient mice have enhanced air- be upregulated in the lungs of patients with heart way reactivity to a muscarinic agonist despite having failure (Thorneloe et al., 2012). reduced BAL inflammation (Sel et al., 2008). In a rat TRPC channels appear to have functional roles in model of pulmonary arterial hypertension, genetic the pulmonary vasculature, particularly with regard to inactivation of TRPC4 also conferred a clear survival responses to hypoxia (reviewed in Moran et al., 2011). benefit (Alzoubi et al., 2013). Regional alveolar hypoxia redirects blood flow to the Menthol is added to certain cigarette brands to well oxygenated areas. This reflex mechanism is reduce airway irritation. There is an increasing impaired in TRPC6 knockout mice because their ex recognition that menthol may make it easier for young vivo lungs develop marked deficits in arterial oxygen people to start smoking and more difficult for smokers saturation after airway instillation of saline to produce to quit (www.cancer.org/cancer/news/fda-investigates- regional ventilatory failure (Weissmann et al., 2006). menthol-in-cigarettes). Even worse, people who smoke Moreover, a SNP (-254C-.G) in the TRPC6 promoter menthol-flavored cigarettes inhale deeper and hold the has been linked to IPAH (Yu et al., 2009b). Consistent smoke longer, which is believed to increase the risk with this hypothesis, pulmonary arterial smooth for cancer by prolonging the exposure of the lung to muscle cells obtained from patients with IPAH showed carcinogens in cigarette smoke. Interestingly, menthol significantly higher protein levels of TRPC6, higher preference among smokers has been linked to TRPA1 resting levels of cytosolic Ca2+, and larger OAG (1- (and not TRPM8) variants (Uhl et al., 2011). oleoyl-2-acetyl-sn-glycerol)-dependent cationic cur- In healthy volunteers, inhaled menthol relieves citric rents than samples from control subjects (Kunichika acid–evoked cough (Morice et al., 1994). Furthermore, et al., 2004; Yu et al., 2004). Of note, hypoxia-induced inhaled menthol elevates the capsaicin cough thresh- proliferation of pulmonary arterial smooth muscle cells old in patients with chronic cough (SHR) (Millqvist was also attenuated in TRPC1 knockdown animals, et al., 2013). Indeed, the menthol receptor TRPM8 is and TRPC1 knockout mice did not develop pulmonary expressed in trigeminal ganglion neurons (McKemy hypertension when kept in a chronically hypoxic et al., 2002), including cold-sensitive afferents serving environment (Malczyk et al., 2013). Taken together, the upper (Abe et al., 2005; Keh et al., 2011; Zhou et al., 752 Nilius and Szallasi

2011) and lower airways (Xing et al., 2008). Using opposite effect, that is, it can block mast cells a combination of functional and neurochemical (immu- degranulation. Unfortunately (as discussed below), nostaining and single-cell RT-PCR) studies, it was TRPM4 is highly expressed in the heart and gain-of- shown that menthol exerts its antitussive effect in the function TRPM4 mutations have been linked to cardiac guinea pig by interacting at TRPM8-expressing (and arrhythmias and hypertension (reviewed in Mills and TRPV1 negative) trigeminal afferents (Plevkova et al., Milan, 2010; Abriel et al., 2012). These findings predict 2013). Cough is believed to be “plastic”: Up- and down- that TRPM4 agonists may exhibit unacceptable car- regulation of the cough reflex may be mediated by diovascular side effects. distinct sensory afferents. Sensitization of the cough reflex appears to be driven by nerves that coexpress C. Transient Receptor Potential Channels as TRPV1 and TRPA1. Conversely, the activation of Therapeutic Targets in Bladder Disorders TRPM8+ afferents may inhibit (down-regulate) the Multiple TRP channels are expressed in the bladder cough response. If this holds true, an ideal antitussive (Fig. 26), urothelium, nerve endings, and detrusor agent should block TRPV1 and TRPA1 and, at the muscle, where they presumably function as sensors of same time, activate TRPM8. Menthol, however, was stretch and chemical irritation (reviewed in Avelino reported to evoke mucus secretion by airway epithelial and Cruz, 2006; Everaerts et al., 2008; Skryma et al., cells (Li et al., 2011c). In patients with COPD, 2011; Avelino et al., 2013; Birder and Andersson, increased TRPM8-like immunoreactivity was found in 2013). Intravesical administration of TRPV1 agonists bronchial epithelium (Li et al., 2011c). Thus, menthol (capsaicin and RTX) has been in use in the manage- may worsen COPD symptoms, especially in “blue ment of the overactive bladder for many years on bloaters.” Conversely, TRPM8 antagonists might pro- a largely empirical basis (reviewed in Ozawa et al., vide symptomatic relief by reducing mucus secretion. 1999; Szallasi and Fowler, 2002). The recent recogni- Of note, TRPM8 appears to mediate the respiratory tion of disease state-related changes in TRP channel depressive action of some volatile anesthetics (Vanden expression has given new impetus to investigate the Abeele et al., 2013) and TRPM7 seems to be involved in roles of these channels in normal bladder function and the proliferation of human lung fibroblasts in response dysfunction. to TGFb1 (Yu et al., 2013). 1. Vanilloid Transient Receptor Potential 1 and Both TRPV2 (Nagasawa et al., 2007; Link et al., Vanilloid Transient Receptor Potential 4. There is 2010) and TRPM2 (Knowles et al., 2011) are expressed a dense, TRPV1-expressing C fiber network in the in alveolar macrophages, where they are believed to suburothelium and muscularis of the human renal play a role in phagocytosis and ROS production pelvis, ureter, bladder, and urethra (reviewed in (reviewed in Knowles et al., 2013). The implications Avelino et al., 2013) but the existence of functional of these findings for respiratory disorders are unclear. TRPV1 in urothelial cells remains controversial. In Chinese children with asthma, elevated TRPV2 Urothelial TRPV1 expression is based on the following expression was found in alveolar macrophages (Cai findings: 1) TRPV1-like immunoreactivity was de- et al., 2013). In a murine model of COPD, no difference scribed in all urothelial layers in the human bladder, was observed between wild-type and TRPM2-null mice and 2) capsaicin (as well as heat and low pH) evokes (Hardaker et al., 2012). Genetic deletion of TRPM2 did Ca2+ currents and ATP release in rat and human not change the shape and chemotaxis of neutrophils urothelial cells in culture (see Avelino et al., 2013). either. By contrast, impaired antigen-induced degran- However, TRPV1-detecting antibodies are probably ulation was observed in mucosal mast cells isolated much less specific than previously thought; indeed, from TRPM2 knockout animals (Oda et al., 2013). This they stain cells in TRPV1 knockout animals (Everaerts observation might be interpreted to imply a therapeutic et al., 2009). Capsaicin itself is not selective for TRPV1 potential for TRPM2 blockers in patients with asthma either (see Szallasi and Blumberg, 1999). There is also (also food allergy) and other allergic airway disorders. a discrepancy between functional data: in particular, For the sake of completeness, it is worth mentioning two independent groups were unable to replicate the here that primary mast cells and mast cell–derived cell capsaicin effects detected in human urothelial cells lines also express TRPC1, TRPC5, TRPM4, and (Charrua et al., 2009b) using mouse (Yamada et al., TRPM7 (reviewed in Freichel et al., 2012). Studies 2009) and guinea pig (Xu et al., 2009b) urothelial cells, with TRPM4-deficient mice suggest that this channel respectively (which may represent another striking plays a critical role in mast cell degranulation species-related difference in TRP expression). Paren- inasmuch bone marrow–derived mast cells isolated thetically, adding to the confusion, increased TRPV1 from the knockout animals exhibited excessive release expression and capsaicin-evoked ATP release was of histamine and other mediators in response to IgE- recently reported in cultured human urothelial cells mediated stimulation (Vennekens et al., 2007). If lack obtained from bladder biopsies of overactive bladder of TRPM4 exacerbates mast cell degranulation, one patients compared with control subjects (Birder et al., might argue that TRPM4 agonism may exert the 2013). Of note, the electrophysiological properties of TRP Channels as Drug Targets 753 capsaicin-evoked currents in the rat urothelium (linear et al., 1997; Silva et al., 2000, 2007). Importantly, this current-voltage relation) were dissimilar to those effect is fully reversible and then it can be achieved detected in rat DRG neurons (outwardly rectifying). again by repeated RTX administration (Silva et al., This controversy aside, an almost complete loss of 2000). In keeping with these findings, biopsies taken specific [3H]RTX binding sites (a neurochemical mea- from the bladder of patients undergoing intravesical sure of TRPV1 receptors) was noted after surgical RTX therapy showed no significant alterations at (Szallasi et al., 1993) or chemical (Goso et al., 1993b) either light microscopic or electron microscopic levels denervation of the rat bladder. This is in keeping with (Silva et al., 2001). the loss of TRPV1 in the human bladder after Intravesical RTX reduces the number of incontinent botulinum toxin injections (Apostolidis et al., 2005). episodes in patients with spinal cord injury (Kim et al., Thus, if nonneuronal TRPV1 exists in the bladder, its 2003; Silva et al., 2005). Likewise, intrathecal RTX expression level relative to neuronal TRPV1 expres- blocks detrusor overactivity in rats with complete sion should be very low. Of note, immunohistochemical chronic spinal cord trans-section (Cruz et al., 2008). analysis also suggests the existence of TRPV1 in other Taken together, these findings raise the possibility nonneuronal cells of the human bladder, including that intrathecal RTX can be used in neurogen- detrusor muscle, fibroblasts, and endothelial cells ic bladder patients in whom catheterization is (Lazzeri et al., 2004). contraindicated. The involvement of neuronal TRPV1 in the physio- The therapeutic value of TRPV1 antagonists in logic micturition reflex is not clear. Rats whose TRPV1- managing detrusor overactivity is unclear since no expressing nerves have been ablated by neonatal endogenous agonist (“endovanilloid”) was identified in capsaicin treatment develop hugely distended bladder the bladder of patients with incontinence. By contrast, (see Szallasi and Blumberg, 1999). The bladder the pain and bladder hyperactivity that accompany phenotype of the TRPV1 knockout mouse is, however, interstitial cystitis are thought to be amenable to less pronounced: these animals showed only mild TRPV1 antagonist therapy. Most (.90%) bladder spotty incontinence (Birder et al., 2002). Furthermore, (lumbosacral and thoracolumbar) DRG neurons are TRPV1 antagonists did not alter micturition in naïve capsaicin responsive (Dang et al., 2013). In rats with animals (Charrua et al., 2009a; Kitagawa et al., cyclophosphamide-induced cystitis, increased current 2013b). When explaining these seemingly conflicting density was observed in these neurons. In a feline results, it should be kept in mind that neonatal model of interstitial cystitis, abnormally enhanced capsaicin administration ablates not only TRPV1 but capsaicin responses were detected secondary to TRPV1 also other receptors coexpressed with TRPV1 on phosphorylation by PKC (Sculptoreanu et al., 2005a). bladder afferents that may be involved in micturition. In mice, genetic manipulation of the TRPV1 gene Clearly, further studies are need to elucidate the role prevents bladder reflex hyperactivity and spinal c-fos (if any) of TRPV1 in the normal bladder reflex activity. overexpression in response to cystitis (Wang et al., By contrast, the role of TRPV1 in micturition reflex 2008b). The TRPV1 antagonist GRC-6211 (structure dysfunction is well established (reviewed in Avelino shown in Fig. 9) ameliorates reflex activity in chron- and Cruz, 2006; Szallasi et al., 2006; Juszczak and ically inflamed bladder (Charrua et al., 2009a). Thor, 2012). In humans, intravesical TRPV1 agonist Combined, these findings imply a therapeutic value (capsaicin and RTX) desensitization therapy is based for TRPV1 antagonists in the symptomatic treatment on the concept that the C fiber–driven micturition of interstitial cystitis. reflex, which is inactive in the adult life, resumes In clinical trials, intravesical RTX yielded no clear control of micturition in both neurogenic and non- benefits over placebo in patients with interstitial neurogenic cases of overactive bladder (reviewed in cystitis (reviewed in Mourtzoukou et al., 2008). This Szallasi and Fowler, 2002; Avelino and Cruz, 2006). In was unexpected and puzzling. RTX is a highly lipo- patients with neurogenic detrusor overactivity disor- philic compound, difficult to keep in aqueous solutions. ders, intravesical capsaicin (Lazzeri et al., 1996; De The apparent affinity of RTX is hugely dependent on Ridder et al., 1997) or RTX (Cruz et al., 1997; Lazzeri assay conditions. For example, low pH was reported to et al., 2000; Kim et al., 2003; Silva et al., 2005, 2007) inhibit specific [3H]RTX binding by 50% at a pH value provides symptomatic relief by increasing bladder of 5.3 and by 90% at a pH value of 4 (Szallasi et al., capacity and decreasing the number of daily inconti- 1995). Although the pH of urine is close to neutral in nent episodes. In some patients, intravesical RTX even a pH-balanced body, dietary changes or certain fully restores continence (Cruz et al., 1997). Intra- medications can make the urine acidic (pH 4 to 5). It vesical RTX appears to be superior to capsaicin in its is not clear to what degree the bioavailability of RTX initial excitation (burning pain) to lasting desensitiza- was controlled in the interstitial cystitis clinical trials tion ratio. At doses at which it evokes only mild [RTX sticks to plastics and is difficult to keep in discomfort, RTX treatment results in an increase in aqueous solution (Szallasi and Blumberg, 1992)]. bladder capacity that lasts for several months (Cruz Another possible confounding factor is TRPV1b, 754 Nilius and Szallasi a dominant negative splice variant (Charrua et al., urothelium (Aizawa et al., 2012).] Importantly, 2008). It was speculated that TRPV1 may be more (or GSK1016790A increased the amplitude of bladder less) active in the bladder of certain patients depending contractions in a rat model of neurogenic underactive on the relative ratio of TRPV1b to TRPV1. In the rat, bladder (Young et al., 2013). As predicted by data from cystitis was shown to decrease the production of the TRPV4(2/2) mice, the TRPV4 antagonist HC- TRPV1b, leading to a more active TRPV1 phenotype 067047 (Fig. 11) improved bladder function in mice (Charrua et al., 2008). It is possible (although not very with cystitis (Everaerts et al., 2010a). Short-term likely) that patients participating in the interstitial dosing did not alter water intake, core body tempera- cystitis trials had reduced RTX sensitivity due to an ture, thermal selection behavior, heart rate, locomo- overproduction of TRPV1b. Finally, it is worth men- tion, or motor coordination in vivo (Everaerts et al., tioning here that in bladder biopsies taken from 2010a). Taken together, these observations imply patients with interstitial cystitis only TRPM2 and a value for TRPV4 agonists and antagonists in the TRPV2 correlated with disease severity (Homma et al., management of underactive and overactive bladder, 2013). Thus, it is entirely possible that (unlike in respectively. Furthermore, TRPV4 antagonists may experimental animals) TRPV1 does not play any major improve bladder function in patients with prostatic role in the pathogenesis of interstitial cystitis. enlargement. In the bladder, TRPV4 is predominantly expressed 2. Melastatin Transient Receptor Potential 8 and in urothelium (Fig. 26), but it is also present in Ankyrin Transient Receptor Potential 1. In the human detrusor muscle (Birder et al., 2007; Gevaert et al., bladder, TRPM8 is present both in urothelium and 2007; Xu et al., 2009b). In transgenic mice that suburothelial myelinated nerve fibers (Fig. 26) (Du overexpress NGF in their urothelium, increased et al., 2008) with increased expression in patients with TRPV4 levels were described in both urothelial cells bladder pain or idiopathic idiopathic detrusor over- and DRG neurons (Girard et al., 2013). The neuronal activity (Mukerji et al., 2006a,c). Of note, some recent expression pattern of TRPV4 is puzzling. Whereas studies confirmed (Kullmann et al., 2009; Shabir et al., TRPV4 is present in the majority (.80%) of lumbar 2013), whereas others questioned, the presence of DRG neurons, no TRPV4-like immunoreactivity was TRPM8 in urothelial cells (Everaerts et al., 2010a). detected in bladder afferents. In human urothelium, The TRPM8 agonist menthol activates the micturition TRPV4 was localized adjacent to the adherence reflex in the rat (Nomoto et al., 2008) and pig (Vahabi junctions (Janssen et al., 2011). Interestingly, TRPV4 et al., 2013). Conversely, the TRPM8 antagonist AMTB seems to be highly coexpressed with TRPV2 and (Fig. 11) decreases the frequency of volume-induced TRPM7 in both mouse (Everaerts et al., 2010a) and bladder contractions (Lashinger et al., 2008). These human (Shabir et al., 2013) urothelial cells. findings imply a therapeutic potential for TRPM8 TRPV4 knockout mice show an altered micturition antagonists in the management of bladder disorders pattern that is characterized by increased intermictur- characterized by pain and/or detrusor overactivity. ition intervals and spotting due to nonmicturition TRPA1 is highly coexpressed with TRPV1 on rat contractions (Gevaert et al., 2007). In other words, bladder afferents, especially in the bladder outflow the TRPV4 knockout mice have an incontinent pheno- region (Gratzke et al., 2009). Indeed, the TRPA1 type. In addition, TRPV4-null mice show reduced agonist allyl-isothiocyanate contracts the rat bladder urinary frequency and increased void volume after (Andrade et al., 2006). Moreover, TRPA1 appears to be bladder damage due to cyclophosphamide (Everaerts expressed in the urothelium (Gratzke et al., 2009). The et al., 2010a). By contrast, a subset of patients with functional role of this dual (neuronal and urothelial) TRPV4 axonal neuropathy spectrum disorders due to TRPA1 expression in the normal micturition reflex gain-of-function TRPV4 mutations show bladder remains to be delineated. urgency/incontinence (reviewed in McEntagart, 2012). In naive rats, the TRPA1 antagonist HC-030031 has On the basis of these findings, it was postulated that no apparent effect on bladder functions (Minagawa TRPV4 plays a crucial role in the mechanosensory et al., 2013). However, after spinal cord injury, TRPA1 pathway in the bladder by detecting changes in mRNA and protein levels are elevated in rat bladder intravesical pressure, and provides an important afferents and their perikarya in L6–S1 DRG (Andrade contribution to the pathogenesis of neurogenic over- et al., 2011). This increase in TRPA1 expression active or underactive bladder (Everaerts and De parallels the development of bladder overactivity. Ridder, 2013). Importantly, both genetic knockdown and pharmaco- Activation of TRPV4 located in detrusor muscle can logical blockade of TRPA1 reduce the number of lead to contractions. In accord, the TRPV4 agonist nonvoiding bladder contractions in these animals. GSK1016790A is able to contract the bladder directly Conversely, intravesical TRPA1 agonist administra- even in the absence of urothelium (Thorneloe et al., tion induces detrusor muscle overactivity (Andrade 2008). [Of note, another study found GSK1016790A- et al., 2006). Of particular interest is the finding that induced bladder contractions to be dependent on H2S, a gasotransmitter produced during inflammation TRP Channels as Drug Targets 755

(e.g., bacterial cystitis), can act as a potent endogenous United States. The majority of death is due to heart TRPA1 agonist (Streng et al., 2008). Taken together, attack, or acute myocardial infarction (AMI), and these observations imply a therapeutic value for cerebral stroke (both ischemic and hemorrhagic). The TRPA1 antagonism in the management of cystitis possible role of TRP channels in the pathogenesis of (Meotti et al., 2013) and patients with overactive cardiac arrhythmias, pulmonary vascular disorders, bladder. cerebral stroke, and ocular neovascularization disor- 3. Canonical Transient Receptor Potential 1 and ders is discussed elsewhere in this review. Here we Canonical Transient Receptor Potential 4. Increased focus on the contribution of TRP channels to cardio- sprouting of sensory afferents and subsequent bladder myopathies (Fig. 32), cardiac fibrosis (Fig. 33), hyper- overactivity was recently observed in wild-type, but not tension (Fig. 39), and atherosclerosis (also reviewed in TRPC1/TRPC4 double-null, mice with cyclophosphamide- Watanabe et al., 2009, 2013; Rowell et al., 2010; induced cystitis (Boudes et al., 2013). These findings Vennekens, 2011; Zholos and Curtis, 2013). We will imply a crucial role for TRPC1/TRPC4 in neuronal also touch on the emerging concept that TRP channels sprouting during cystitis that causes overactive bladder. might play an important role in thrombotic disorders by regulating platelet aggregation (see Authi, 2007; D. Transient Receptor Potential Channels in Dionisio et al., 2012). Cardiovascular Disorders 1. Transient Receptor Potential Channels and Heart Cardiovascular disease includes a broad range of Disease. The heart beats about 100,000 times daily disorders (ranging from heart disease through vascular and two and a half billion times over a 70-year lifetime. diseases of the brain, kidney, and lung to peripheral In a much simplified manner, the heart can be thought arterial disease) that affect the cardiovascular system. of as a pump with an energy supply (coronary vessels) The causes of cardiovascular disease are diverse but and an electrical conduction system that connects the atherosclerosis and hypertension are the most com- SA node (that orchestrates the pacemaker activity) to mon. Although smoking remains the most important the cardiomyocytes (that do the pumping) via special- risk factor for cardiovascular disease, nonsmokers who ized muscle fibers known as the bundle of His, the are diabetic and/or obese or lead a sedentary lifestyle fascicles of Tawara, and the fibers of Purkinje. are also at increased risk. Cardiovascular disease kills Disorders that affect this conduction system are called an estimated 17 million people worldwide each year. heart block and can be subdivided into separate Indeed, cardiovascular disease is the leading cause of categories based on the anatomic location of the death in most developed countries, including the damage. Heart rhythm abnormalities (arrhythmias)

Fig. 32. Schematic illustration of the roles that TRP channels are believed to play in the development of cardiac hypertrophy. See the text for details. Reprinted with permission from Watanabe et al. (2013). 756 Nilius and Szallasi

display premature ventricular contractions that are exacerbated by OAG (1-oleoyl-2-acetyl-sn-glycerol) (Hirose et al., 2011), a DAG analog known to activate a functional subset of TRPC channels, namely TRPC3, TRPC6, and TRPC7 (Venkatachalam et al., 2003). Interestingly, these animals showed increased expres- sion of TRPC3 and TRPC6 in their heart. Furthermore, the premature ventricular contractions were prevented by SK&F-96356 [1H-pyrrolo[3,2-c]quinolin-4-amine,2,3- dihydro-N,6-dimethyl-1-(2-methylphenyl)], a nonspecific inhibitor of receptor-mediated calcium entry, originally described as a reversible inhibitor of the gastric H+/K+- ATPase (Leach et al., 1992), which is often used as a biochemical tool to define functional TRPC channels. However, it should be noted here that SK&F-96356 also blocks voltage-gated T-type Ca2+ channels (Singh et al., 2010). On the basis of these observations, one can argue that TRPC3 and TRPC6 might be part of the pacemaker activity in the SA node. The evidence for this is, however, very circumstantial. Clearly, we need better tools (e.g., selective TRPC3 and TRPC6 antag- onists) to scrutinize this concept. By contrast, the involvement of TRPM4 in cardiac conduction anoma- lies is well documented (see the above section on TRP channelopathies; Abriel et al., 2012). A number of TRP channels (including TRPC1, TRPC5, TRPC6, TRPV1, TRPV2, TRPP1, TRPP2, and TRPM4) have been detected in human cardiomyo- cytes, of which particular attention was paid to TRPC3 Fig. 33. Illustration depicting the putative roles of TRP channels in the and TRPC6 as attractive targets in cardiac hypertro- development of cardiac fibrosis. See the text for details. Reprinted with phy (Fig. 32; recently reviewed in Watanabe et al., permission from Yue et al. (2013). 2013). Generally speaking, cardiac hypertrophy devel- ops when the heart works against increased resistance occur when the electrical impulses that coordinate (e.g., valvular stenosis or hypertension). Among the the heart beats become disorganized. Diseases of the neurohumoral factors that may trigger the intracellu- cardiomyocytes that result in deterioration of the lar signaling cascade in cardiomyocytes leading to pumping function are grouped together as cardiomy- hypertrophy, previous studies focused on calcineurin opathies. Alternatively, the heart may be weakened by (Fig. 32) (see Wang et al., 2014b). Indeed, in the mouse, increased deposition of cellular matrix proteins by overexpression of calcineurin induces massive heart fibroblasts, a process known as cardiac fibrosis. Abrupt hypertrophy (Molkentin et al., 1998). Calcineurin blockage of the coronary vessels that deprive cardio- dephosphorylates NFAT; NFAT then translocates into myocytes from their O2 supply can cause sudden the nucleus where it activates the hypertrophic re- cardiac death (AMI), whereas sustained deprivation sponse genes (Fig. 32; reviewed in Watanabe et al., may lead to chronic ischemic cardiomyopathy. Various 2013). Although most authorities agree that NFAT is TRP channels have been implicated in all of these the shared downstream target for both mechanical disorders (reviewed in Watanabe et al., 2009, 2013). stress and humoral factors (e.g., angiotensin II and Under physiologic conditions, the heart rate is set by endothelin-1) to cause cardiac hypertrophy, opinions the rate of systolic depolarization that occurs in the vary as to the molecular link between NFAT and the pacemaker cells of the SA node. Although the existence hypertrophic signals. of a number of TRP channels (TRPM4 and all TRPCs There is emerging evidence to implicate TRPC3 and with the exception of TRPC5) have been reported in SA TRPC6 in cardiac hypertrophy (see Eder and Molkentin, node cells (see Watanabe et al., 2009; Dietrich and 2011; Watanabe et al., 2013). Genetic overexpression Gudermann, 2011), it is unclear whether any of these of Gaq proteins in the mouse causes heart failure and channels is involved in the cation conductance that elevated TRPC3 and TRPC6 expression (Hirose et al., regulates the pacemaker activity. Transgenic mice 2011). TRPC3 is also upregulated in several animal that overexpress Gaq (a mouse model of heart failure) models of cardiac hypertrophy (see Watanabe et al., TRP Channels as Drug Targets 757

2013). In the mouse heart, TRPC6 expression is the wild type when subjected to pressure overload by increased in response to pressure overload (Fig. 32; thoracic aortic constriction (Buckley and Stokes, 2011). Kuwahara et al., 2006). Silencing of TRPC6 by siRNA The authors of this article speculated that TRPV1 blocked the hypertrophic signal elicited by either antagonists may be repurposed clinically as antihyper- angiotensin II or endothelin-1. Conversely, TRPC6 trophic agents. Indeed, BCTC has recently been shown to transgenic mice displayed striking cardiomyopathy be cardioprotective in a preclinical model of heart failure secondary to NFAT activation. Interestingly, the (Horton et al., 2013). Since BCTC is not selective for TRPC6 gene promoter region contains two conserved TRPV1, among others, it blocks TRPV4 and TRPM8 NFAT consensus sites, creating the framework for channels; this experiment needs to be replicated by a positive feedback loop in which TRPC6 activation a selective TRPV1 antagonist. As a cautionary note, stimulates NFAT which, in turn, further enhances postischemic cardiac recovery was impaired in the TRPC6 activity. Somewhat confusingly, gene silencing TRPV1-null mice (Wang et al., 2005). Finally, TRPV2 of TRPC1 and TRPC3 mimicked the effect of TRPC6 was found to be increased in the sarcolemma of patients knockdown on cardiac hypertrophy (Ohba et al., 2007). with dilated cardiomyopathy (Iwata et al., 2013). The The expression of TRPC1 is increased in the myocar- TRPV2 inhibitor tranilast slowed down the progression of dium of rats with isoproterenol-induced cardiac hyper- dilated cardiomyopathy in both hamster (d-sarcoglycan– trophy (Chen et al., 2013d). Furthermore, TRPC1-null deficient animals) and mouse (transgenic animals over- mice did not develop maladaptive cardiac hypertrophy expressing sialyltransferase) models of the human when subjected to hemodynamic stress (Seth et al., disease. 2009). One might speculate that TRPC1, TRPC3, and In TRPM2-null mice, myocardial necrosis and contrac- TRPC6 form a heteromultimer in the (mouse) heart, tile dysfunction was ameliorated after left main coronary and the blockade of any of these three constituents is artery ligation/reperfusion injury (Hiroi et al., 2013). sufficient to disrupt the signaling cascade that acti- Myeloperoxidase staining revealed much fewer neutro- vates NFAT. Indeed, only combined genetic deletion or phils in the infarcted area. These findings imply an pharmacological blockade of TRPC3 and TRPC6 important role for neutrophil TRM2 channels in myocar- channels protected against pressure overload-induced dial ischemia/reperfusion injury. Of note, in a second cardiac hypertrophy (Seo et al., 2014). study, no difference in infarcted area was noted between Klotho (named after the youngest of the three Fates wild-type and TRPM2-knockout mice (Miller et al., 2013) in Greek mythology who was responsible for spinning and, surprisingly, the knockout animals even showed the thread of human life) is a worse cardiac function after injury. The TRPM4 inhibitor related to b-glucuronidases. Klotho-deficient mice show 9-phenanthrol also protected rat hearts from ischemia/ premature aging (reviewed in Nabeshima, 2002). Of reperfusion damage (Wang et al., 2013b). relevance to our discussion, these mice (although they 2. Transient Receptor Potential Channels and do not show baseline cardiac abnormalities) respond to Hypertension. Hypertension is a chronic medical hemodynamic stress by developing exaggerated patho- condition in which the pressure in the arteries is logic cardiac hypertrophy (Xie et al., 2012). This elevated (currently defined as .140/90 mm Hg). hypertrophy is absent in the TRPC6(2/2) animals. By Hypertension affects one in four American adults. contrast, transgenic mice with heart-specific TRPC6 Most hypertension cases are “essential” (as opposed to overexpression develop spontaneous cardiac hypertro- secondary hypertension in which a well defined phy. Finally, soluble Klotho inhibits TRPC6-mediated hypertensive factor such as renovascular occlusion currents in cardiomyocytes. Taken together, these can be identified) and may be attributed to an findings imply that TRPC6 mediates the cardioprotec- unhealthy lifestyle. Indeed, lifestyle changes (e.g., tive action of Klotho and thus strengthen the case for restriction of salt intake and increased physical targeting TRPC6 in patients with cardiac hypertrophy. activity) may restore normal blood pressure is some TRPM4 (Guinamard et al., 2006), TRPV1 (Thilo individuals. In a much simplified manner, blood et al., 2010), and TRPV2 (Iwata et al., 2013) may also pressure is determined by the strength of the heart be involved in the development of cardiac hypertrophy. stroke, the volume of the circulating blood, and the In spontaneously hypertensive rats, increased levels of elasticity (diameter) of the blood vessels. Indeed, TRPM4 were reported (Guinamard et al., 2006). b-blockers (to weaken heart stroke), (to Interestingly, TRPM4-null mice are also hypertensive reduce blood volume), and angiotensin-converting secondary to increased catecholamine secretion enzyme inhibitors (vessel relaxants) are all clinically (Mathar et al., 2010). TRPV1 was likewise increased useful antihypertensive drugs. Below we focus on TRP in the heart of mice suffering from heart failure secondary channels that are implicated in setting the vascular to hypertrophic cardiomyopathy (Thilo et al., 2010). tone by regulating the activity (constriction state) of Cardiac TRPV1 mRNA in these animals showed good vascular smooth muscle cells (Fig. 39). correlation to ventricular thickness. The TRPV1(2/2) Six members of the mammalian TRPC family are animals revealed less cardiac hypertrophy compared with expressed in vascular smooth muscle cells (see 758 Nilius and Szallasi

Watanabe et al., 2013). In freshly isolated rabbit disease. Despite decades of intensive research, the mesenteric artery smooth muscle cells, TRPC1 and pathogenesis of atherosclerosis remains only partially TRPC6 show activation in response to angiotensin II understood. The prevalent theory postulates a central exposure (Saleh et al., 2006). Another vasoactive role for endothelial damage (possibly initiated in some substance implicated in the pathobiology of essential patients by sustained, low-grade inflammation) that hypertension is endothelin-1. Both silencing of the paves the way for plaque (lipid, calcium, and collagen TRPC1 gene and an antibody directed against the deposition in the intima) formation. These plaques may TRPC1 protein were shown to reduce arterial smooth either limit blood flow (e.g., peripheral vascular muscle contraction evoked by endothelin-1 (Bergdahl disease) or dislodge and travel (embolize) to distant et al., 2003). In TRPC6-null mice, increased vascular locations. An example of the latter phenomenon with smooth muscle contractility was demonstrated (Dietrich potentially devastating consequences is ischemic stroke et al., 2005b). caused by carotid artery disease. Smoking, hyperten- Vascular smooth muscle cells also express four of the sion, and hypercholesterolemia are all known risk six members of the vanilloid family of TRP channels factors for developing atherosclerosis, probably by (TRPV1–TRPV4) as well as all TRPM family members, damaging endothelial cells one way or another. Mono- with the exception of TRPM1 (Yang et al., 2006; cytes then adhere to the site of endothelial damage Earley, 2010). Capsaicin was reported to constrict and penetrate into the intima of the vessel. Finally, smooth muscle cells isolated from the rat gracilis vascular smooth muscle cells get recruited, after a arterioles via TRPV1 (Kark et al., 2008). The signifi- phenotypic switch from contractile to proliferative. cance of this finding is unclear since TRPV1 knockout TRP channels have been implicated in the major steps animals have no relevant phenotype and capsaicin (endothelial damage/dysfunction, monocyte adhesion/ actually prevents the development of hypertension in penetration, and vascular smooth muscle phenotypic rats kept on a high-salt diet (Hao et al., 2011). Dietary switch) leading to atherosclerosis (reviewed in Kwan capsaicin also delays the onset of stroke in spontane- et al., 2007; Liu et al., 2008; Tano et al., 2012). ously hypertensive rats (Xu et al., 2011). Anecdotal Ossabaw island pigs (established from feral swine evidence suggests a beneficial (blood pressure- who had adapted to the unique conditions in this island lowering) effect for dietary capsaicin consumption in on the Georgia coast) have insular dwarfism and humans (www.sciencedaily.com/releases/2010/08/ develop early onset T2DM and atherosclerosis when 100803132734.htm). fed an atherogenic diet (Etherton, 1980; Sturek et al., The case for TRPV4 in blood pressure regulation is 2007; Neeb et al., 2010). They also show high TRPC1, much stronger. In cerebral artery smooth muscle cells, TRPC3, TRPC5, and TRPC6 expression (compared activation of TRPV4 by endothelium-derived arachi- with lean pigs) that is proportional to the increase in donic acid metabolites (e.g., 11,12-epoxyeicosatrienoic serum levels (Hu et al., 2009a). Strikingly, acid) causes hyperpolarization and resultant vaso- physical exercise reverses this increase in TRPC1 relaxation (Earley et al., 2005). This effect is clearly mRNA and protein levels (Edwards et al., 2010). This mediated by TRPV4 because it was absent in the is the first (and thus far only) example of a TRP TRPV4 knockout mice (Earley et al., 2009). If so, channel being regulated by physical activity. TRPC1/TRPC6 and TRPV4 exert opposite effects on Of TRP channels expressed in endothelial cells vascular tone (constriction versus relaxation). (TRPC1, TRPC3–TRPC7, TRPV1, TRPV4, and TRPM7), The recognition of the important role that Mg2+ plays TRPC3 has attracted the most attention as a poten- in hypertension (in the clinics, intravenous tial therapeutic target in atherosclerosis based on its was used to reduce blood pressure in patients with abundance (relative to other TRP channels) in coronary severe hypertension) has brought TRPM6 and TRPM7 artery endothelial cells of human origin (HCAECs) (see (two TRP channels involved in Mg2+ homeostasis) into Smedlund et al., 2012). Unfortunately, results obtained attention, although to date there is no convincing with HCAECs should be interpreted with caution since evidence to implicate TRM6 and/or TRPM7 in the TRP channel expression in these cells varies dramat- pathobiology of high blood pressure (see Watanabe ically with passage number and assay conditions. et al., 2013). As mentioned above, TRPM4-null mice Parenthetically, similar considerations apply to other develop hypertension secondary to increased catechol- cultured cells such as urothelial cells and odontoblasts amine secretion (Mathar et al., 2010) and TRPM4 has in which conflicting TRP channels expressions were been implicated in the Bayliss effect (see Vennekens, reported by different laboratories. Since TRPC3 is 2011). believed to be constitutively active, its upregulated 3. Transient Receptor Potential Channels and expression may have profound effects on Ca2+ homeo- Atherosclerosis. Atherosclerosis, defined as the nar- stasis (see Vazquez et al., 2004). Oxidative stress may rowing of the lumen of an artery due to lipid and provide an important contribution to endothelial collagen deposition, is the leading cause of coronary damage. Using a dominant negative N-terminal frag- artery disease/AMI, stroke, and peripheral vascular ment of TRPC3, preliminary evidence was obtained TRP Channels as Drug Targets 759 that TRPC3 (maybe in combination with TRPC4) may diet, atherosclerotic plaques occurred later and were be part of the redox-sensitive channel that mediates smaller with a reduced necrotic core compared with oxidative stress in endothelial cells (Balzer et al., control subjects (Tano et al., 2014). 1999). In HCAECs, proinflammatory mediators (e.g., Taken together, these findings imply that TRPC3 ATP) induce the expression of VCAM-1 that, in turn, may be upregulated in monocytes of patients with risk attracts leukocytes to adhere to the endothelium factors for atherosclerosis (e.g., essential hypertension (Smedlund et al., 2010). There is emerging evidence and/or T2DM) and this upregulated TRPC3 expression that TRPC3 is an obligatory component of the may promote monocyte migration/foam cell formation signaling cascade that connects the P2Y2 receptor and, at the same time, block efferocytosis. The (that recognizes ATP) to VCAM-1 (see Smedlund et al., potential role of TRPV2 and TRPM2 in monocyte 2012). A role for TRPC3 in endothelial NO release was functions is mentioned elsewhere (section III.B.2). also postulated (Huang et al., 2011). Of note, TRPC1 A phenotypic switch between the contractile and was implicated in mediating the endothelial damage proliferative phenotype of vascular smooth muscle cells caused by oxidized phospholipids but the relevance is believed to contribute to the narrowing of the of this observation was questioned by the down- vascular lumen during atherosclerosis. This molecular regulation of TRPC1 to almost undetectable levels in switch is a potential therapeutic target. Angiotensin II HCAECs under proinflammatory conditions (reviewed induces vascular smooth muscle proliferation via in Smedlund et al., 2012). TRPC1 activation (Saleh et al., 2006). In a vascular The increasing recognition of atherosclerosis as injury model, TRPC1 appeared to mediate the switch a low-grade, maladaptive inflammatory disease has from the contractile to the proliferative phenotype brought attention to monocytes/macrophages as poten- (Golovina et al., 2001; Kumar et al., 2006). This is in tial therapeutic targets. Atherosclerotic lesions are keeping with the postulated role of TRPC1 in vascular now believed to have a microenvironment that either graft restenosis (Kumar et al., 2006), maladaptive promotes healing or, conversely, accelerates lesion cardiac hypertrophy (Ohba et al., 2007), and IPAH growth and plaque instability. A decisive lesional (Zhang et al., 2007). More recently, the TRPC3 factor may be efferocytosis, the ability of resident antagonist Pyr3 was reported to inhibit smooth muscle macrophages to clear pathogenic monocytes. There is proliferation and prevent stent-induced remodeling preliminary evidence to implicate TRPC3 in efferocy- (Koenig et al., 2013). tosis (“burying the dead cells”). Bone marrow–derived Other potential TRP channels that may operate this macrophages obtained from TRPC3(2/2) mice showed phenotypic switch include TRPM3, TRPM7, TRPV1, decreased survival and impaired efferocytosis (Tano and TRPV4 (Yang et al., 2006). TRPM3 is present both et al., 2011, 2014). If so, TRPC3 may play an important in proliferating and contractile vascular smooth muscle role not only in disease initiation (endothelial damage) cells with low-level constitutive activity (Naylor et al., but also in disease progression (see Tano et al., 2012). 2010). In freshly isolated aorta smooth muscle cells, Accumulation of apolipoprotein (Apo) B–containing activation of TRPM3 stimulated contractile responses. lipoproteins in the subintima is thought to be a major Loading the experimental system with cholesterol to triggering event in atherosclerosis. These lipoproteins generate foam cells suppressed TRPM3 activity to attract monocytes from the circulation that, in turn, undetectable levels. Smooth muscle cells isolated from differentiate into lesional foam cells. The atheroscle- the ascending aorta also express functional TRPM7 rotic plaque will progress if the formation of foam cells channels (Baldoli et al., 2013). In these cells, angio- (set by monocyte influx and differentiation into macro- tensin II increased TRPM7 expression via the Pyk2- phages) outpaces their clearance (due to apoptosis and ERK1/2-Elk1 signaling pathway (Inoue and Xiong, efferocytosis). TRPC3 was found to be upregulated in 2009). TRPM7 silencing with siRNA attenuated cell monocytes isolated from hypertensive rats compared proliferation after angiotensin II administration. As with normotensive control subjects (Zhao et al., 2012b). expected, TRPM7 silencing also mimicked the effects of Importantly, a similar increase in TRPC3 was detected Mg2+ deficiency on human microvascular endothelial in monocytes obtained from the blood of patients with cells (HMVECs) (Baldoli and Maier, 2012). Pulmonary essential hypertension and/or T2DM. When challenged artery smooth muscle cells also express TRPV1 and with the chemotactic peptide formyl-Met-Leu-Phe, TRPV4. TRPV1 and TRPV4 agonists (capsaicin and these monocytes showed accelerated in vitro migration 4a-PDD, respectively) induced migratory responses in in correlation with their TRPC3 expression. Further- these cells in a manner that was inhibited by both more, monocytic TRPC3 mRNA strongly correlated capsazepine (a first-generation TRPV1 antagonist) with the IL-1 and TNF transcripts. Finally, monocytes (Wang et al., 2008a) and the TRPV4 blocker HC- isolated from TRPC3(2/2) animals exhibited im- 067047 (Martin et al., 2012). paired efferocytotic activity. Of note, TRPV1 was implicated as the target In ApoE-deficient mice transplanted with the bone mediating the protective effect of evodiamine on the marrow of TRPC3-null animals and kept on a high-fat development of atherosclerosis (Wei et al., 2013). In 760 Nilius and Szallasi

ApoE knockout mice, long-term administration of synthesize and deposit extracellular matrix proteins. evodiamine attenuates hyperlipidemia, reduces the There is increasing evidence (summarized in Fig. 33) size of atherosclerotic lesions, and alleviates fatty liver that TRP channels are involved in the activation and disease. Evodiamine was shown to function as a TRPV1 differentiation of cardiac fibroblasts (see Yue et al., agonist (Pearce et al., 2004). Indeed, genetic deletion of 2013). This is important because existing therapeutic TRPV1 abrogated the protective effect of evodiamine options to halt (or reverse) cardiac fibrosis are against atherosclerosis. Interestingly, double-knockout unsatisfactory. (ApoE/TRPV1-null) mice showed severe atherosclero- There are at least three major signaling pathways sis and fatty liver disease (Wei et al., 2013). involved in the biochemical cascade of fibrogenesis, Endothelial damage also stimulates the formation of namely the renin-angiotensin system, the TGFb thrombi. There is a growing body of evidence support- signaling pathway, and the oxidative stress pathway ing a role for abnormal Ca2+ signaling by TRP channels (Fig. 33). Cultured human cardiac fibroblasts express in the pathogenesis of platelet dysfunction (reviewed in a number of TRP channels, including TRPC1, TRPC4, Dionisio et al., 2012; Mahaut-Smith, 2013). Indeed, TRPC6, TRPV2, and TRPV4, as well as TRPM7 TRPC6 knockout mice showed prolonged bleeding time (Nishida et al., 2007; Rose et al., 2007; Du et al., after tail injury, as well as an increased time for 2010). In addition, mouse cardiac fibroblasts also occlusion in the carotid artery injury thrombosis model express TRPC3, TRPM4, and TRPM6. This expression (Paez Espinoza et al., 2012). These findings suggest pattern is based on RT-PCR experiments and many that TRPC6 may be a new therapeutic target for observations lack functional correlate. However, there managing thrombotic disorders. Elevated TRPC3 is indirect evidence that cardiac fibroblasts express (Zbidi et al., 2009) and TRPC6 (Liu et al., 2008) abundant functional TRPC3 and TRPM7 channels. It expression was reported in isolated from the was speculated that TRPM7 is a target for C-type blood of patients with T2DM. Apparently, this effect is natriuretic peptide (Rose et al., 2007). TRPM7 is also secondary to the increase in blood glucose and is a downstream target for the TGFb signaling pathway mediated by the PI3K pathway. (Du et al., 2010). Indeed, knockdown of TRPM7 by As an example of puzzling (and troubling) species- shRNA inhibits TGFb-induced fibroblast proliferation related differences in TRP channel expression, func- and collagen production (Du et al., 2010). Conversely, tional TRPV1 was detected in human, but not in TRPM7 is upregulated in TGFb-stimulated cardiac mouse, platelets (Sage et al., 2013). Capsaicin evoked fibroblasts. 5-hydroxytryptamine release from human platelets in From cardiac fibroblasts, TRPM2-like and TRPV4- a manner that was blocked by the TRPV1 antagonist like currents were also recorded. The TRPM2-like 5-iodoresiniferatoxin (I-RTX) (Harper et al., 2009). It current was evoked by hypoxia and was eliminated in was speculated that TRPV1 might be a useful target to the presence of gene knockdown by TRPM2 siRNA explore in patients with unstable coronary plaques (Li (Takahashi et al., 2012). On the basis of these and Huang, 2011). observations, it was speculated that TRPM2 may be 4. Transient Receptor Potential Channels and Car- involved in the pathogenesis of hypoxia (ischemia)- diac Fibrosis. Cardiac fibrosis is not a distinct entity; induced cardiac fibrosis (reviewed in Yue et al., 2013). rather, it is a common pathologic pathway for various Using the TRPV4 agonist 4a-PDD, a TRPV4-like diseases (e.g., AMI and chronic ischemic heart disease, current can be measured in fibroblasts (Hatano et al., inflammatory and hypertrophic cardiomyopathies, and 2009). This is interesting because TRPV4 may be aging-associated heart disease) that damages the activated by mechanical stretch, a known etiological myocardium (reviewed in Yue et al., 2013). The factor in the pathogenesis of cardiac fibrosis. damaged myocardium is then replaced by connective tissue synthesized by fibroblasts and myofibroblasts. E. Transient Receptor Potential Channels in Obesity This is a problem because cardiac fibrosis interferes and Metabolic Disorders with electrical conduction and impairs the pumping In the United States, the prevalence of diabetes has function of the heart. Cardiac fibrosis is subdivided reached epidemic proportions. Almost 26 million into reparative (e.g., after AMI) and reactive types, Americans (7% of the population) have diabetes and although this distinction is somewhat arbitrary. the prevalence of T2DM exceeds 20% in elderly Fibroblasts are abundant and quiescent in the individuals. Furthermore, an estimated 65% of Amer- normal heart. By contrast, myofibroblasts are rare in ican adults are overweight or obese. The prevalence of the healthy heart but become enriched during disease. obesity is approaching 20% in the adolescent popula- In response to pathogenic stimuli (e.g., myocardial tion, which is even more worrisome. The Healthy injury, oxidative stress, and/or mechanical stretch), People 2010 Goal aimed at an obesity prevalence of fibroblasts get activated and a subset differentiates 15%; as yet, no state has met this target. into myofibroblasts (see Yue et al., 2013). In turn, Obesity is an increasingly significant medical prob- activated fibroblasts and myofibroblasts excessively lem in the developing world as well. For example, an TRP Channels as Drug Targets 761 estimated 40% of individuals living in urban areas in easier said than done. Sadly, compulsive overeating India are overweight or obese. This is important (popularly referred to as food addiction) can be difficult because this rampant obesity is thought to bear at to overcome without the help of therapeutic interven- least partial responsibility for the rapidly increasing tion. Despite breakthrough discoveries in our under- prevalence of T2DM as well as heart disease and standing of the control of ingestive behavior (e.g., the possibly cancer worldwide. In women, being obese is role of leptins and their receptors), the success of drug a well established risk factor for breast and endome- therapy for obesity to date has been modest at best, trioid adenocarcinoma. beset with problems associated with adverse effects. A The combined cost of obesity and diabetes is stagger- recent disappointment was the cannabinoid receptor 1 ing. In 2010, the United States spent an estimated $146 (CB1) inverse agonist rimonabant that caused de- billion on diabetes- and obesity-linked illnesses, ac- pression and suicidal thoughts in many patients counting for approximately 6% of the $2.6 trillion (reviewed in Di Marzo and Szallasi, 2008; Di Marzo healthcare expenditure. Obese Americans spend $1400 et al., 2008). more annually on health care than their normal weight 1. Therapeutic Intervention for Appetite Control and compatriots, and the estimated lifetime cost to Medicare Weight Loss: Central versus Peripheral Transient of treating obesity-related diseases exceeds $160,000. If Receptor Potential Targets. the current trend continues, obesity- and T2DM-related a. Vanilloid transient receptor potential 1. healthcare spending may reach $1 trillion in 10 years, Connoisseurs of hot spicy food are intimately familiar potentially bankrupting Medicare. Clearly, there is with the predominant pharmacological actions of a dire need for novel therapeutic interventions in capsaicin from personal experience: it induces profuse weight loss and blood glucose control. perspiration (known as gustatory sweating) as well as There is no shortage in speculation as to what is a hot, burning sensation in the tongue and oral mucosa responsible for the worldwide explosion in obesity that dissipates upon repeated challenge (reviewed in rates. There can be no dispute that we are living in Szallasi and Blumberg, 1999). Evolutionary selective an “obesogenic” environment. Meal and sugary drink pressure seems to have maximized the pungency of sizes have become larger and we are bombarded with capsaicin. It was speculated that the compound’s ads promoting food overconsumption (as memorialized pungency is able to deter ambulatory animals from in the documentary “Supersize Me!”). On the other side eating fruits, favoring those plants whose of the calorie equation is the reduced energy expendi- seeds were dispersed widely by birds (see Szallasi and ture due to a sedimentary lifestyle. Evolution may also Blumberg, 1999). Indeed, the avian TRPV1 receptor is bear some responsibility for this trend. At times of food not activated by capsaicin (Jordt and Julius, 2002), and shortages, slow metabolism (effective calory utiliza- hence birds are undeterred from ingesting chili pepper tion) may promote survival. When food is plentiful, fruits and can excrete the pepper seeds large distances these biochemical traits may promote rapid weight away. This forms the basis of the development of hot gain. Ossabaw island pigs (as discussed above, used as pepper–flavored “squirrel-free” bird feed (Szallasi and a model to study the potential link between TRP Blumberg, 1999). It is still a mystery, however, why the channels and atherosclerosis) are a good example of same pungency that repels squirrels is perceived as the phenomenon. These pigs have adapted to the harsh pleasurable by many human beings. environment of the island (they have a “thrifty pheno- Several lines of evidence have established a link of type”) and they gain weight rapidly when they are TRPV1 to the regulation of body weight, although the allowed to eat ad libitum. exact nature of this link (whether TRPV1 agonism or A much debated question is the role of food additives blockade protects against obesity) remains controver- in altering metabolism and promoting obesity. High- sial. There is anecdotal evidence that consumption of corn syrup and “high-intensity” non-nutritive hot, spicy food is associated with a lower prevalence of sweeteners have been used with increasing frequency obesity (see Szallasi and Blumberg, 1999). In accord, over the years, mirroring the trend in obesity. rats kept on a diet containing 0.014% capsaicin showed Paradoxically, rats fed diets containing saccharin and no change in calory intake but a significant (approx- acesulfame potassium show increased food intake, imately 30%) reduction visceral fat weight compared weight gain, and adiposity compared with control with control subjects fed standard chow (Kawada et al., subjects. It was speculated that these non-nutritive 1986). In long-term (120 days) feeding experiments, sweeteners might activate sweet taste receptors in the dietary capsaicin prevented obesity in wild-type, but gut, dissociating the chemosensory signal from caloric not TRPV1 knockout, mice assigned to a high-fat diet consequences, and setting off countercompensatory (Zhang et al., 2007b). Dietary capsaicin also sup- processes. If this also hold true in men, diet soda pressed appetite and weigh gain in rabbits (Yu et al., might not be “diet” at all. 2012). A meta-analysis of 20 trials involving 563 Although sensible lifestyle changes (a combination of participants also found a modest benefit (50 kcal per diet and exercise) can no doubt lead to weight loss, it is day increase in energy expenditure) for daily capsaicin 762 Nilius and Szallasi consumption (Ludy et al., 2012). This forms the neonatal capsaicin treatment of TRPV1-expressing experimental foundation of using Cheyenne pepper nerves yielded conflicting reports (reviewed in Szallasi capsules as dietary supplements for weight manage- and Blumberg, 1999). Some authors (e.g., van de Wall ment. It was speculated that capsaicin may suppress et al., 2006) found no change in food consumption, body appetite and increase energy expenditure. Indeed, weight, and energy balance in the capsaicin-treated capsaicin was reported to increase O2 consumption animals (attributed to compensatory mechanisms), and thermogenesis in rat skeletal muscle (Cameron- whereas others described lower weight predominantly Smith et al., 1990). In healthy volunteers, dietary due to atrophied BAT and a marked (approximately capsaicin boosted thermogenesis by 50% with no effect 40%) decrease in thermogenic response to infused on satiety (Clegg et al., 2013). Interestingly, non- after neonatal capsaicin administra- pungent capsaicin analogs (e.g., evodiamine from the tion (Cui et al., 1990). Adding to the confusion, fruit of Evodia rutaecarpa; see Fig. 5 for structure) also capsaicin-treated animals did not lose weight after boosted energy consumption and prevented weight weight loss surgery (intragastric implantation of gain both in mice kept on a high-fat diet (Kobayashi silicone bubbles) (Northway et al., 1992). On the basis et al., 2001) and in human volunteers (Snitker et al., of the latter finding, it was postulated that TRPV1 is 2009). an important mediator of satiety. Indeed, N-OEA, Rats with chemically ablated (by high-dose systemic a fatty acid metabolite that suppresses appetite (see capsaicin) sensory neurons overeat when fed chow Thabuis et al., 2008; Sarro-Ramirez et al., 2013), supplemented with glucose; however, on a fat- functions as a TRPV1 agonist (Ahern, 2003; Almási containing diet, they consume amounts similar to et al., 2008). controls (Ritter and Taylor, 1989) after an initial A recent study found no difference in weight gain overconsumption (Chavez et al., 1997). Somewhat between TRPV1 knockout and wild-type mice kept on paradoxically, when kept on a high-fat diet, TRPV1 a high-fat diet (Marshall et al., 2013). These seemingly knockout mice accumulate less visceral (intra- contradictory findings may be reconciled by a recent abdominal) adipose tissue than their wild-type litter- study in which the phenotype of TRPV1 knockout mates (Figs. 34 and 35) despite the equivalent food mouse was 1) dependent on the age of the animals and consumption and dietary lipid uptake (Motter and 2) was also heavily influenced by environmental Ahern, 2008). These animals also appear to be variables (diet, room temperature, etc) that can impact protected from fatty liver disease (Fig. 35). If so, thermoregulation (Wanner et al., 2011). For instance, TRPV1 antagonists may represent a novel strategy young TRPV1 knockout mice exhibited increased for intervention in patients with nonalcoholic steato- locomotor activity, which could account for increased hepatitis. Unfortunately, recent articles present a very energy expenditure and protect against gaining different picture. Dietary capsaicin was found to weight. As the genetically TRPV1-deficient mice age, increase hepatic uncoupling protein (UCP)-2 expres- their motor activity declines and body weight concom- sion and reduce fat accumulation in hepatocytes in itantly increases. Parenthetically, aging also seems to wild-type, but not in TRPV1-null, mice (Li et al., 2012, reverse the role of TRPV1 in systemic inflammation 2013). Activation of TRPV1 by dietary capsaicin in from anti-inflammatory to proinflammatory (Wanner hepatocytes also improved the liver function tests et al., 2012). If these observations hold true in humans, (Tani et al., 2004; Li et al., 2013). young individuals could benefit from TRPV1 blockers It was suggested that the resistance to weight gain for weight management, whereas older individuals in TRPV1 knockout mice was due to an increased may want to supplement their diet with TRPV1 thermogenic capacity. In rodents, chemical ablation by agonists. Of note, the loss-of-function TRPV1 variant V585I appears to confer some protection against diet- induced obesity (Zhang et al., 2007b). An interesting (and controversial) hypothesis postu- lates a central role for TRPV1- expressing adipocytes in regulating body weight and questions the feasibility of targeting TRPV1 for weight loss in individuals who are already obese (see Saito and Yoneshiro, 2013). By a combination of immunoblotting and RT-PCR, TRPV1 was detected in the visceral adipose tissue of mice and humans, with decreased expression in both obese humans and mice (ob/ob and db/db) relative to lean control subjects (Zhang et al., 2007b). In mice on a high-fat or high- diet, supplementation of food Fig. 34. TRPV1-null mice stay lean on a high-fat diet compared with their wild-type (WT) littermates. Reprinted with permission from Motter with the TRPV1 agonist monoacylglycerol increased and Ahern (2008). mitochondrial UCP1 expression in BAT and prevented TRP Channels as Drug Targets 763

Fig. 35. Compared with their wild-type (WT) littermates, TRPV1-null mice accumulate less intra-abdominal (A and B) and subcutaneous fat (B) and are protected from obesity-induced fatty liver disease (C and D). Reprinted with permission from Motter and Ahern (2008). white fat accumulation (Iwasaki et al., 2011). In with CGRP, and mice whose NK1Rs were deleted by preadipocyte 3T3-L1 cells, a model of adipocyte genetic recombination show protection against diet- differentiation, capsaicin evoked Ca2+ influx and induced weight gain comparable to that seen in the prevented adipogenesis (Zhang et al., 2007b). Con- CGRP knockout animals (Karagiannides et al., 2011). versely, knockdown of TRPV1 by RNA interference This protective effect was replicated by NK1R antago- restored adipogenesis. Capsaicin responses were at- nists (Karagiannides et al., 2008; Ramalho et al., 2013). tenuated in fat cells isolated from obese individuals It is worth mentioning here that obesity is a recognized (Zhang et al., 2007b). Combined, these results suggest risk factor for migraine and it was speculated that that capsaicin may prevent weight gain via UCP1 CGRP may be the molecular link between these two upregulation in subjects with normal body weight, but disorders (Recober and Goadsby, 2010). this beneficial effect may be lost in obese individuals An interesting (and somewhat neglected) weight due to down-regulation/loss of TRPV1 receptors on management strategy is the use TRPV1 agonists to adipocytes. reduce energy consumption by altering (speeding up) In rodents, BAT is a major determinant of energy gastrointestinal motility. Because the rate of gastric expenditure through thermic responses. BAT is rich in emptying directly impacts nutrient uptake, facilitating sensory afferents. Chemical ablation by capsaicin of gastric motility by pharmacologic agents could have TRPV1-expressing afferents renders young rats re- a positive impact on satiety. Indeed, there is good sistant to age-related obesity (Melnyk and Himms- evidence that capsaicin can increase the rate of gastric Hagen, 1995). Of note, a major neuronal mediator of motility in humans (Gonzalez et al., 1998; Debreceni this effect on BAT appears to be CGRP. CGRP was et al., 1999). This also implies a therapeutic potential shown to stimulate thermogenesis in BAT, and for TRPV1 agonists in the pharmacotherapy of gastro- disruption of CGRP gene in mice resulted in protection paresis (Barquist et al., 1996). against the metabolic effects and weight gain caused b. Vanilloid transient receptor potential 4. A pivotal by a high-fat diet (Walker et al., 2010). As discussed connection was recently established between TRPV4 below, CGRP has also been linked to T2DM, a frequent and UCP1 (also known as thermogenin), a key modu- complication of obesity. For example, in the Zucker rat, lator of nonshivering thermogenesis in BAT (Ye et al., elevated circulating CGRP levels were detected prior to 2012). In BAT, UCP1 is constitutively active. Mito- the onset of obesity and insulin resistance, presumably chondrial UCPs divert electrons to minimize ROS as a result of increased sensory neuron activity (Gram production and generate heat. The naked mole rat, et al., 2005). CGRP is, however, probably not the only which lives in subterranean burrows with constant important player in the sensory neuron-adipose tissue ambient temperature and therefore has little need for interaction. Capsaicin-sensitive nerves coexpress SP heat generation, has a unique UCP1 that maintains 764 Nilius and Szallasi

ATP synthesis with essentially no ROS production (reviewed in Rodriguez et al., 2011; Lewis et al., 2013). These rats are the longest-living rodents (up to 32 years), at least when kept at the zoo (zoo.sandiegozoo. org/animals/naked-mole-rat). Because adult humans have virtually no BAT, until recently, the role of UCP1 in body weight regulation was unclear (Kozak and Annunciado-Koza, 2008). In 2012, “beige adipocytes” (UCP1-positive fat cells) were demonstrated in human white adipose as the thermogenic equivalent of murine BAT (Wu et al., 2012). The white to beige adipose tissue molecular switch is operated by irisin (Boström et al., 2012), the “magic exercise hormone” encoded by the “human exercise gene” (Kelly, 2012; Timmons et al., 2012). During strenuous physical exercise, skeletal muscle produces irisin (via AMP kinase) as the membrane cleavage product of FNDC5. In turn, Fig. 36. Schematic illustration of the participation of TRPV4 channels in irisin facilitates thermogenesis by upregulating UCP1 obesity. See the text for details. Courtesy of Drs. Li Ye and Bruce and thereby transforming white fat cells into beige Spiegelman. (reviewed in Elbelt et al., 2013). Of note, irisin is already popularized as a “miracle weight loss pill” Ye et al., 2012). The TRPV4 antagonist GSK205 (www.mensfitness.com/nutrition/supplements/miracle- changed the phenotype of 3T3 F442A adipocytes (by weight-loss-pill-irisin-allows-for-easy-workouts). upregulating factors that increase thermal responsive- The transcription of UCP1 is under the control of ness) and improved glucose tolerance in healthy mice. peroxisome proliferator–activated receptor-g coactiva- Moreover, the TRPV4 knockout mice were protected tor 1a (PGC1a), originally identified as a cold-inducible from obesity when kept on a high-fat diet despite the factor in BAT. PGC1a was postulated to play a pivotal similar food intake and physical activity compared role both in obesity and T2DM (reviewed in Attie and with their wild-type littermates. Importantly, GSK205 Kendziorski, 2003). Indeed, PGC1a gene polymor- increased both PGC1a and UCP1 activity in obese phism has been linked to T2DM in Asian Indians mice. Unfortunately, a subsequent independent study (Vimaleswaran et al., 2005). PGC1a activators improve reported the opposite phenotype for TRPV4 knockout exercise endurance and promote weight loss in mice mice. In this study, the knockout mice became obese on (Luo et al., 2012b). The UCP1 gene has a peroxisome a high-fat diet and, even worse, developed severe OA proliferator–activated receptor-g (PPARg) responsive (O’Conor et al., 2013). Parenthetically, in a different element in the enhancer region (Barbera et al., 2001). study, bone mass was increased by approximately 20% PPARg is a known regulator of adipogenesis. The in male TRPV4-null mice compared with sex-matched knockout mice do not deposit adipose tissue even if wild-type animals (van der Eerden et al., 2013). As we kept on a high-fat diet (Jones et al., 2005b). The saw above, similarly discrepant observations were variant allele G482S of PPARgC1a confers an in- published in the TRPV1 knockouts. One might argue creased risk of obesity in elderly men (Ridderstråle that both the genetic background of the knockout et al., 2006). Targeted PGC1a overexpression upregu- animals (e.g., B6 mice are prone to weight gain, whereas lates UCP1 and protects against obesity (see Liang and B129 animals are not) and uncontrolled environmental Ward, 2006). Conversely, PGC1a knockout mice gain factors can influence the apparent phenotype. weight and show early exhaustion during physical c. Ankyrin transient receptor potential 1. In mice, exercise. In aging mice, white adipose tissue (WAT) is the TRPA1 agonist cinnamaldehyde was reported to redistributed from the subcutis to the viscera with increase UCP1 expression in BAT (Tamura et al., a concomitant loss of UCP1 (Rogers and Smith, 2012). 2012). Cinnamaldehyde also reduced visceral adipose This phenomenon might explain why older individuals tissue deposition in mice fed a high-fat or high-sucrose find it difficult to lose weight and are prone to develop diet although it had no effect on total calorie intake. If insulin resistance (see Rogers and Smith, 2012). Loss- so, TRPA1 agonists may promote beige fat formation of-function polymorphism in the PGC1a gene was and increased energy utilization in humans. The recently linked to obesity and T2DM (Oberkofler TRPA1 agonist methyl syringate (a pungent ingredient et al., 2004). Mitofusin 2 is the downstream target of in the castor oil tree Kalopanax) was reported to PGC1a. Taken together, these findings highlight suppress food intake and gastric emptying (Kim et al., PGC1a as a promising target for weight control. 2013). TRPA1 also appears to be expressed on enter- Most recently, a chemical screen identified TRPV4 as ochromaffin cells that contain CCK (Nozawa et al., a negative modulator of PGC1a in adipocytes (Fig. 36; 2009). Upon TRPA1 activation, these cells may release TRP Channels as Drug Targets 765

CCK that, in turn, may accelerate gastric emptying content. This is important because regular sodas are and promote satiety. In vitro, TRPA is activated by thought to be a main cause of obesity and, as discussed PUFAs (Motter and Ahern, 2012). In feeding studies, above, diet sodas may cause paradoxical weight gain. the wild-type animals consumed much less PUFA- TRPM5 is also expressed in a well defined subset of containing chow than did TRPA1-null mice. The typical enteroendocrine cells, the so-called L cells (Kokrashvili US diet contains approximately 8% calorie-equivalent et al., 2009). These cells secrete the incretin hormone PUFA. In mice, prenatal PUFA exposure leads to adult GLP-1 that, in turn, influences satiety via three obesity. On the basis of these studies, one might mechanisms: 1) it suppresses food consumption; 2) it speculate that TRPA1 agonists can induce taste facilitates gastric emptying, and 3) it promotes insulin aversion to PUFAs. In relation to dietary TRPA1 release in response to glucose (reviewed in De Silva agonist consumption (although not obesity, the topic and Bloom, 2012). Moreover, in response to glucose, of our discussion), it was suggested that TRPA1 TRPM5-expressing L cells release peptide YY, which is agonism promotes longevity (at least in the worm, believed to suppress appetite in humans. Some gastric C. elegans) (Xiao et al., 2013), leading to the headline and duodenal L cells in humans also coexpress TRPM5 “Age Slowly by Eating Sushi Outside!” (www.express. with gustducin (see Liman, 2010). Gustducin functions co.uk/news/health/378380/age-slowly-by-eating-sushi- as a bitter taste receptor in the tongue. The biologic outside). function of gustducin in gastric and duodenal L cells is d. Melastatin transient receptor potential 5. The key unclear, but it was hypothesized that these TRPM5+/ role of TRPM5 in taste signaling is well documented gustducin+ cells protect the body from toxins by (reviewed in Sprous and Palmer, 2010; Medler, 2011; evoking vomitus when stimulated. If this model holds Palmer and Lunn, 2013). Generally speaking, the true, it might be a fine line to synthesize such TRPM5 “taste” sensation arises from recognition of a “tasty” agonists that control appetite by releasing GLP-1 and ligand by a cognate taste receptor. These taste peptide YY but do not cause vomiting as an on-target receptors are expressed in highly specialized sensory side effect. Perhaps surprisingly, the number of cells in the tongue. TRPM5 is a downstream target in TRPM5+ cells is increased in the stomach of obese type 2 taste receptor cells for GPCR cognate taste individuals (Widmayer et al., 2012). It is unclear receptors that mediate the basic taste sensations of whether this is a disease-causing or compensatory sweet, bitter, and (savory) (see Sprous and mechanism. Palmer, 2010; Palmer and Lunn, 2013). Indeed, genetic Most people would consider fat as tasteless; yet ablation of TRPM5 severely impairs the ability of mice TRPM5-deficient mice lose their ability to distinguish to detect sweet, umami, and bitter, as well as “fat” (Liu LA-containing solutions from physiologic saline (Liu et al., 2011a), taste (Zhang et al., 2003). When et al., 2011a). Moreover, TRPM5 mediates CCK release stimulated, these taste receptors initiate a second- from enterochromaffin cells (I cells) in small intestinal messenger signaling cascade that culminates in the tissue in response to LA (Shah et al., 2012). CCK is release of intracellular Ca2+. In turn, the elevated a peptide hormone that (as its name implies) stim- intracellular Ca2+ opens TRPM5 to allow Na+ enter the ulates gallbladder contractions. CCK also suppresses taste cells and propagate the taste signal. hunger. TRPM5 knockdown by siRNA blocks LA- A possible strategy for weight control is to make food induced CCK release by at least 60%. less palatable (less tasty) by blocking TRPM5 in taste TRPM5-like immunoreactivity was also detected in cells (reviewed in Sprous and Palmer, 2010; Palmer brush cells lining the gastrointestinal tract, particu- and Lunn, 2013). As proof of concept, TRPM5 knockout larly in the ileum and colon (Bezençon et al., 2008). mice gain less weight than their wild-type littermates Brush cells share some morphologic characteristics when kept on a carbohydrate-rich diet (Glendinning with taste cells. The functions of brush cells are not et al., 2012). In a different study, however, the TRPM5- well understood, but their highly differentiated apical deficient mice developed a robust preference for sucrose villi positioned toward the intestinal lumen suggest (probably based on the calory content since these that they detect the presence of dietary nutrients. The animals cannot taste sweet), questioning the feasibility presence of TRPM5 in brush cells was confirmed in of this approach (de Araujo et al., 2008). genetically engineered mice in which the expression of Quinine (a TRPM5 blocker) reduces weight gain in TRPM5 was coupled to green fluorescent protein to mice kept on standard chow (Cettour-Rose et al., 2013). visualize the distribution of TRPM5 protein. In brush This “antihedonistic” approach, however, may not be cells, TRPM5 seems to be colocalized with the opiates popular with the general population. Alternatively, b-endorphin and Met-enkephalin. In wild-type mice, TRPM5 may be stimulated by positive allosteric the duodenum released b-endorphin into the lumen in modulators in the hope that people will eat less from response to hyperosmotic solutions. This response was, “supersweet” desserts (see Palmer and Lunn, 2013). however, attenuated in the duodenum of TRPM Moreover, positive allosteric modulators may maintain knockout mice, indicating that TRPM5 was required the of sugary beverages with reduced calorie for optimal b-endorphin release. Opiates are known to 766 Nilius and Szallasi influence intestinal motility, thus it can be speculated macrophage chemotactic protein-1 (MCP1) that, in that TRPM5 affects nutrient processing in the small turn, attracts M1 macrophages to infiltrate the fat (see and large intestine by controlling b-endorphin and Johnson et al., 2012). M1 macrophages, in turn, secrete Met-enkephalin release. TNFa, which disrupts normal insulin signaling. In- e. Melastatin transient receptor potential 8. The deed, obese individuals have high plasma TNFa levels TRPM8 agonist menthol increases locomotor activity that precede the development of T2DM (Mishima et al., and prevents diet-induced weight gain in wild-type, 2001). Of note, inflammation induces the upregulation but not TRPM8 knockout, mice (Ma et al., 2012a). In of TRPV2 in sensory neurons (Shimosato et al., 2005). humans, the TRPM8 variant L250L has been linked to TRPV2 might also play some role in weight control truncal obesity. Menthol upregulates upstream tran- since the TRPV2 knockout mice show slightly reduced scription factor-1, although it is not clear whether this body weight (Park et al., 2011). is mediated by TRPM8. Upstream transcription factor- 3. Targeting Transient Receptor Potential Channels 1, a member of the helix-loop-helix leucine zipper in Metabolic Disorders and Diabetes. Type 1 diabetes family, is involved in the regulation of several genes mellitus (T1DM) is an autoimmune disease that that affect glucose and lipid metabolism. results from the destruction of insulin-secreting pan- f. Canonical Transient Receptor Potentials. Mature creatic b cells by autoreactive cytotoxic T cells. By adipocytes were reported to express TRPC1 and contrast, T2DM is a consequence of insulin resistance TRPC5 in a way so that they negatively regulate (cells do not respond to insulin properly even if it is adiponectin (Sukumar et al., 2012). Adiponectin is present at normal plasma levels) and is closely a “fat hormone” that is believed to play an important associated with obesity and a sedentary lifestyle. Of role in the pathogenesis of atherosclerosis. Indeed, note, b cells will try to compensate for this insulin plasma levels of adinectin are inversely correlated resistance by increasing insulin secretion (compensa- with body fat. Transgenic mice overexpressing tory hyperinsulinemia) that may, eventually, lead to b adiponectin show increased energy expenditure and stay cell exhaustion (reviewed and Suri and Szallasi, 2008). lean. If TRPC5 blockade increases plasma adiponectin Thus, patients with T2DM may also develop insulin levels by eliminating the negative control, it may be dependence and become similar to those with T1DM. beneficial in patients with metabolic syndrome/T2DM The nonobese diabetic (NOD) mouse is an attractive secondary to obesity. TRPC5 channels are also overex- model to study human T1DM because it shares many pressed in a porcine model of metabolic syndrome (Hu of its salient features, including spontaneous onset and et al., 2009a). expression of disease-susceptible MHC molecules Given the rather ubiquitous presence of TRPCs in (reviewed in Suri and Szallasi, 2008). Indeed, human contractile cells, the reports of TRPC expression in and mouse class II MHC molecules, the function of gastrointestinal smooth muscle are hardly unexpected which is to present peptide antigens to CD4+ helper (reviewed in Guibert et al., 2011; Holzer, 2011a,b). T cells, share a number of genetic, structural, and Furthermore, TRPC4 was detected in the interstitial functional properties. cells of Cajal (Walker et al., 2002). These cells are In 2006, a crucial role for TRPV1-expressing sensory a part of the pacemaker apparatus of the gastrointes- afferents was demonstrated in the pathogenesis of tinal tract, responsible for regulating smooth muscle T1DM (Razavi et al., 2006). In the mouse, neurogenic contraction, and TRPC4 has been proposed as a candi- inflammation is clearly involved in maintaining islet date for the nonselective cation channel that is central inflammation (“insulitis”) and b cell death. With the to the pacemaker activity. The presence of these TRPC recently recognized importance of adipose tissue- channels within the interstitial cells suggests a poten- infiltrating lymphocytes in T2DM, it was postulated tial common control point for the cells involved in that sensory innervation of the fat may play an enteric motor neurotransmission and gastrointestinal analogous role to pancreatic afferents (see Tsui et al., tract peristalsis. 2011). This unifying neuroimmune model blurs the line 2. Obesity as a Low-Grade Inflammatory Disease: between T1DM and T2DM and places TRPV1 in Implications for Transient Receptor Potential Channel a central position in the pharmacotherapy of both Targeting. It was proposed that obesity is in fact (see Tsui et al., 2007, 2011; Suri and Szallasi, 2008). a low-grade chronic inflammatory disorder (Johnson Moreover, a number of functional TRP channels were et al., 2012). Indeed, obesity is associated with shown to be expressed on pancreatic b cells in which macrophage accumulation in adipose tissue (Weisberg they are believed to play a key role in the regulation of et al., 2003). In humans, the density of CD68+ macro- insulin secretion, opening up new avenues for the phages in subcutaneous adipose tissue correlates with development of novel antidiabetic drugs (see Uchida the body mass index. Obese mice reveal a proinflam- and Tominaga, 2011; Colsoul et al., 2011, 2013). matory gene expression profile (Xu et al., 2003). It was 4. Targeting Transient Receptor Potential Channels hypothesized that “obese” adipose tissue (secondary to in Sensory Afferents in the Pharmacotherapy of this molecular switch) starts producing the chemokine Diabetes. TRP Channels as Drug Targets 767

a. A role for vanilloid transient receptor potential 1 in T cells in the presence of mitogens, including human autoimmune diabetes. In NOD mice, TRPV1 maps to T cells (Payan et al., 1984); this effect is abolished in the the idd4 locus, a unique hypofunctional (hyposecre- presence of the NK1R blocker SR140333 [(S)1-2-[3-(3,4- tory) mutant resulting from two amino acid substi- dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin- tutes. High-dose capsaicin administered to neonatal 3-yl]ethyl-4-phenyl-l azonia-bicyclo[2.2.2]octane] (Santoni NOD mice to ablate TRPV1-expressing neurons abro- et al., 1999). These pharmacological actions are in gated islet-specific autoimmunity (and thus protected accord with the presence of mRNA for NK1R in the animals from the signs of autoimmune diabetes) human T cells (see Bost, 2004). SP was reported to be despite the infiltration of other tissues (e.g., salivary present in human T cells with upregulated expression glands) by autoreactive T cells (Razavi et al., 2006). In in the presence of HIV infection (Ho et al., 2002), other words, the absence of TRPV1 protected the which adds another layer to this complexity. pancreatic islets without eliminating the diabetogenic Unfortunately, no clear picture is emerging regard- T cell population. ing the role of TRPV1 in neuroimmune regulation After neonatal capsaicin treatment, there is a per- and dysfunction. As we saw above, genetic deletion of manent loss of SP-like immunoreactivity in sensory TRPV1 protects NOD mice against T1DM (Razavi neurons (see Szallasi and Blumberg, 1999). Since SP is et al., 2006). Likewise, capsaicin-induced depletion of a well established mediator of neurogenic inflamma- sensory afferents protects SCID mice against T cell– tion (see Szallasi and Blumberg, 1999), it can be easily mediated colitis (Gad et al., 2009). By contrast, TRPV1 visualized how loss of SP may protect pancreatic islets knockout mice show 1) enhanced Th2-biased immune from insulitis. Unexpectedly, SP administered directly responses in the airways after ovalbumin sensitization into the pancreas of newly diabetic NOD mice was (Mori et al., 2011) and 2) reveal diminished protection shown to inhibit T cell proliferative responses and against experimental autoimmune hepatitis (Hegde restore normoglycemia (Razavi et al., 2006). To et al., 2011), as well as autoimmune encephalitis reconcile these conflicting observations, it was argued (Paltser et al., 2013). In patients with MS, the that SP has a dual action on insulitis. A relatively low missense rs877610 TRPV1 SNP conferred a higher level and sustained SP release promotes neurogenic risk for progressive disease. On the basis of these pancreatitis, whereas higher, bolus-like SP doses findings, it was postulated that TRPV1 is a critical inhibit (non-neurogenic) T cell responses (see Suri disease modifier and potential therapeutic target in and Szallasi, 2008). Unfortunately, this model is at patients with MS (Paltser et al., 2013). variance with some of the literature (see below) that The participation of TRPV1 in atopic dermatitis is suggests a tonic action for SP in T cell responses. even more confusing. Although TRPV1(2/2) mice are The contribution of sensory nerve fibers to immune less susceptible to dermatitis (Bánvölgyi et al., 2005; regulation is long recognized (reviewed in Cortright Usuda et al., 2012), TRPV1 antagonists relieve only and Szallasi, 2009) although its significance is still the pruritus with no effect on the underlying in- underappreciated. Rats whose TRPV1-expressing sen- flammation in mice (Lim and Park, 2012). In humans, sory neurons were destroyed by neonatal capsaicin topical capsaicin ameliorates allokinesis (itched in- administration show reduced ability to mount a pri- duced by usually nonpruritic stimuli) in healthy mary antibody response to sheep red blood cell volunteers but not in patients with atopic eczema antigens. Of interest to this section, T cell proliferation (Weisshaar et al., 1998). A primary drawback of many in response to mitogens or IL-2 was also attenuated in of these studies is the lack of a relationship between an capsaicin-treated rats, and this effect was reversed by altered immune response and antigen specificity. SP administration (Santoni et al., 1996). The mecha- Immunomodulation at the level of antigen-specific nisms underlying this altered immunoregulation re- cellular responses is clearly more desirable than the main to be understood. A subset of thymic T cells was global dampening of the immune response. Unfortu- reported to express TRPV1 (Wenning et al., 2011); nately, manipulation by TRPV1 antagonists of neuro- therefore, the possibility that capsaicin may kill immune interactions amounts to global dampening. On lymphocytes directly via TRPV1 activation cannot be the basis of the available literature, it is impossible to discounted (Farfariello et al., 2012). Indeed, neonatal predict the net effect of TRPV1 blockade on autoim- capsaicin treatment was shown to impair T cell mune diseases. If the mouse data hold true for maturation and induce apoptosis (Santoni et al., humans, TRPV1 antagonists will improve T1DM, 2000). This possibility notwithstanding, a more likely IBD, and rheumatoid arthritis but may worsen asthma explanation is depletion by capsaicin of neuronal SP and autoimmune hepatitis. since exogenous SP is able to fully restore antibody In Ashkenazi Jews, T1DM was significantly associ- response to sheep red blood cell antigens and prevent ated with the rs222747 (M3151) genotype. Individuals capsaicin-induced apoptosis in T cells (Santoni et al., who are homozygous for this allelic TRPV1 variant 1996, 2004). Thymocytes express NK1Rs (Santoni et al., were found to have a 67% increase in the odds for 1999). Indeed, SP augments the proliferation of CD4+ developing the disease (Sadeh et al., 2013). 768 Nilius and Szallasi

b. Vanilloid transient receptor potential 1 and type 2 incorrect. WAT is not simply a fat (energy) depot; diabetes mellitus: is type 2 diabetes mellitus a low-grade rather, it is a complicated endocrine organ involved in inflammatory disorder? Given the high prevalence of the regulation of glucose and insulin homeostasis T2DM, surprisingly little is known about the etiology (reviewed in Adamczak and Wiecek, 2013). For exam- of this rampant disorder and the available pharmaco- ple, lean adipocytes secrete adiponectin, a fat hormone therapy is mostly symptomatic. In brief, T2DM is that plays an important role in physiologic glucose and a long-term disorder that results from the inability of lipid metabolism in insulin-sensitive cells. This WAT- cells to react to circulating insulin (insulin resistance). driven biochemical cascade can malfunction in the This is perceived by the body as insufficient insulin presence of nutrient excess, creating a prodiabetic production and triggers compensatory mechanisms to environment. If so, understanding the signals that secrete more insulin (hyperinsulinemia). Hyperglyce- cause this malfunction may lead to novel, disease- mia ensues when the pancreas is unable to keep up modifying pharmacological intervention in patients with the increased insulin demand. Eventually, b cells with T2DM. are exhausted and the patient becomes insulin re- In rodent models of T2DM, there is increasing sistant. Most patients with T2DM are obese, are evidence to imply an important, multifaceted role for hypertensive, and have dyslipidemia. Collectively, TRPV1 in the development and maintenance of insulin these related metabolic disorders are often referred to resistance (see Suri and Szallasi, 2008; Tsui et al., as “syndrome X.” Despite recent advances in treating 2011; Zsombok, 2013). Trpv1(2/2) mice are resistant diabetic complications (cardiovascular disease, kidney to diet-induced obesity and associated impaired glu- failure, retinopathy, neuropathy, etc), T2DM remains cose tolerance (Motter and Ahern, 2008), at least when the fifth leading cause of death in the industrialized they grow older (Wanner et al., 2011). Importantly, world. There is an increasing recognition that TRP chemical ablation by capsaicin of TRPV1-expressing channels are involved in the development of metabolic sensory afferents (Gram et al., 2007) and pharmaco- syndrome (reviewed in Colsoul et al., 2013). logical blockade of TRPV1 by antagonists such as Low-grade inflammation appears to precede the BCTC (Tanaka et al., 2011) recapitulate the phenotype onset of T2DM and is believed to play a role in the of Trpv1(2/2) mice. Of note, in a different study, development of insulin resistance (see Jin and Flavell, BCTC was proposed to keep mice lean by inhibiting 2013). Indeed, markers of chronic low-grade inflam- TRPV4 channels (Ye et al., 2012). TRPV1-positive mation (e.g., C-reactive protein [CRP]) are persistently sensory neurons possess high-affinity insulin receptors high in patients with T2DM. Many antidiabetic drugs (Baiou et al., 2007). Insulin was suggested to facilitate used in the management of patients with T2DM (e.g., the redistribution of TRPV1 from intracellular organ- the PPARg agonists rosiglitazone and pioglitazone) elles to the plasma membrane and thereby sensitize possess anti-inflammatory activity and reduce CRP the neurons to TRPV1 agonists (see Bishnoi and (Hanefeld et al., 2011). In fact, blood glucose decreases Premkumar, 2013). This may be an important molec- in tandem with CRP after pioglitazone treatment. ular mechanism underlying the development of di- In an effort to link obesity and T2DM to sensory abetic neuropathic pain (Bishnoi et al., 2011; Miura afferents and adipose inflammation, it was postulated et al., 2011). Of note, insulin-secreting 1E cells express that hypercaloric diets create a permissive milieu for functional TRPV1 (Jabin Fågelskiöld et al., 2012), T cells and bone marrow–derived macrophages to hindering the interpretation of results obtained with infiltrate the visceral fat (see Bouloumié et al., 2008). TRPV1 ligands. There is good evidence that the number of fat-resident Upon stimulation, TRPV1-expressing afferents re- macrophages positively correlates with the body mass lease SP and CGRP. The role of these neuropeptides in (Weisberg et al., 2003; Lê et al., 2011). It is not clear pain transmission and neurogenic inflammation was what attracts the macrophages in the adipose tissue detailed elsewhere. Perhaps surprisingly, adipocytes but a correlation among calprotectin, macrophage express both CGRP receptors and SP (NK1) receptors, infiltration, and obesity was reported (Catalán et al., making them amenable to sensory neuronal control 2011). Macrophage infiltration appears to be the (Karagiannides et al., 2011). Furthermore, macro- primary event that attracts the T cells to the fat phages possess a SP recognition site that is different (Morris et al., 2012). In lean adipose tissue, macro- from NK1Rs (Hartung et al., 1986). Thus, SP released phages are sparse and scattered (Weisberg et al., from adipose tissue afferents may attract macrophages 2003). By contrast, macrophages in obese individuals both directly (by interacting at macrophage SP recep- are abundant and form crown-like structures around tors) and indirectly (by facilitating the release of the large adipocytes. These fat-resident macrophages macrophage-attracting chemokines from adipocytes). can produce cytokines such as TNFa (Xu et al., 2003) Obese adipocytes have a proinflammatory phenotype: that, in turn, may disrupt the insulin signaling They secrete factors such as the MCP1 that attracts cascade, leading to insulin resistance. According to macrophages. In support of the key role that fat- this hypothesis, our traditional thinking of WAT is infiltrating macrophages play in the pathogenesis of TRP Channels as Drug Targets 769

T2DM, MCP1-deficient mice are resistant to diet- might argue that overactive sensory afferents make an induced diabetes (see Johnson et al., 2012). Indeed, early and pivotal contribution to T2DM pathobiology MCP1 is an emerging target for weight loss and T2DM by fueling islet cell inflammation (via SP release) and (see Panee, 2012). antagonizing insulin actions (via CGRP). It is not clear In pancreatic islets, CGRP was reported to inhibit how these afferents “sense” the prodiabetic environ- insulin release (Kogire et al., 1991). Conversely, after ment. One possible link is by Toll-like receptors. glucose challenge, attenuated insulin secretion was TRPV1-positive trigeminal ganglion and DRG neurons noted in the SP-deficient mice. These findings imply are known to express Toll-like receptor-4 (Diogenes that CGRP and SP may play opposing roles in the et al., 2011), which might be activated by fatty acids in pancreas, in which CGRP antagonizes insulin release the presence of nutrient excess. Another possible and SP stimulates insulin release (see Suri and connection is oxidative stress. Sensory C fiber afferents Szallasi, 2008). Indeed, persistently high circulating of the pancreas coexpress TRPV1 with TRPA1, an CGRP levels were detected in obese humans with oxidative stress sensor. As discussed above, insulin insulin resistance. The participation of SP in the itself (which is released in excess during the early pathogenesis of T2DM is, however, less clear. The phase of T2DM) may directly increase SP and CGRP phenotype of SP-null mice is confusing. These animals release from sensory nerves by interacting at neuronal are more insulin sensitive than their wild-type litter- insulin receptors. Thus, a pathogenic positive feedback mates but their glucose tolerance is paradoxically loop is created in which SP promotes insulitis and impaired (Karagiannides et al., 2011). Adding to this inflammatory mediators enhance neuropeptide complexity, SP also has a proinflammatory action in release. the pancreas. Indeed, recent evidence suggests that SP In diabetic obese (ob/ob) mice, the TRPV1 antago- is critical for the development of chronic pancreatitis nist BCTC reduced fasting glucose, triglyceride, and (reviewed in Di Sebastiano et al., 2004). insulin levels (Tanaka et al., 2011). Furthermore, The opposing effects of SP and CGRP in inflamma- BCTC increased insulin secretion in response to tory disorders (and neuroimmune interactions) are glucose in the oral glucose tolerance test. Thus, BCTC somewhat puzzling, yet not unprecedented. For exam- has a dual beneficial action: it acts both as insulin ple, in biopsies taken from the large intestine of secretagogue and sensitizer. This is important because patients with IBD, elevated SP and reduced CGRP most per os antidiabetic agents function either as levels were documented (see Engel et al., 2011a,b, insulin secretagogues or sensitizers, but not both. 2012). In accord, SP(2/2) mice were protected in Thus, TRPV1 antagonists may represent a novel class oxazolone colitis, whereas CGRP-null animals ex- of antidiabetic drugs. As an added benefit, TRPV1 hibited exaggerated inflammatory responses. These antagonists may also help patients lose weight, amelio- observations imply that after a simultaneous loss of SP rate diabetic neuropathic pain, and protect against and CGRP, the SP-null phenotype will predominate. cardiovascular complications. Of note, in preclinical TRPV1 is also elevated in the colonic mucosa of studies and phase I/II clinical trials, blood glucose patients with IBD and a positive correlation between levels remained within normal limits in the TRPV1 TRPV1 and pain was noted (Keszthelyi et al., 2013). antagonist-treated groups. Furthermore, there is good evidence that deletion of 5. Targeting Transient Receptor Potential Channels TRPV1 by genetic manipulation (Szitter et al., 2010) or in b Cells for Controlling Insulin Release. Mouse and neonatal capsaicin treatment (Kihara et al., 2003) is rat primary b cells and/or insulin-secreting cell lines protective in colitis models. Confusingly, in adult rats, express a variety of TRP channels belonging to the capsaicin treatment achieved the opposite effect: It vanilloid (TRPV2 and TRPV4), ankyrin (TRPA1), worsened experimental colitis (McCafferty et al., 1997). canonical (TRPC1, TRPC4, and TRPC6), and melastatin However, a higher incidence of colonic adenomas was (TRPM2–TRPM5) subfamilies (reviewed in Jacobson recently reported in TRPV1(2/2) mice, leading to the and Philipson, 2007; Colsoul et al., 2013). In human controversial proposal that neurogenic inflammation islets, TRP channel expression appears to be restricted is, in fact, protective against colorectal carcinogenesis, to four members of the melastatin family: TRPM2, at least in patients with IBD (Vinuesa et al., 2012). TRPM4, TRPM5, and TRPM6. We focus on these Clearly, there is a delicate (and currently only channels below, but we first briefly review the molecular partially understood) balance between TRPV1- mechanism of insulin secretion. positive sensory afferents and glucose homeostasis in Insulin secretion is a complex process driven by which a sustained, TRPV1-dependent neuropeptide electrical activity and oscillations in intracellular Ca2+ release may exert a tonic action on b cells (see Suri and concentration in pancreatic b cells (see Colsoul et al., Szallasi, 2008). Both increased and decreased neuro- 2013). The main driver of insulin secretion is plasma peptide release seem to impair glucose tolerance. glucose. Glucose enters the b cells via the high Km Because the gamut of evidence points to the therapeu- glucose transporter-2. Glucose metabolism produces tic potential of TRPV1 blockade in diabetic models, one ATP, which, in turn, closes ATP-sensitive K+ channels. 770 Nilius and Szallasi

This increases the input resistance, allowing a small clarify whether this lysosomal TRPM2 is involved in inward current, the molecular identity of which is still regulating insulin release. elusive, to generate depolarization. A complex inter- As discussed elsewhere (e.g., Miller and Zhang, play between ion channels (e.g., ATP-sensitive K+ 2011), TRPM2 is a death channel operated by ROS channels and voltage-dependent Ca2+ channels) results during oxidative stress. Apoptosis of b cells is a prom- in a unique pattern of depolarizations in which bursts inent feature of diabetes (see Rhodes, 2005). In rat of action potentials are interrupted by hyperpolarized insulinoma cells, activation of TRPM2 by H2O2 intervals. The resultant oscillatory increase in in- initiates Ca2+ influx and resultant cell death (Ishii tracellular Ca2+ levels causes exocytosis of insulin- et al., 2006). Conversely, insulinoma cells with sup- 2 containing vesicles. In principle, Cl efflux or cation pressed TRPM2 expression are protected from H2O2- influx could provide the depolarizing current that induced cell death (Lange et al., 2009). drives the b cells toward the initiation of electrical b. Melastatin transient receptor potential 4. activity. TRP channels are interesting candidates for TRPM4isabundantlyexpressedinanumberof this background depolarizing current (see Qian et al., insulinoma cell lines where it is involved in the control 2002). of insulin release (Cheng et al., 2007a). Indeed, in these a. Melastatin transient receptor potential 2. neoplastic cells, blockade of TRPM4 was shown to TRPM2 is natively expressed and forms a functional decrease the magnitude of the Ca2+ signal and to channel in b cells (see Togashi et al., 2006). There is inhibit insulin release in response to glucose, arginine- good evidence that TRPM2 contributes to insulin vasopressin (a Gq-coupled receptor agonist in b cells), release in response to glucose and incretin hormones and glibenclamide (Cheng et al., 2007a; Marigo et al., (Togashi et al., 2006; Uchida and Tominaga, 2011). 2009). However, the role of TRPM4 in physiologic Knockdown of TRPM2 by siRNA reduces insulin insulin secretion remains unclear. Although TRPM4 release evoked by (an activator of adenylyl expression was detected both in human b cells and cyclase) and exendin-4 (a GLP-1 receptor agonist) mouse pancreatic islets, studies on Trpm4(2/2) mice (Togashi et al., 2006). Furthermore, 2-APB inhibits revealed no difference in glucose-induced insulin exendin-4–evoked insulin release from rat pancreatic secretion compared with pancreatic islets obtained islets (Togashi et al., 2008). In the Trpm2(2/2) mouse, from wild-type animals (Vennekens et al., 2007). insulin secretion induced by glucose and GLP-1 was Moreover, the TRPM4 knockout mice did not exhibit impaired, whereas the response to tolbutamide, a KATP any sign of impaired glucose tolerance. These data channel inhibitor, was unchanged (Uchida et al., 2011). suggest that TRPM4 is probably not involved in the Indeed, Trpm2(2/2) mice show hypoinsulinemia and signal mechanism after glucose stimulation. By con- resultant hyperglycemia/impaired glucose tolerance, trast, TRPM4 appears to be involved in glucagon a diabetic phenotype. However, no correlation was secretion by the pancreatic a-cell line aTC1-6 (Nelson detected between TRPM2 variants and the risk for et al., 2011). It is noteworthy that TRPM4 was reported developing T2DM (Romero et al., 2010). to form a heteromer with Sur1 (Woo et al., 2013). Sur1 Unexpectedly, glucose-stimulated insulin secretion gene polymorphism may be partially responsible for evoked under conditions of diazoxide and high K+ individual differences in response to sulfonylurea (designed to “clamp” intracellular Ca2+ and inactivate antidiabetic drugs (reviewed in Glamoclija and the KATP channel-mediated pathways) was lost in Jevric-Causevic, 2010). It is unclear what role (if any) Trpm2-deficient islets (Uchida et al., 2011). Since the Sur1/TRPM4 heteromers play in T2DM. intracellular Ca2+ under these conditions was not c. Melastatin transient receptor potential 5 and different between wild-type and knockout islets, these melastatin transient receptor potential 6. TRPM5 is data suggest that TRPM2 mediates insulin secretion in abundantly expressed in pancreatic b cells where it is a way that is independent of its role as a Ca2+ entry highly colocalized with insulin (Colsoul et al., 2010). In channel. rodent b cells, a Ca2+-activated nonselective mono- Of note, TRPM2 has been reported to play a role in valent cation channel was detected that was largely intracellular Ca2+ release in pancreatic b cells (Lange reduced in Trpm5(2/2) mice. Normal islets respond to et al., 2009). Internally applied ADPR gives rise to glucose stimulation with three distinct types of a single Ca2+ transient in both insulin-secreting 1 cells oscillations: slow, mixed, and fast. Islets obtained from and primary mouse b cells, which was completely Trpm5(2/2) animals show a curious phenotype in that 2+ abolished in Trpm2(2/2) primary mouse b cells. the fast glucose-induced oscillations in Vm and Ca are Moreover, TRPM2 colocalizes with lysosome- markedly reduced. This implies that TRPM5 contrib- associated membrane protein-1, a specific marker for utes to the slow depolarization in the slow interburst lysosomes. On the basis of these results, it might be interval of the glucose-induced electrical activity, postulated that ADPR-dependent TRPM2-mediated thereby shortening the interburst interval and leading 2+ 2+ Ca release occurs predominantly from a lysosomal to faster glucose-induced oscillations in Vm and Ca .In store. Clearly, further experiments are needed to remains unclear why TRPM5 is only functionally TRP Channels as Drug Targets 771 relevant in the fast-oscillating subpopulation of the intracellular compartments. Addition of serum induces islets. translocation of TRPV2 to the plasma membrane It was speculated that TRPM5-mediated depolariza- (Kanzaki et al., 1999; Hisanaga et al., 2009). Genetic tion is coupled to the glycolytic rate of the cell (see inactivation (knockdown) of the insulin receptor atten- Colsoul et al., 2011, 2013). According to the dual uated insulin-induced translocation of TRPV2 (Hisanaga oscillator model, fast oscillations are characterized by et al., 2009). Interestingly, inhibition of TRPV2 reduced a high glycolytic rate and a resulting high ATP glucose-induced insulin secretion. These data indicate production. Under these conditions, TRPM5 should that insulin released from b cells may create a positive be able to depolarize Vm in the interburst interval since feedback loop by recruiting TRPV2 to the plasma mem- the hyperpolarizing KATP current is largely inactive brane and thereby accelerating insulin secretion (see (see Colsoul et al., 2013). This is in contrast with the Uchida and Tominaga, 2011). situation in slow oscillating islets in which the TRPV4 is expressed both in insulinoma cell lines and oscillating glycolytic rate and the resulting high mouse primary b cells (Casas et al., 2008). Knockdown activity of KATP in the interburst interval would render of Trpv4 by siRNA treatment protected MIN6 insuli- 2+ TRPM5 insufficient to depolarize Vm. Of note, fast Ca noma cells against human islet amyloid polypeptide oscillations are more efficient than the slow ones in (hIAP)–induced Ca2+ elevation and resultant apopto- triggering insulin release. In accord, glucose-induced sis. hIAPP, the main component of amyloid, is insulin release was reduced in isolated pancreatic associated with the loss of pancreatic b cells during islets from Trpm5(2/2) mice (Brixel et al., 2010). T2DM (Lorenzo et al., 1994). It was speculated that Moreover, Trpm5(2/2) mice display low plasma TRPV4 is the main sensor for detecting the physical insulin levels and impaired glucose tolerance during changes in the plasma membrane induced by hIAPP oral and intraperitoneal glucose tolerance tests, con- aggregation. sistent with a prediabetic phenotype caused by b cell Finally, TRPA1 is abundantly expressed in freshly dysfunction. Of note, TRPM5 expression is negatively isolated rat pancreatic b cells (Cao et al., 2013a). correlated with blood glucose concentrations in the Activation of TRPA1 in b cells by mustard oil and 4- small intestine from patients with diabetes (Young hydroxy-2-nonenal evokes insulin release. Interest- et al., 2009). An association was recently found ingly, the TRPA1 antagonist HC-030031 inhibited between TRPM5 variants and prediabetic phenotypes glucose-induced insulin release, implying a role for in individuals who are at risk for developing T2DM TRPA1 in physiologic insulin control (Leibiger et al., (Ketterer et al., 2011). 2002). For the sake of completeness, it should be Low serum Mg2+ (,1.6 mg/dl) confers increased risk mentioned here that TRPM8(2/2) mice were reported for T2DM as well as other diseases (reviewed in Wälti to exhibit prolonged hypoglycemia in response to et al., 2003; Rosanoff et al., 2012). TRPM6 is a gate- insulin (McCoy et al., 2013a). It was suggested that keeper of human Mg2+ metabolism (see Montell, 2003). TRPM8-expressing afferents may control insulin clear- Insulin was reported to enhance surface expression of ance in the liver. In diabetic Han Chinese patients, TRPM6 in human b cells. Two TRPM6 variants TRPC1 polymorphism was linked to diabetic nephrop- (V1393I and K1584E) were recently described in athy (Chen et al., 2013c). women with gestational DM (Nair et al., 2012). d. Melastatin transient receptor potential 3, vanilloid F. Transient Receptor Potential Channels in the Brain transient receptor potential 2, vanilloid transient re- as Novel Targets for Neurologic and ceptor potential 4, and ankyrint transient receptor Psychiatric Disorders potential 1. TRPM3 is expressed in mouse pancreatic Despite the plethora of reports on TRP channel islets (Wagner et al., 2008). However, the TRPM3 expression in the brain (reviewed in Reboreda, 2012; blocker did not block glucose-induced Vennekens et al., 2012), our understanding of the Ca2+ increase (Klose et al., 2011), indicating that participation of these channels in physiologic functions TRPM3 is not involved in physiologic insulin release. and disease states is still rudimentary. However, there TRPM3 was recently proposed to constitute a regulated is no shortage in speculations linking TRP channels to Zn2+ entry pathway in pancreatic b cells (Wagner the whole spectrum of neurologic and psychiatric et al., 2010). Zinc is important for insulin release. Since diseases from the common (e.g., stroke, anxiety dis- Zn2+ ions are coreleased with insulin, pancreatic b cells orders, and AD) to the esoteric (e.g., Western Pacific have to replenish their Zn2+ stores. Indeed, Zn2+ ALS-G/dementia complex). On the basis of a combina- deficiency has been linked to impaired insulin synthe- tion of immunostaining and molecular studies, many sis and the development of T2DM (see Chausmer, TRP channels appear to be expressed by brain tissue, 1998). some highly and broadly (e.g., TRPC3 and TRPC5) and TRPV2 is expressed in murine pancreatic b cells others at low levels in a few nuclei (e.g., TRPV1). The (Hisanaga et al., 2009). Under serum-free condi- function of these channels remains to be elucidated, tions, TRPV2-like immunoreactivity is localized to but there is evidence that multiple TRPs may 772 Nilius and Szallasi contribute to neuronal excitability and neurotransmit- In addition to neurons, TRPV1 expression was also ter signaling in the brain. It was speculated that TRP described in astrocytes (Doly et al., 2004; Verkhratsky channels (when they “go bad”) may be involved in the et al., 2013). Indeed, the selective TRPV1 agonist RTX development and maintenance of addiction (see was reported to induce Fos expression in astrocytes in Wescottetal.,2013). wild-type, but not TRPV1 knockout, mice (Mannari 1. Vanilloid Transient Receptor Potential 1 and et al., 2013). It was speculated that astrocytic TRPV1 Canonical Transient Receptor Potential 5 in Anxiety might be involved in the pathogenesis of neurodegen- Disorders. Ongoing controversy surrounds the ex- erative disorders (Ho et al., 2012). pression and role of TRPV1 in the brain. Early studies The TRPV1-null mice adapt more easily than their described widespread TRPV1 expression (albeit at wild-type littermates to aversive light and they explore much lower levels than in sensory neurons) throughout freely the open arm of the elevated maze, indicative of the whole neuroaxis of the rat, using a combination of reduced unconditional fear response (Marsch et al., immunohistochemical, molecular (RT-PCR), and [3H]RTX 2007). Furthermore, they exhibit less freezing in binding studies (Sasamura et al., 1998; Mezey et al., 2000; auditory fear conditioning assays. These findings imply Szabó et al., 2002; Roberts et al., 2004; Tóth et al., 2005; a therapeutic potential for TRPV1 antagonists as novel Cristino et al., 2006). Neonatal capsaicin treatment was agents (see Moreira et al., 2012). reported to induce widespread neurodegenerative changes Anxiety is a common affective (mood) disorder, in the rat brain (reviewed in Szallasi and Blumberg, treated by minor tranquilizers and anxiolytic drugs. 1999). Indeed, no TRPV1 was detected by [3H]RTX Although these agents are effective, they are associ- autoradiography in the brain of these animals when they ated with common side effects (e.g., sedation, impo- reached adult age (Roberts et al., 2004). TRPV1 expres- tence, and ), have an abuse potential, and sion was also absent in the brain of TRPV1(2/2)mice. can be even lethal if overdosed. “Atypical” anxiolytic In basal ganglia, TRPV1 was highly colocalized with agents such as are not but can tyrosine hydroxylase, suggesting that TRPV1 might be cause paradox nervousness and asomnia. It is clear involved in the pathogenesis of Parkinson disease that there is a great need for novel anxiolytic drugs (Mezey et al., 2000). In the mouse, unilateral injection with improved efficacy and safety. of the neurotoxin 6-hydroxydopamine into the striatum Cannabinoids exert a well-documented biphasic is known to cause locomotor disturbances (e.g., shuf- action on mood, anxiety, and fear, both in humans fling gait and short steps) that are characteristic of (relaxation and at low doses, followed by Parkinson disease (Metz et al., 2005). In these animals, panic and anxiety—a “bad trip”—at high doses) and the TRPV1 agonist O-arachidonoylethanolamine was experimental animals (as studied in the unconditioned found to reduce L-DOPA–induced dyskinesia and this fear test) (see Di Marzo et al., 2008). The cannabinoid beneficial effect was absent in mice whose TRPV1 CB1 receptor is among the most abundant GPCRs in receptors were eliminated by high-dose capsaicin the brain. The anxiolytic effect of tetrahydrocannabi- treatment (González-Aparicio and Moratalla, 2014). diol (the active ingredient in marijuana) is well known, Importantly, neurons in brain slices and/or neurons and the anandamide transport inhibitor AM404 [N-(4- obtained form brain nuclei and kept in culture showed hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide] (which Ca2+ fluxes in response to capsaicin that were pre- increases the concentrations of bioavailable ananda- vented by the first-generation TRPV1 antagonist mide, an “endocannabinoid” in the brain, in the vicinity capsazepine (Sasamura et al., 1998; Marinelli et al., of cannabinoid CB1 receptors) mimics the anxiolytic 2002; Gibson et al., 2008). In the amygdala, capsaicin effect of cannabis (see Di Marzo et al., 2008). In affects LTP via TRPV1 activation (Zschenderlein et al., keeping with these findings, the CB1 receptor knock- 2011). Rodents, whose brain TRPV1 was eliminated by out mice display increased anxiety behavior under genetic recombination (Caterina et al., 1997) or neo- emotional stress, as well as sustained fear response natal capsaicin treatment (see Szallasi and Blumberg, (reviewed in Valverde et al., 2005). On the basis of 1999), showed no obvious deficits in behavioral assays. these observations, it was postulated that the endo- This is surprising since the brain of TRPV1(2/2) mice cannabinoid system suppresses the fear response. This was reported to show multiple electrophysiological model begs the following question: what target is anomalies (e.g., reduced tetanus-induced LTP after responsible for the paradox anxiogenic effect of electrical stimulation (Brown et al., 2013), whereas the cannabinoids? TRPV1 knock-in animals (Cre-loxP–based conditional Capsaicin microinjected into the periaqueductal gray expression) showed stereotyped behavior in response matter, a midbrain structure strongly implicated in to capsaicin (Arenkiel et al., 2008). Furthermore, anxiety, evoked anxiety-like behavior in mice subjected TRPV1 was reported to facilitate glutamate release to the elevated maze test (Mascarenhas et al., 2013). in the striatum (Musella et al., 2009), and to control So, what is the endovanilloid that regulates brain synaptic plasticity in the superior colliculus (Maione TRPV1? Anandamide (at concentrations much higher et al., 2009a). than those required to active CB1 receptors) was TRP Channels as Drug Targets 773 reported to activate TRPV1 (reviewed in Di Marzo a single fear conditioning paradigm. These data are et al., 2002). Although it is unlikely that anandamide consistent with observations made in brain slice in the brain can reach sufficiently high concentrations recordings in which Trpc5(2/2) mice showed normal to activate TRPV1, it migh do so in concert with other membrane excitability and synaptic function but re- related brain-derived endovanilloids such as NADA duced synaptic responses to activation of mGluRs and (Huang et al., 2002). It was speculated that cannabi- CCK receptors. These data provide a potential link noids are anxiolytic by interacting with presynaptic between TRPC5 and the CCK-4 signaling pathway, the CB1 receptors but can exert the opposite (anxiogenic) activation of which induces anxiety behaviors in both effect when they activate postsynaptic TRPV1 recep- rodents and humans. Steroids are known to influence tors (see Moreira et al., 2012). In fact, CB1 and TRPV1 mood and anxiety. In this context, it is worth were reported to be colocalized in the hippocampus mentioning that neuroactive steroids inhibit TRPC5 (Cristino et al., 2006). Thus, TRPV1 may mediate the channels (Majeed et al., 2011). Combined, these “dark side” of endocannabinoids (Cernak et al., 2004). findings suggest that TRPC5 antagonists might be A recent work relying on a powerful combination of useful . Additional work will be required to reporter mice, in situ hybridization, electrophysiological determine whether a pharmacological agent recapit- recordings, and Ca2+ imaging suggests that TRPV1 ulates genetic deletion. Further analysis of the role on expression is restricted to very few brain regions, most TRPC5 in learning and memory is also needed. notably the caudate nucleus of the hypothalamus, as A recent study (Puram et al., 2011) has raised some well as endothelial cells (Cavanaugh et al., 2011). At concerns about the phenotype of the Trpc5(2/2) present, it is unclear how one can reconcile these mouse. Morphologically, neurons in the brain of these strikingly different findings. Clearly, additional work animals reveal highly branched dendrites with im- will need to be done to explain the differences that have paired dendritic claw formation (Puram et al., 2011). In been noted between wild-type and TRPV1(2/2)mice. behavioral assays, the Trpc5(2/2) mice showed In a much broader context, this raises the following deficits in gait and motor coordination. uncomfortable question: when do we target experimen- 2. Canonical Transient Receptor Potential 3 and tal artifacts, especially when findings are unexpected Cerebellar Ataxia. TRPC3 is the most abundant and contradict entrenched dogmas? In the TRPV1 TRPC channel in cerebellar Purkinje cells (Hartmann field, reports of functional TRPV1 channels in non- et al., 2008). Similar to TRPC6 and TRPC7, DAG neuronal tissues (e.g., urothelium, skin, and lymphoid activates TRPC3, and Gq-coupled receptors are likely tissues) were even more unexpected than the finding of crucial regulators of channel activity. Consistent with TRPV1 in the brain. We would be curious to see what a role in the cerebellum, TRPC3-null mice show vastly reporter mice would reveal in nonneuronal TRPV1 attenuated inward currents in response to activation of expression patterns. mGluR1 (Hartmann et al., 2008). These mice also TRPC5 is robustly expressed in the hippocampus display distinct impairment in walking behavior; and amygdala (Strübing et al., 2001; Chung et al., however, as predicted, the TRPC3 knockout phenotype 2006). In the hippocampus of young rats, TRPC5 is is less severe than the ataxia observed in mGluR1(2/2) expressed on the growth cone where it interacts with animals. stathmin-2 (Greka et al., 2003). Inhibition of TRPC5 A screen in chemically mutagenized mice also activity by a dominant negative variant resulted in identified a crucial role for TRPC3 in regulating longer neurits. The semaphorin 3A (a “chemorepulser” Purkinje cell survival. The so-called Moonwalker mice that guides migrating axons)–induced growth cone (mice with a T635A mutation in TRPC3) showed collapse is attenuated in TRPC5 knockout mice progressive Purkinje cell degeneration (Becker et al., (Kaczmarek et al., 2012). In many neurons, TRPC5 is 2009, 2011). These animals are characterized by in- coexpressed with the neurotrophin-3 receptor TrkC. In creased gait width, irregular step alteration, and a these cells, activation of TRPC5 seems to mediate the tendency to fall from the rotarod device; that is, they negative control of neurotrophin-3 on dendritic growth show changes similar to those seen in patients with (He et al., 2012b). In adults, TRPC5 in hippocampal ataxia. Perplexingly, the phenotype of TRPC3-deficient pyramidal neurons is suggested to play a role in (knockout) and hyperactive (gain-of-function mutation) cholinergic plateau potential formation (Tai et al., mice are quite similar (Trebak, 2010). In cerebellum 2011). Furthermore, TRPC5 is present both in the slices, this mutation resulted in larger currents (a gain- frontal (Fowler et al., 2007) and temporal (von Bohlen of-function mutation) in response to low concentration Und Halbach et al., 2005) cortex, where it might be of an mGluR1 agonist (Becker et al., 2009). Morpho- involved in seizure generation (Phelan et al., 2013). logically, the cerebellum of Moonwalker mice reveal Trpc5(2/2) mice show less innate fear behavior short dendrites with reduced arborization. Together, than wild-type littermates in an open-field test, these data indicate that TRPC3 channels play an elevated plus maze, and nose bumping assay (Riccio important role not only in synaptic transmission in et al., 2009). Conditioned fear memory is unaffected in cerebellar Purkinje cells, but also in their survival. 774 Nilius and Szallasi

Identification of selective pharmacologic agents will be TRPM2 by shRNA enhances blood flow and reduces useful in determining whether TRPC3 plays a broader stroke volume after experimental stroke in male (but role in ataxias. not in female) mice (Jia et al., 2011), which is a puzzling Staggerer mutant (sg/sg) mice were named after example of sex-related differences in TRP channel their unsteady (staggering) gait. They also show functions. In the hippocampus, TRPM2 appears to be muscle hypotonia and tremor and die prematurely. involved in NMDA-mediated metaplasticity (Xie et al., These mice have an underdeveloped cerebellar cortex 2011). with a reduced number of abnormal Purkinje cells. The TRPM2 (Verma et al., 2012), TRPM4 (Loh et al., genetic defect that is responsible for the phenotype of 2014), and TRPM7 have been implicated in ischemic these mice is the loss of the -related orphan brain injury (reviewed in Aarts and Tymianski, 2005; receptor-a gene. Human spinocerebellar ataxia type-1 Bae and Sun, 2013). In vivo siRNA-mediated silencing is associated with retinoid-related orphan receptor-a of TRPM4 reduces brain infarct volume by half and dysfunction that manifests in the absence of mGluR- facilitates functional recovery in rats with middle mediated slow EPSPs. It is noteworthy that TRPC3 is cerebral artery occlusion (Loh et al., 2014). Knockdown a downstream target for mGluR signaling, and the of TRPM7 also reduces cell death due to oxygen- expression of TRPC3 is markedly reduced in the sg/sg glucose deprivation in isolated neuronal cultures, mice (Mitsumura et al., 2011). implying a role for TRPM7 antagonists in the treat- Paradoxically, TRPC3 knockout mice also exhibit an ment of stroke. Indeed, lentiviral delivery of TRPM7 ataxia phenotype. Similar to the Staggerer mutant shRNA prevents ischemic neuronal cell death after mice, these animals show absent mGluR-mediated experimental stroke in the rat (Sun et al., 2009). slow EPSPs. However, unlike in the Staggerer mice, However, the expectations toward TRPM7 as a thera- the Purkinje cell morphology is normal. To reconcile peutic target in stroke have been tempered by the lack these findings, it was postulated that cerebellar of association between TRPM7 variants and the risk TRPC3 is constitutively active and the ion flux is for suffering ischemic stroke (Romero et al., 2009). partially controlled by channel trafficking (Trebak, Rupture of blood vessels in the brain, with the 2010). In Moonwalker mice, the hyperactive TRPC3 subsequent accumulation of blood in the parenchyma, channel causes Purkinje cell death due to Na+ and Ca2+ causes accumulation of blood-derived factors, induces overload. In the Staggerer and Trpc3(2/2) mice, the excessive inflammatory changes, and is involved in the mGluR signaling is impaired. It is noteworthy that progression of intracerebral hemorrhage. Thrombin PKCg negatively regulates TRPC3 and human spino- leaks into the brain parenchyma and induces brain cerebellar ataxia type-14 has been linked to a mutant injury and astrogliosis. Thrombin typically upregulates (S119P) PKCg gene. TRPC3, which in turn contributes to pathologic Of note, TRPC3 is a downstream target for BDNF (Li astrogliosis (Shirakawa et al., 2010). Administration et al., 1999), and BDNF-mediated survival of cerebellar of the specific TRPC3 blocker Pyr3 in a mouse model granular cells depends upon functional TRPC3 (Li reduced neurologic deficits, neuronal injury, brain et al., 2005b; Jia et al., 2007). It was speculated that edema, and perihematomal accumulation of astrocytes TRPC3 might be involved in the pathogenesis of Rett and ameliorated intracerebral hemorrhage brain in- syndrome (a neurodevelopmental disorders that jury (Munakata et al., 2013). Therefore, TRPC3 is mainly affects girls) (Amaral et al., 2007) and bipolar a potential therapeutic target for the treatment of disorder (Roedding et al., 2013). Almost all cases of hemorrhagic brain injury (Munakata et al., 2013). The Rett syndrome are attributable to a mutation in the TRPC6 activator hyperforin attenuated brain infarct methyl CpG-binding protein (MECP2) gene. The link volume in rats with middle cerebral artery occlusion in (if any) between this mutation and TRPC3 is unclear. a CREB-mediated fashion (Lin et al., 2013). Interest- 3. Melastatin Transient Receptor Potential 2, Mela- ingly, TRPC6 also seems to link neuroprotectin D1 (an statin Transient Receptor Potential 7, and Canonical endogenous compound that limits ischemic brain in- Transient Receptor Potential 3: Potential Targets in jury) to CREB activation via the Ras/MEK/ERK path- Stroke Patients. In the United States, stroke is way (Yao et al., 2013). a leading cause of disability and the second leading 4. Transient Receptor Potential Channels in Neuro- cause of death (over 1 million strokes are responsible degenerative Disorders and Mental Retardation. for close to 200,000 death). In the brain, TRPM2 is AD is a devastating neurodegenerative disorder that expressed by neurons and microglia. In keeping with affects an estimated 2.5 to 5.1 million Americans (a the concept that TRPM2 functions as a redox sensor firm diagnosis of AD can only be made by postmortem (Jiang et al., 2010; Nazıroglu, 2011a), TRPM2(2/2) examination of the brain), placing severe emotional mice show protection against various pathologies burden on the affected families and draining the related to oxidative stress, including the focal ischemia financial resources of the healthcare system. Sadly, model of stroke. In stroke models, TRPM2 expression there is no effective treatment of AD. A histologic is increased (Fonfria et al., 2006a), and knockdown of hallmark of AD is b-amyloid deposition in the TRP Channels as Drug Targets 775 striatum. TRPM2 is expressed in the rat striatum, b-methyl-amino-alanin, a known neurotoxin (see Banack where it is activated under oxidative stress by ROS and Murch, 2009). The human striatum expresses (Hill et al., 2006). In AD models, striatal TRPM2 (a a novel, N-terminal truncated TRPM2 variant, the neuronal death channel) is upregulated, and knock- biologic function of which is unclear (Uemura et al., down of this channels by siRNA was reported to 2005). ameliorate b-amyloid–induced neurotoxicity (Fonfria 5. Transient Receptor Potential Channels and et al., 2005). These findings offer a glimmer of hope Neuroregeneration. As detailed above, TRP channels that TRPM2 antagonists may halt (or at least slow (especially TRPC5, but also TRPC1, TRPC6, TRPV1, down) the relentless progression of AD. and TRPV4) are thought to play a crucial role in the Astrocytic TRPA1 was recently implicated in the brain in the regulation of synaptic plasticity, neuronal pathogenesis of AD. Indeed, the TRPA1 agonist activity, and neurogenesis, as well as growth cone acrolein, a highly electrophilic a,b-unsaturated alde- guidance. More in-depth studies on the brain architec- hyde generated during the combustion of organic ture in developing TRPC5 mice, as well as experiments materials (Kehrer and Biswal, 2000), induces an AD- looking at receptor-activated neurite extension in brain like condition in the rat (Huang et al., 2013). It was slices should help clarify the relative contribution of speculated that TRPA1 channels regulate astrocytic TRPC5 to axonal pathfinding (see for detailed reviews, resting Ca2+ levels and inhibitory synapse efficacy via Nilius, 2012; Vennekens et al., 2012). GAT-3 (GABA transporter-3) (Shigetomi et al., 2012). Hippocampal LTP, a form of synaptic plasticity in- G. Transient Receptor Potential Channels in Cancer volved in learning and memory formation, depends on The “war on cancer” initiated by President Nixon in the release of D-serine from astrocytes (Henneberger 1971 is one of the most overused slogans in medical et al., 2010), which, in turn, is regulated by TRPA1 research. If this is a war, it is one with no clear victory (Shigetomi et al., 2013). The astrocytic hypothesis of in sight and the opening of a new front in cancer AD states that astrocytes convert into a chronically research that may offer a glimmer of hope is most activated phenotype. Switching off these astrocytes by welcome progress. Although molecular biology has sub- genetic manipulation stops cognitive decline in pre- stantially advanced our understanding of cancer biology, clinical AD models (Furman et al., 2012). One might certain cancers (e.g., metastatic melanoma and pancre- argue that TRPA1 antagonists could be beneficial in atic adenocarcinoma) are still as deadly as ever. Indeed, patients with AD by blocking astrocytic TRPA1. It is with over half million victims annually, cancer remains noteworthy that in cerebral arteries of transgenic mice a leading cause of death in the United States, second that overexpress a mutated form of the human amyloid only to heart disease. The increasingly confusing and precursor protein (APP mice) abnormal responses to controversial literature on TRP channels and cancer acetylcholine were observed, attributed to TRPV4 should be read with these sobering thoughts in mind. dysfunction in the endothelium (Zhang et al., 2013b). In a much simplified manner, cancer can be thought TRPC5 is located in the locus for nonsyndromic, of as an uncontrolled growth of tumor cells with X-linked mental retardation (Sossey-Alaoui et al., seemingly limitless replicative potential that is either 1999). TRPC5 is robustly expressed in the hippocam- due to enhanced proliferation (e.g., Burkitt lymphoma) pus (Strübing et al., 2001). As discussed above, the or resistance to apoptotic death (e.g., follicular lym- phenotype of the TRPC5-null mouse in behavioral phoma overexpressing the antiapoptotic protein BCL- assays is controversial (no phenotype in one study and 2). Carcinogenesis appears to be a multistep process in impaired gait and motor coordination in another). It which mutations that confer selective growth advan- was proposed that TRPC5 plays a key role in normal tage accumulate, allowing the progeny of mutated cells neurodevelopment; abnormal TRPC5 activity might to outgrow their normal counterparts. A critical step in lead to autism, mental retardation, and other brain this process is the acquisition by cancerous cells of the disorders (reviewed in Reboreda, 2012). Of note, potential to invade surrounding tissues and metasta- TRPC6 has been linked to normal exploratory behavior size to distant locations. When it happens, it trans- in the mouse (Beis et al., 2011). forms a disease that is potentially curable by surgical Loss-of-function TRPM2 (P1018L) and TRPM7 excision into one that requires systemic chemotherapy (T1482I) mutants have been linked to PD-G and and may prove deadly. Western Pacific ALS-G (Hermosura et al., 2005, Now it is well recognized that tumor cells can 2008). The mutant TRPM7 has normal kinase activity produce factors to create a microenvironment that but shows increased sensitivity to Mg2+ blockade. The promotes their growth. For example, many cancers mutation by itself is not sufficient to produce the depend on VEGF production for acquiring and main- disease: It needs coexistent environmental factors. In taining adequate blood supply (from a clinical point of Guam, the drinking water is extremely low in Mg2+ view, reviewed in Hicklin and Ellis, 2005). Indeed, and the diet of natives used to include fruit bat as bevacizumab (Avastin; Genentech, South San Francisco, a delicacy. Fruit bats feast on cycad nuts containing CA), a humanized anti-VEGF antibody, is broadly used 776 Nilius and Szallasi in the management of glioblastoma and metastatic contrast, TRPV1 appears to be highly expressed in colonic adenocarcinoma to starve these cancers from O2 glioblastoma and only weakly expressed (or absent) in and nutrients (www.avastin.com). It is noteworthy that surrounding normal brain (Stock et al., 2012). This is a number of TRP channels (e.g., TRPV4 as well as a potential problem for drug development as an agent TRPC1 and TRPC6) seem to play an important role in that may inhibit the growth of one kind of tumor might neoangiogenesis. Indeed, TRPV4 is expressed in the conversely promote the formation of a different kind of vascular endothelium (see Nilius et al., 2003) and has malignancy. This behavior is, however, not unprece- been implicated as a potential therapeutic target in dented and did not prevent the use of other chemother- ocular neovascularization disorders (e.g., diabetic apeutic agents. For example, the use of alkylating retinopathy). TRPC1 is essential for VEGF-induced agents has been linked to the development of secondary angiogenesis in the zebrafish (Yu et al., 2010). In U87 hematologic malignancies (high-grade myelodysplastic glioma cells, a role for TRPC1 in hypoxia-induced syndrome and/or acute myeloid leukemia) in some VEGF expression was reported (Wang et al., 2009). patients. For such a deadly disease such as glioblastoma Furthermore, TRPC6 has been implicated as an (see below), the possibility that the patient may develop important downstream target in VEGF-induced angio- another, late malignancy after a TRP channel-targeting genesis (Pocock et al., 2004; Ge et al., 2009). Indeed, in treatment is of a lesser concern. HMVECs, overexpression of a dominant negative Given the broad expression of TRP channels in mutant of TRPC6 was shown to inhibit migration, normal tissues, the finding of altered TRP channel sprouting, and proliferation in response to VEGF expression (including TRPV1, TRPV2, TRPV6, TRPC3, administration (Hamdollah Zadeh et al., 2008). Con- TRPC5, TRPC6, TRPM1–TRPM4, TRPM7, and versely, overexpression of a wild-type TRPC6 construct TRPM8) in various cancers was hardly unexpected enhanced the proliferation and migration of HMVECs. (reviewed in Prevarskaya et al., 2010; Lehen’kyi and It is tempting to speculate that targeting vascular TRP Prevarskaya, 2011; Liberati et al., 2013; Morelli et al., channels (in particular TRPC6) may aid in depriving 2013). For most TRP channels and cancers, it is unclear cancers from their blood supply. whether the observed changes are therapeutically Although no clear picture has yet emerged, there is relevant (i.e., involved in the proliferation and migration increasing evidence that certain TRP channels may of tumor cells and/or in their resistance to chemothera- play opposing roles (oncogenic versus tumor suppres- peutic agents) or simply an epiphenomenon of cancer sor) during carcinogenesis (reviewed in Prevarskaya progression. A notable exception is TRPM8. Increased et al., 2010; Lehen’kyi and Prevarskaya, 2011; Liberati TRPM8 expression was detected in aggressive prostatic et al., 2013; Morelli et al., 2013). To resolve some of the adenocarcinoma (Tsavaler et al., 2001; Zhang and discrepancies in the literature, one might even postu- Barritt, 2006; Gkika et al., 2010; Gkika and Prevarskaya, late that this effect is cell type dependent; that is, 2011). In vitro, menthol, a TRPM8 agonist, inhibits a TRP channel that is oncogenic in one cell type may be proliferation of prostate cancer cells (Wang et al., conversely tumor suppressor in another. If this 2012b). D-3263 [3-(2-amino-ethyl)-1(R)-[(2(S)-isopropyl- hypothesis holds true, TRPV2 could be a dramatic 5(R)-methyl cyclohexanecarbonyl)]-5-methoxy-1,3- example of this phenomenon. It is virtually absent in dihydro-benzoimidazol-2-one hydrochloride], a TRPM8 non-neoplastic prostate tissue but is upregulated in agonist, was recently advanced to a phase I clinical trial metastatic, castration-resistant prostatic adenocarci- (ClinicalTrials.gov identifier NCT00839631) in patients noma (Monet et al., 2010). When transplanted into with metastatic prostatic carcinoma with a strategy to nude mice, the growth of the xenograft is inhibited by kill TRPM8-expressing tumor cells by Ca2+ and Na+ knockdown of the Trpv2 gene. By contrast, TRPV2 is overload through TRPM8 activation. Below are a few highly expressed in normal, but not in neoplastic selected cancers in which, at least in our opinion, (glioblastoma), brain tissue (Morelli et al., 2012). aberrant TRP channels expression may have impor- Overexpression of TRPV2 inhibits glioblastoma growth tant clinical ramifications. in a mouse xenograft model and promotes differentia- 1. Glioblastoma Multiforme. Glioblastoma multi- tion toward a more mature glial phenotype (Morelli forme (GBM) is a highly aggressive (World Health et al., 2012; Nabissi et al., 2013). In other words, Organization grade IV) and deadly form of brain cancer TRPV2 may function as an oncogene in the prostate (malignant glioma) with a 3-month median survival and a tumor suppressor gene in the brain. Another time from the diagnosis without treatment (Fig. 37). puzzling example is TRPV1. TRPV1 expression was Even with standard treatment regimens, the life reported in normal urothelial tissue (Lazzeri et al., expectancy of patients with GBM is less than a year, 2004) with a decrease in urothelial dysplasia and a loss with only 2% of patients living longer than 3 years of expression in invasive urothelial carcinoma (Lazzeri after diagnosis. Although some investigational treat- et al., 2005). On the basis of these findings, it was ment modalities (e.g., and gene therapy) postulated that TRPV1 plays an antioncogenic role showed initial promise, the medium survival has in the bladder (Santoni and Farfariello, 2011). By showed only very modest (approximately 3-month) TRP Channels as Drug Targets 777

brain tissue) and it was speculated that TRPV1 agonism may be used for therapeutic purposes (Stock et al., 2012). Indeed, TRPV1 agonists induced apoptosis in glioma cells in vitro via a subdivision of the endoplasmic reticulum stresspathwaythatiscontrolledbyactivatingtranscrip- tion factor-3. On the basis of their observations, the authors argue that endovanilloids (endogenous brain- derived TRPV1 agonists) released from neural precursors cells patrol the juvenile brain by eliminating TRPV1- expressing glioma cells. When this endogenous defense mechanism is impaired (neural precursors cells decline Fig. 37. Glioblastoma multiforme. Reprinted with permission from with age and eventually disappear), high-grade gliomas Liberati et al. (2013). may arise. These TRPV1-expressing cancers may be killed (or at least controlled) by either stimulating improvement during the past 3 decades. GBM is endovanilloid release or TRPV1 agonist therapy. Indeed, a greatly infiltrative tumor (not amenable to complete arvanil, a synthetic TRPV1 agonist given systemically surgical removal with clean margins) that is also (via intraperitoneal injections), prolonged survival of highly resistant to chemotherapy and/or radiation SCID mice injected with GBM cells. This is an elegant therapy. A number of recent observations in the TRP theory with potential clinical ramifications. Although it is channel field offer a new glimmer of hope for patients rather doubtful that patients will tolerate systemic with GBM. TRPV1 agonist (arvanil or other) injections, intrathecal As mentioned above, TRPV2 is highly expressed in administration represents a viable alternative. Indeed, normal brain tissue (mostly in astrocytes); indeed, intrathecal RTX (an ultrapotent TRPV1 agonist) injec- TRPV2 was originally cloned as a vanilloid receptor tions have been tried both in preclinical models (e.g., dogs 1-like protein from rat brain (Caterina et al., 1999). with osteosarcoma) and a small number of patients to Although the physiologic role of TRPV2 in normal relieve cancer pain with tolerable side effects. brain functions remains enigmatic, emerging evidence The high level of expression of TRPC1 (Bomben and implicates an important role for TRPV2 in GBM Sontheimer, 2010) and TRPC6 (Chigurupati et al., tumorigenesis. In U87MG glioma cells (a human 2010; Ding et al., 2010) both in glioma cell lines and primary GBM cell line), silencing of TRPV2 by siRNA patient-derived GBM samples is well documented. promotes growth by stimulating cell cycle (upregulated These channels are interesting in that they appear to cyclin E1 and CDK2 and overexpressed Raf-1 and be involved in multiple steps of GBM tumorigenesis. BCL-XL) and protecting tumor cells from apoptosis (via The potential role of TRPC1 and TRPC6 in VEGF- down-regulation of Fas/CD95 and caspase-8 expres- induced neoangiogenesis was previously mentioned in sion) (Nabissi et al., 2010). In keeping with these in this work. Bevacizumab is one of the few therapeutic vitro findings, TRPV2 siRNA accelerated GBM pro- options in GBM, and one might speculate that TRPC1 gression in a mouse xenograft model (Morelli et al., and/or TRPC6 blockade may boost the therapeutic 2012). Importantly, the reserve is also true: 1) over- efficacy of bevacizumab. It is worth mentioning that expression of TRPV2 in glioma cells decreases viability knockdown of TRPC6 (by overexpression of a negative by increasing proapoptotic Fas/CD95 expression, and dominant mutant or via siRNA interference) in GBM 2) reduces the tumor volume and mitotic rate when cells reduced tumor volume in a murine xenograft GBM is xenografted into nude mice. Intriguingly, the model and increased survival in the intracranial mouse residual tumor shows evidence of differentiation to- model (Ding et al., 2010). ward a more mature (i.e., lower grade) astrocytic 2. Breast Cancer. With the exception of non- phenotype. Provided that TRPV2 can be overexpressed melanoma skin cancers, breast carcinoma is the most in human GBM via gene therapy, the above observa- common type of cancer among American women. In tions may represent the first real breakthrough in fact, one in eight women (approximately 12%) in the GBM research because the prognosis of grade 2 (or United States will develop invasive breast carcinoma even grade 3) glioma (5-year survival of 27% and 65%, during their lifetime. The American Cancer Society respectively) is far superior to that of GBM (practically estimates that about 230,000 new cases of invasive no patient survives for 5 years). breast carcinoma will be diagnosed in 2013 and close to In contrast with TRPV2, which is highly expressed 40,000 women will die from it. Although death rates throughout the whole neuroaxis, the expression of TRPV1 from breast cancer have been declining since the late in normal brain is controversial. The conflicting reports 1980s (attributed to a combination of early detection by are discussed elsewhere in this review. Here it suffices to screening and advances in therapy such as herceptin), mention that a high level of TRPV1 expression was re- breast carcinoma remains the second leading cause of ported in GBM (but not in surrounding non-neoplastic cancer death among women, exceeded only by lung 778 Nilius and Szallasi cancer. Breast cancer is a heterogenous group of diseases, TRPC4, TRPC6, and TRPM8 was reported in prostatic including both relatively indolent cancers (e.g., tubular adenocarcinoma (reviewed in Gkika and Prevarskaya, carcinoma and mucinous carcinoma) that almost never 2011). The significance of TRPV1 expression in prostate metastasize and highly aggressive cancers (e.g., such as carcinoma cells (Sanchez et al., 2005) is unclear since the so-called triple negative carcinoma that is resistant to the TRPV1 agonist capsaicin was reported to stimulate both tamoxifen and herceptin therapy because it does not the growth of androgen-responsive cancer cells derived express estrogen receptors and is also negative for Her2/ from lymph node metastasis of prostate carcinoma neu overexpression). (Malagarie-Cazenave et al., 2009), a most puzzling ob- Overexpression of a number of TRP channels (in- servation since TRPV1 agonism is usually advocated to cluding TRPC1, TRPC5, TRPC6, TRPM7, TRPM8, and kill TRPV1-expressing cancerous cells. Indeed, capsai- TRPV6) was described in breast carcinomas compared cin induces apoptosis in the PC-3 prostate cancer cell with normal breast tissue (reviewed in Ouadid-Ahidouch line (Sánchez et al., 2006). TRPV2 and TRPV3 are more et al., 2013). Of these channels, TRPC5, TRPM7, and interesting since they are predominantly expressed in TRPV6 appear to be the most interesting. TRPC5 was castration-resistant carcinomas. Silencing of TRPV2 by shown to render breast cancer cell lines resistant to siRNA reduced tumor volume and increased survival in chemotherapy (adriamycin) through NFAT isoform mice with xenografted prostate cancers (Monet et al., c3-mediated P-glycoprotein upregulation, enabling the 2010). cancer cells to pump cytotoxic drugs out of the cells (Ma In contrast with vanilloid and canonical TRP chan- et al., 2012b). TRPM7 seems to be exclusively expressed nels whose function in physiologic prostate function is in high-grade [Scarff-Bloom-Richardson (SBR) grade essentially unknown, TRPM8 functions as an androgen- 3/3] breast carcinomas (Guilbert et al., 2009; Dhennin- responsive element that is involved in protein secretion Duthille et al., 2011). In a mouse xenograft model, (in acinar cells) and proliferation (in smooth muscle) TRPM7 expression is required for metastasis formation (see Zhang and Barritt, 2006). In PC3 cells, TRPM8 is (Middelbeek et al., 2012). If so, it is hardly surprising overexpressed in both the plasma membrane and the that a high level of TRPM7 expression is an indepen- endoplasmic reticulum. Interestingly, PSA can stimu- dent adverse prognostic factor in patients with breast late the redistribution of TRPM8 from the endoplasmic cancer (see Ouadid-Ahidouch et al., 2013). Interestingly, reticulum to the plasma membrane (Gkika et al., 2010). it was speculated that this risk can be minimized by It was speculated that plasma membrane–bound TRPM8 dietary Mg2+ supplementation (Sahmoun and Singh, plays a protective role against prostate cancer pro- 2010). Microarray analysis of breast cancer samples gression, whereas the shift toward the endoplasmic identified TRPV6 overexpression as a feature of estrogen reticulum indicates cancer progression. The experi- receptor–negative carcinomas (see Bolanz et al., 2008). mental findings are, however, confusing. In PC3 cells, Similar to TRPM7, TRPV6 overexpression portends PSA treatment (presumably by enhancing TRPM8 an adverse prognostic significance in breast cancer. In activity in the plasma membrane) reduced cell motility MCF7 breast cancer cells, tamoxifen inhibits TRPV6 and proliferation, implying a therapeutic value for activity in a way that is independent of estrogen receptor TRPM8 agonism in the treatment of prostate cancer activation (Bolanz et al., 2008). On the basis of these (Gkika et al., 2010). However, in another study, it were findings, it was proposed that TRPV6 may be a viable the TRPM8 antagonists AMTB and JNJ41876666 that therapeutic target in estrogen receptor–negative breast reduced the proliferation rateofprostatecancercells, cancer (see Peters et al., 2012). whereas the TRPM8 agonist icilin was without any effect 3. Prostatic Adenocarcinoma. With an estimated (Valero et al., 2012). Moreover, TRPM8 silencing induced 240,000 new cases and 30,000 deaths in 2013, prostate apoptosis in lymph node metastasis-derived prostate cancer remains the most common cancer among American carcinoma cells (Thebault et al., 2005). This finding was men (aside from nonmelanoma skin cancers). Prostate- interpreted to imply that TRPM8 expression is required specific antigen (PSA) screening dramatically increased for the survival and growth of metastatic prostate prostate cancer detection but resulted in disappointingly carcinoma. little reduction in death. Indeed, most prostate cancers are TRPM8 expression appears to correlate with the indolent and patients die with, and not from, their disease. grade of prostate cancer: the higher the grade, the Identifying the cancers that will spread and kill patients higher the expression. It is, however, unclear whether via a simple laboratory marker is the ultimate goal in TRPM8 expression is an independent adverse prog- prostate cancer research. Gleason grading works well in nostic factor in biopsies. It also remains to be seen representative tumor samples but often has limited value whether serum TRPM8 (with or without PSA) can be in limited specimens (e.g., minute focus of adenocarcinoma used as a marker to screen individuals for possible in a core biopsy). Until such a marker is identified, many high-grade prostate carcinoma. Of note, serum TRPM8 men will continue undergoing unnecessary prostatectomy. mRNA was advocated as a useful diagnostic test to Compared with non-neoplastic prostate, increased identify patients with metastatic prostate cancer (Bai expression of TRPV1–TRPV4, TRPV6, TRPC1, TRPC3, et al., 2010). TRP Channels as Drug Targets 779

4. Ovarian, Pancreatic, and Gastric Adenocarcinoma. steadily increasing over the past 3 decades, attributed Ovarian, pancreatic, and gastric adenocarcinomas are to increased sun (UV) exposure. In 2013, an estimated deadly diseases that are often discovered in an advanced 77,000 new cases of melanoma will be diagnosed and stage when they are no longer amenable to complete 10,000 patients will die of the disease. Of note, survivors surgical removal. In fact, the 5-year survival rate in of melanoma are about nine times more likely to develop ovarian carcinoma drops from 90% (cancer still confined another melanoma than the general population, indicative totheovary)to30%orlesswhenthecancerhasspread of genetic predisposition. Whereas localized melanoma outside the pelvis. Pancreaticcancerhasanextremely portends a good prognosis (if removed surgically with poor prognosis, with a 6% 5-year survival rate with all clean margins), metastatic melanoma (especially the BRAF stages combined. The stage IV gastric cancer survival mutation negative cases) remains a deadly disease with rate is also dismal (,10%). Clearly, there is a large, few treatment options. Nonmelanoma skin cancer is the unmet need in the medical management of late stage most common form of cancer in the United States, with (widely metastatic) ovarian, pancreatic, and gastric one in five Americans developing skin cancer in the carcinomas. course of a lifetime. An increase in mRNA levels encoding TRPC1 and The melastatin subfamily of TRP channels was named TRPC3–TRPC5 was recently described in ovarian after a protein (now called TRPM1) that is highly adenosarcoma cells (Zeng et al., 2013), correlating with expressed in benign nevi, including Spitz nevi (Erickson the grade of the carcinoma. A novel spliced isoform of et al., 2009), with reduced expression in primary TRPC1 with exon 9 deletion was also detected (Zeng melanoma and absence of expression in metastatic et al., 2013). Transfection of cancer cells with siRNA melanoma (Duncan et al., 1998). These observations targeting these TRPC channel genes (Zeng et al., 2013), led to the suggestion that the TRPM1 gene is a tumor or application of TRPC channel-specific blocking anti- suppressor. In melanocytes, TRPM1 expression appears bodies (Zeng et al., 2013), reduced the proliferation of to be regulated by MITF. Indeed, the promoter region cancer cells in vitro. A second, independent study also of the TRPM1 gene has a MITF-recognition site. In- demonstrated a therapeutic potentialforsilencing terestingly, TRPM2 has two isoforms (TRPM2-AS and TRPC3 in the ovarian carcinoma cell line SKOV3 TRPM2-TE) that are upregulated in melanoma (see Guo (Yang et al., 2009). It remains to be seen whether these et al., 2012). These isoforms are thought to function as in vitro findings also hold true for in vivo tumors. antisense transcripts that negatively regulate TRPM2. In pancreatic ductal adenocarcinoma, TRPM7 (Rybarczyk Indeed, overexpression of wild-type TRPM2 and knock- et al., 2012) and TRPM8 (Yee et al., 2012a) have been down of TRPM2-TE exert the same effect: increased identified as potential therapeutic targets. Compared susceptibility of melanoma cells to apoptosis (reviewed with non-neoplastic pancreatic tissue, TRPM7 is highly in Guo et al., 2012). Although not strictly related to expressed (13-fold) in pancreatic cancer. Importantly, an melanoma, it is worth mentioning here that an anti- inverse correlation was found between patient survival TRPM1 autoantibody was described in patients with and TRPM7 expression. In BxPC3 cells, silencing of occult melanoma and paraneoplastic syndrome, namely TRPM7 by siRNA interference inhibited Mg2+ fluores- melanoma-associated retinopathy (Dhingra et al., 2011; cence and blocked the migration, but (disappointingly) Dalal et al., 2013). not the proliferation, of cancer cells (Rybarczyk et al., Local excision is curative in most patients with BCC 2012). On a more positive note, in a second independent or SQCC of the skin. However, both BCC and SQCC study, the combination of anti-TRPM7 siRNA and have subtypes (e.g., morphea-like BCC and basaloid gemcitabine produced enhanced tumor cell killing com- SQCC) that are difficult to manage surgically. Multifocal- pared with gemcitabine alone (Yee et al., 2012b). A superficial BCC may involve large areas in sun-exposed subset of patients with pancreatic carcinoma aberrantly skin. Some of these areas (e.g., eyelid) allow limited surgical expresses TRPM8 (Yee et al., 2012a). Targeted inhibition intervention. BCC is thought to represent a maturation of TRPM8 in these patients may increase survival. defect in keratinocyte differentiation (the carcinoma cells In gastric carcinoma, TRPC6 and TRPM7 are being resemble the normal basal layer of the epidermis, hence the investigated as potential therapeutic targets. Expression name BCC). TRPC1/TRPC4 was recently implicated in the of TRPC6 is greatly elevated in human gastric carcinoma Ca2+ signaling that regulates keratinocyte differentiation compared with non-neoplastic gastric epithelium (Cai (Beck et al., 2008). One might speculate that creams et al., 2009). Silencing of TRPC6 was reported to inhibit containing TRPC1 and TRPC4 agonists may restore the formation of gastric carcinomas in nude mice. Human normal differentiation in the epidermis and thereby prevent gastric adenocarcinoma cells also express abundant the arising of further BCC. Invasive SQCC often arises in TRPM7. In these cells, anti-TRPM7 siRNA inhibits a background of dysplasia attributed to sun damage (called growth and promotes apoptosis (Kim et al., 2008c). actinic keratosis) that can be quite extensive. The role of 5. Melanoma and Nonmelanoma Skin Cancers. TRPV1 in skin carcinogenesis is controversial. The TRPV1 Malignant melanoma is a potentially deadly form of antagonist AMG9810 [(2E)-N-(2,3-dihydro-1,4-benzodioxin- skin cancer. The incidence of melanoma has been 6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide] was 780 Nilius and Szallasi reported to promote skin tumor (papilloma) formation melanocytes, and hair follicle cells, as well as endothelial in mouse skin (Li et al., 2011d). In similar experiments, cells, fibroblasts, Langerhans cells, adipose cells, and TRPV1 knockout mice also showed an increased smooth muscle cells) express a number of TRP channels number of skin papillomas compared with their wild- that are implicated in a variety of key cutaneous type littermates (Bode et al., 2009). However, mice in functions, including skin-derived pruritus, prolifera- which TRPV1 was chemically deleted by neonatal RTX tion, differentiation, apoptosis, and inflammatory administration showed no increase in skin papilloma processes (summarized in Fig. 38; reviewed in Denda formation (A. Szallasi and P.M. Blumberg, unpub- and Tsutsumi, 2011; Moran et al., 2011; Valdes- lished observations). Regardless of the interpretation Rodriguez et al., 2013). The role of TRP channels in of these conflicting results, TRPV1 appears to be over- itch and skin cancer was discussed elsewhere in this expressed in SQCC and thus may represent a therapeutic review. Here we focus on dermatological disorders (e.g., target for tumor cell killing (Marincsák et al., 2009). It alopecia/hirsutism, rosacea, atopic dermatitis/eczema, would be interesting to see whether a topical cream and ) in which targeting TRP channels may containing capsaicin or RTX can eliminate actinic have therapeutic value. keratosis. Of note, capsaicin appears to kill oral SQCC The growth of hair is a cyclic process that involves cells in culture independent of TRPV1 (Gonzales three phases: the growth phase (anagen), the transi- et al., 2014). tion phase (catagen), and the resting phase (telogen). In ex vivo human skin explants, restored The physiologic regulation of hair growth cycle is not normal keratinocyte differentiation in the sun-damaged completely understood but IGF-1 appears to play a areas (actinic keratosis, a precursor for SQCC) by activating critical role in promoting hair growth (reviewed in Su TRPC6 (Woelfle et al., 2010). In head and neck SQCC, et al., 1999). Indeed, finasteride (Propecia; Merck, TRPC6 is highly expressed and plays a role in carcino- Whitehouse Station, NJ) is believed to stimulate hair genesis as evidenced from knockdown of Trpc6 in head growth by upregulating IGF-1 in dermal papillae. The andneckSQCC-derivedcelllines.siRNA-inducedknock- target for IGF-1 is a receptor tyrosine kinase that down of Trpc6 expression in head and neck SQCC- supports cell growth (mitogen) and survival (antiapop- derived cells dramatically inhibited tumor invasiveness. totic) in many cell types. Indeed, mice carrying null Thus, TRPC6 is likely to be a promising therapeutic mutations in the Igf-1 and Igf-1r genes remain small target in the treatment of head and neck SQCC (Bernaldo (dwarfism) if they survive (many die shortly after de Quirós et al., 2013). Broadly speaking, head and neck birth) due to general organ hypoplasia, including the SQCC comprises at least two distinct subsets of cancer, epidermis (Liu et al., 1993). Surprisingly, the hetero- one driven by human papillomavirus and the other zygous Igf1r(+/2) knockout mice live longer and have related to smoking. It is unclear whether TRPC6 over- an otherwise unremarkable phenotype (Holzenberger expression characterizes either or both cancers. More- et al., 2003). over, TRPC6 overexpression appears to be an adverse In the hair follicle, IGF-1 is believed to maintain the prognostic marker is esophageal SQCC (Zhang et al., anagen stage and postpone the catagen stage. In 2013d). support of this concept, transgenic mice that over- express IGF-1 in their skin have early, accelerated hair H. Transient Receptor Potential Channels as follicle development (Semenova et al., 2008). By con- Therapeutic Targets in Dermatology trast, proinflammatory cytokines (e.g., IL-1a,IL-1b, The skin is the largest organ of the human body. For TNFa, and IFNg) are thought to promote hair loss by the average adult, the skin has a surface area of 1.5– inducing apoptosis in the hair bulb. 2m2 and weighs 3–4 kg. Functions of the human skin There is emerging evidence that TRPV1 may be are extremely diverse and often underappreciated. involved in the regulation of human hair growth (Bodó For example, the skin serves as a sensory organ and et al., 2005). In organ-cultured human hair follicles, mechanical barrier that connects us to, and separates activation of TRPV1 by capsaicin markedly suppressed us from, our environment. Moreover, the skin is an hair shaft elongation and induced apoptosis-driven immunologic organ. It also participates in water and catagen regression. Interestingly, this effect was accom- electrolyte regulation and in maintaining normal body panied by an alteration in gene expression profiles, temperature. Finally, the skin is an important organ of promoting the intrafollicular production of cytokines sensuality and psychologic well-being. Indeed, in the (e.g., IL-1b and TNFa) that inhibit hair growth. Indeed, United States alone, women spend over $7 billion on TRPV1-null mice exhibit a significant delay in hair cosmetics and beauty products and another $2 billion on follicle cycling compared with wild-type animals, sup- laser hair removal, facelifts, and other cosmetic proce- porting the concept that TRPV1 may exert negative dures such as microdermabrasion. control (i.e., a growth-inhibitory function) in mamma- The skin is divided into three layers: the epidermis lian skin (Bíró et al., 2006). These observations imply (with the skin appendages), the dermis, and the a therapeutic value for topical TRPV1 agonist prepara- subcutaneous adipose tissue. Skin cells (keratinocytes, tions in eliminating unwanted hair growth. One can TRP Channels as Drug Targets 781

Fig. 38. TRP channels in the human skin: implications for dermatological disorders. See the text for details. Reprinted with permission from Moran et al. (2011). also argue that TRPV1 antagonist hair lotions may so, topical TRPV1 agonist application may improve reverse hair loss. acne vulgaris, possibly in combination with existing Of note, TRPV1 is also expressed in human seba- therapeutic options such as , hormonal ceous glands as well as the immortalized SZ95 sebocyte treatment, and . The involvement of TRPV1 cell line (Tóth et al., 2009). In these cells, activation of in solar aging of the skin is controversial (Lan et al., TRPV1 selectively inhibits both basal and arachidonic 2013). acid–induced lipid synthesis (a hallmark of sebocyte TRPV3 is abundantly expressed in the skin (Peier differentiation) and alters the expression profiles of et al., 2002; Xu et al., 2002; Chung et al., 2003), multiple genes involved in cellular lipid homeostasis. If including the hair follicles (Asakawa et al., 2006). 782 Nilius and Szallasi

Indeed, TRPV3 knockout mice have a characteristic 20%. In the skin of NC/nga mice, a model of atopic hair phenotype that includes wavy hair coat and curly dermatitis, an increased density of nerve fibers that whiskers (they also have a very thin stratum corneum) coexpress GRP with TRPV1 was detected (Tominaga (Cheng et al., 2010a). Apparently, normal hair growth et al., 2009). Indeed, desensitization to capsaicin requires tightly controlled TRPV3 activity. For exam- relieves scratching behavior in NC/nga mice. Moreover, ple, the constitutively active, gain-of-function chemical ablation of GRP+/TRPV1+ nerves by neonatal gene mutation (TRPV3G573S) is associated with a hair- capsaicin administration prevents the development of less phenotype both in DS-Nh mice and WBN/Kob-Ht atopic dermatitis–like symptoms in these animals rats (Asakawa et al., 2006; Xiao et al., 2008). In these (Mihara et al., 2004). Importantly, the TRPV1 antag- animals, histologic analysis revealed impaired hair onist PAC-14028 ameliorates the atopic dermatitis– follicle morphogenesis and hair cycling (Imura et al., like symptoms in the NC/nga mice (Lim and Park, 2007). In rodents, TRPV3 is thought to form a signal- 2012). These observations have paved the way to the plex with TNFa and the EGFR that regulates hair phase I clinical trials with PAC-14028 in patients with morphogenesis. In human hair follicle organ cultures, atopic dermatitis. Of note, artemin is increased in activation of TRPV3 also inhibited hair shaft elonga- atopic dermatitis skin lesions. In mice, intradermal tion and promoted apoptosis-driven catagen regression artemin injections cause abnormal sensory nerve sprout- (Borbíró et al., 2011). Combined, these observations ing and thermal hyperalgesia. In transgenic mice, skin- suggest that TRPV3 agonists may control unwanted expressed artemin is retrogradely transported into the hair growth (e.g., hirsutism); conversely, TRPV3 antag- sensory ganglia, where it increases TRPV1 expression onists may promote hair growth in patients with (Murota et al., 2012). These findings give further alopecia (see Moran et al., 2011; Nilius and Bíró, credence to the PAC-14028 trials. Somewhat confusingly, 2013). in NC/Tnd mice, another model of human atopic derma- It is noteworthy that DS-Nh mice carrying the gain- titis, the stimulation of TRPV1, but not the blockade, of-function trpv3 gene mutation (TRPV3G573S) not only relieved the scratching behavior (Amagai et al., 2013). show hair loss but also develop a skin disorder that Human keratinocytes in culture express various resembles human atopic dermatitis (Yoshioka et al., TRPC channels, including TRPC6. In psoriatic skin 2009). Moreover, TRPV3G573S transgenic mice that lesions, the expression of TRPC6 is markedly reduced overexpress the constitutively active mutant TRPV3 (Leuner et al., 2011). Activation of TRPC5 by hyperforin in epidermal keratinocytes also spontaneously de- partially restores the disturbed keratinocyte differentia- velop an atopic dermatitis–like skin disorder. tion in psoriatic lesions (Müller et al., 2008). This implies A widely held concept states that atopic dermatitis a therapeutic potential for TRPC6 agonist creams in the requires a breached skin barrier due to scratching and/ pharmacotherapy of psoriasis. TRPV1 is another poten- or impaired barrier formation. Interestingly, skin barrier tial drug target in psoriasis. It was speculated that formation is impaired both in TRPV3-null and gain- neuropeptides released from TRPV1-positive sensory of-function mutant mice (Cheng et al., 2010a). It was nerve endings innervating the skin (in particular, SP) postulated that TRPV3 forms a complex with ADAM17, exert trophic functions on keratinocytes (reviewed in EGFR, and TGFa to regulate terminal keratinocyte Szallasi and Blumberg, 1999). Overactivity of these differentiation (reviewed in Nilius and Bíró, 2013). nerves (manifesting in increased SP release) may Apparently, both increased and decreased TRPV3 pro- impair keratinocyte differentiation and contribute to duction interferes with this signaling pathway. Hairless the pathogenesis of psoriasis (Divito et al., 2011). rats fed a Mg2+-deficient diet develop dermatitis. It was Indeed, on a largely empirical basis, topical capsaicin speculated (Luo et al., 2012a) that TRPV3 in keratino- was tried in a small number of patients with psoriasis cytes is under the tonic inhibitory control of Mg2+. with conflicting reports as to clinical benefit (see Dietary Mg2+ deficiency liberates TRPV3 from this Hautkappe et al., 1998). negative control and results in a constitutively active Skin biopsies taken patients with rosacea reveal channel. It is noteworthy that in many patients, an increase in TRPV3-positive dermal (but not in pregnancy (i.e., often associated with subclinical Mg2+ epidermal) cells (Sulk et al., 2012). With an estimated deficiency) aggravates atopic dermatitis. Of note, TRPV4 18 million Americans affected, rosacea (watery eyes contributes to intercellular bridge formation between and small visible blood vessel in facial skin) is a keratinocytes (Sokabe et al., 2010) and skin barrier is common but poorly understood disease. Although it also compromised in TRPV4 knockout mice. Human is clinically indolent, rosacea may cause significant keratinocytes reveal abundant TRPV4 expression (Facer psychologic and social problems such as low self- et al., 2007). Administration of a topical TRPV4 agonist esteem and avoidance of social interactions. There is (GSK1016790A) protects skin barrier formation (Kida no known cure for rosacea. If the TRPV3+ cells in the et al., 2012). dermis are involved in the pathogenesis of rosacea, The prevalence of atopic dermatitis is increasing in they may represent the first real breakthrough for American pediatric populations already approaching finding a cure. TRP Channels as Drug Targets 783

I. Transient Receptor Potential Channels and have not yet been fully elucidated. Since growth factors Eye Diseases such as VEGF and platelet-derived growth factor guide Dry eye disease (also known as xerophthalmia or angiogenic sprouting during physiologic angiogenesis, it keratoconjunctivitis sicca) is a common condition, the is a reasonable assumption that aberrant neovasculari- “ ” prevalence of which is increasing with age, affecting zation may be due to a dysorchestrated production of 10% of the elderly population. The management of dry these growth factors. A shared feature of ocular neo- eye disease can be frustrating because artificial tear vascularization disorders is ischemia, which, in turn, drops provide only temporary relief. Symptoms are was shown to upregulate VEGF production. Indeed, it often worse during the summer months when patients was suggested that VEGF may be the predominant angiogenic stimulus in proliferative retinopathies, pav- sleep in air-conditioned rooms with reduced humidity. ing the way to clinical trials with VEGF antagonists. Although dry eye disease is indolent, it has been Although VEGF inhibitors (e.g., ranimizumab and associated with sleep disturbances/deprivation and bevacizumab) have no doubt revolutionized the care resultant depression. In mice with allergic keratocon- of patients with age-related macular degeneration, junctivitis, the (nonselective) TRPV1 antagonist cap- they appear to be less effective in other forms of sazepine reduced tearing and blinking (Acosta et al., ocular neovascularization disorders, indicating that 2013). VEGF is not the only agent that drives the pathology The mouse cornea is densely innervated by TRPM8- in these patients. positive, cold-responsive afferents (Robbins et al., TRPV4 is highly expressed in the mouse and human 2012). Indeed, in some studies, the vast majority (up retina, where it is involved in mediating Ca2+ currents to 90%) of corneal afferents were activated by cold and/ (Gilliam and Wensel, 2011; Ryskamp et al., 2011). or menthol (Hirata and Oshinsky, 2012). Low concen- Importantly, retinal endothelial cells also express trations of menthol increased basal lacrimation in functional TRPV4 channels. Indeed, VEGF evokes wild-type, but not in TRPM8 knockout, mice (Robbins 2+ Ca currents in retinal endothelial cells that are et al., 2012). Importantly, at concentrations at which it blocked by the TRPV4 antagonists RN1734 [2,4-dichloro-N- increased tear production, menthol did not evoke isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide] nocifensive behavior, nor did it interfere with pro- and HC-067046. It was speculated that TRPV4 might tective eye reflexes. Taken together, these findings function in retinal endothelial cells as a shared down- imply that a TRPM8 agonist-containing eye drop may stream target for VEGF and other stimuli of abnormal be a novel therapeutic approach to stimulate tear angiogenesis. If this hypothesis holds true, TRPV4 production in patients with dry eye disease (Parra antagonist-containing eye drops may supplement (or et al., 2010; Kurose and Meng, 2013). even supplant) VEGF inhibitors in the medical man- Glaucoma is a group of diseases that can damage the agement of patients with ocular neovascularization optic nerve. The cause of the nerve damage is usually disorders. an increase in the fluid pressure inside the eye. Without treatment, first the peripheral (or side) vision is lost and then the straight ahead vision decreases IV. Conclusions until no vision remains. In fact, glaucoma is the Many great success stories open with humble leading cause of blindness in the United States. It beginnings. The TRP channel story began in 1969 when was recently speculated that TRPC6, a hyperosmotic Cosens and Manning identified a spontaneously formed solution and pressure-sensitive channel, may be an Drosophila mutant that behaved as if it was blind under appealing target in glaucoma patients who are not bright illumination (Cosens and Manning, 1969). candidates for eye surgery (Fan et al., 2012). In Turkish Twenty years later, the gene responsible for this patients, the TRPM5 polymorphism rs34551253 was abnormal light response was identified and termed trp reported to confer increased risk for developing open- for “transient receptor potential” (Montell and Rubin, angle glaucoma (Okumus et al., 2013). 1989). This seminal discovery paved the way to the Ocular neovascularization disorders (including pro- isolation of the first mammalian homologs of the liferative retinopathy of prematurity, abnormal growth Drosophila TRP channels, the so-called TRPCs (Wes of blood vessels and associated vascular leakage in et al., 1995; Zhu et al., 1995). Since then, mammalian diabetic retinopathy, and exudative age-related macular TRP channels have grown in number to form a unique degeneration) represent a leading cause of blindness (different from traditional ligand-gated ion channels) worldwide. Angiogenesis is the physiologic process by superfamily of 28 proteins that can be subdivided into which new blood vessels (capillaries) develop from six subfamilies: TRPA, TRPC, TRPM, TRPML, TRPP, preexisting ones. Aberrant growth of blood vessels and TRPV. (neovascularization) is believed to constitute the final, TRP channels are conserved during evolution and common pathway in severe retinopathies. The mecha- are expressed in most cell types, where they exert strong nisms that govern this pathologic neovascularization effects on cellular functions and signaling pathways. Not 784 Nilius and Szallasi surprisingly, mutations in genes encoding human TRP et al., 2007). Concentrated efforts by approximately 50 channels are responsible for hereditary diseases, which pharmaceutical companies have yielded a plethora are referred to as TRP channelopathies. Diseases caused (.1000) of patents and compounds, none of which has by gain-of-function mutations (e.g., TRPV3 in Olmsted progressed thus far beyond stage 2 of clinical de- syndrome) may be more amenable to therapeutic in- velopment due to lack of clinical efficacy and/or on- tervention because overactive channels could be targeted target adverse effects, most important, hyperthermia by small molecule antagonists. For diseases caused by and burns secondary to impaired noxious heat pain loss-of-function mutations, reversal of the abnormal chan- sensation (reviewed in Moran et al., 2011; Kort and nel function is likely to require a less validated approach Kym, 2012; Szallasi and Sheta, 2012; Brederson et al., such as gene therapy. 2013; Szolcsányi and Pintér, 2013). For other TRP The rapid progress in TRP channel research has channels, on-target side effects may represent an even brought the understanding of the pathogenic roles that bigger hurdle. For example, blockade of TRPM4 may these channels play in the development and mainte- be beneficial in the treatment of MS (Schattling et al., nance of acquired diseases within reach. Whereas the 2012) and anaphylaxis (Vennekens et al., 2007, but it initial emphasis was on “painful” TRP channels ex- may cause potentially fatal cardiac arrhythmias (Abriel pressed on nociceptive neurons (Patapoutian et al., et al., 2012). TRPV4 is an even more problematic drug 2009), recent research has expanded TRP channel drug target because both gain-of-function and loss-of-function discovery efforts into new disease areas such as re- TRPV4 mutations have been linked to human disease spiratory disorders (chronic cough, COPD, and asthma), (Nilius and Voets, 2013). Indeed, systemic activation of chronic itch, obesity, diabetes, overactive bladder, endothelial TRPV4 by the agonist GSK1016790A caused anxiety, addiction, stroke, cardiac hypertrophy, and catastrophic cardiovascular collapse (Willette et al., 2008). cancer (Fig. 39) (reviewed in Moran et al., 2011; To a limited degree, systemic side effects may be Kaneko and Szallasi, 2013). This is an exciting de- prevented by organ-specific drug delivery (e.g., topical velopment since many of these disorders represent creams for dermatological disorders and nebulizers for large, unmet medical needs. airway diseases). An innovative alternative strategy After the molecular cloning of the capsaicin receptor to circumvent side effects is by selectively targeting TRPV1 in 1997 (Caterina et al., 1997), it took less than TRP channels in diseased tissues. For example, TRPV1 a decade to develop the first TRPV1 antagonists to be is sensitized by chemical messengers or signaling tried in the clinics as novel analgesic drugs (Szallasi pathways during inflammation (Szallasi et al., 2007).

Fig. 39. Schematic model of the complex role of TRP channels in blood pressure regulation and in the development and maintenance of hypertension. Courtesy of Dr. Ryuji Inoue. TRP Channels as Drug Targets 785

Augmented permeation of sensitized TRPV1 by the should be compared with currently available therapeutic activity-dependent agonist Cap-ET allows the targeted options. It is important to temper our expectations and delivery of the cationic anesthetic Na+ channel blocker remember that not a single category of drugs will ever do QX-314, taming neurons with hyperactive TRPV1 but the trick. Indeed, we must accept the need for drug sparing normal nociception (Li et al., 2011a). Sensiti- combinations. For most indications, TRP channel drugs zation itself seems to depend on the interaction of will not be “magic bullets,” but may be useful (although TRPV1 with the scaffolding protein AKAP79 (Zhang imperfect) parts of therapeutic regimens. The obstacles et al., 2008d); indeed, molecules that interfere with this facing the development of clinically useful TRP channel interaction exhibit analgesic potential (Btesh et al., drugs are real but are probably not insurmountable. 2013; Fischer et al., 2013). Clearly, there is a dire need for effective translational There is increasing evidence for redundancy in TRP research by getting the clinicians together with the basic channel functions; therefore, selectively blocking a researchers. With this review, we hope to facilitate channel may not achieve the desired clinical effect. this process. One is tempted to remember Churchill’s For example, Belvisi and colleagues (2011) recently description of El-Alemein, when he stated “This is not suggested that clinically meaningful cough relief may the beginning of the end, but the end of the beginning” require the simultaneous inhibition of TRPV1 and (www.brainyquote.com/quotes/quotes/w/winstonchu163144. TRPA1 (Grace et al., 2012). This implies that a func- html). tionally integrated constellation of signaling molecules (in our example, TRPV1 and TRPA1 coexpressed in vagal afferents mediating cough) is a better target for Appendix effective intervention than a single signaling molecule, a concept referred to as “constellation pharmacology” Suggested Reading: Books on TRP Channels: (Teichert et al., 2012). Chronic neuropathic pain is an even better example. Although the loss-of-function Flockerzi V and Nilius B, eds. (2007) Transient receptor TRPV1 variant I585V was reported to confer decreased potential (TRP) channels, in Handb Exp Pharmacol, 179, risk for painful OA (Valdes et al., 2011), neither the Springer Berlin-Heidelberg-New York. selective TRPV1 antagonist AMG9810, nor the TRPA1 Islam MS, ed. (2011) Transient receptor po- blocker HC-030031, ameliorated ongoing spontaneous tential channels, in Advances in Experimental pain in a model of advanced OA (Okun et al., 2012). Medicine and Biology Series, Vol. 704, Springer, Moreover, the TRPV1 antagonist ABT-116 failed to New York. provide pain relief in client-owned dogs with hip OA Liedtke WB and Heller S, eds. (2007) TRP Ion (Malek et al., 2012), and a clinical trial with AZD1386 Channel Function in Sensory Transduction and Cellular involving 241 patients with OA in seven different Signaling Cascades, CRC Press, Boca Raton, FL. countries had to be terminated prematurely for lack of Nilius B and Flockerzi V, eds. (2014) Mammalian efficacy (Svensson et al., 2010; Miller et al., 2014). transient receptor potential (TRP) cation channels (vol. These disappointing results contrast with the power- 1), in Handb Exp Pharmacol, 222, Springer, Berlin- ful and lasting analgesic action of intrathecal or intra- Heidelberg-New York. articular RTX in dogs with severe OA (Iadarola and Nilius B and Flockerzi V, eds. (2014) Mammalian Gonnella, 2013). RTX silences the whole TRPV1- transient receptor potential (TRP) cation channels expressing nerve, providing the ultimate example of (vol. 2), in Handb Exp Pharmacol, 223,Springer, constellation pharmacology. Berlin-Heidelberg-New York. The more details we learn about TRP channels, the Novartis Foundation (2004) Mammalian TRP Chan- more questions arise. Most of our knowledge was nels as Molecular Targets, John Wiley & Sons Ltd, obtained in knockout mice and rodent models of human Chichester, UK. disease. It is becoming increasingly clear that these Sherkheli A (2010) Trip through the Pharmacol- models do not necessarily reflect the human disease as ogy of Hot- and Cold-Sensing TRP Channels: A exemplified by the lack of analgesic effect of the TRPV1 Hope for Better Painkiller and Antiinflammatory antagonist AZD1386 in clinical trials enrolling patients Drugs, VDM Verlag Dr. Muller, Saarbruecken, with pain related to GERD (Krarup et al., 2013) or OA Germany. (Svensson et al., 2010). Yet, TRPV1 remains a viable Szallasi A (2011) TRP Channels in Health and therapeutic target. Intravesical RTX dramatically Disease: Implications for Diagnosis and Therapy.Nova improved bladder function in patients with overactive Science Publishers, Hauppauge, New York. bladder (Cruz et al., 1997), and intrathecal RTX Szallasi A and Bíró T, eds. (2012) TRP channels in provided permanent relief of intractable cancer pain drug discovery, in Methods in Pharmacology and (Iadarola and Mannes, 2011). Toxicology Series, Springer, New York. In summary, this is a rapidly evolving field. The Zhu MX, ed. (2011) TRP Channels, CRC Press, Boca benefits and side effects of drugs targeting TRP channels Raton, FL. 786 Nilius and Szallasi

Note Added in Proof Ahern GP (2003) Activation of TRPV1 by the satiety factor oleoylethanolamide. J Biol Chem 278:30429–30434. Most recently, differential methylation of the TRPA1 gene pro- Ahn MH, Park BL, Lee SH, Park SW, Park JS, Kim DJ, Jang AS, Park JS, Shin HK, moter has been linked to discordant heat pain sensitivity in humans and Uh ST, et al. (2011) A promoter SNP rs4073T.A in the common allele of the (Bell et al., 2014), suggesting that not only TRP gene variants but also interleukin 8 gene is associated with the development of idiopathic pulmonary fibrosis via the IL-8 protein enhancing mode. Respir Res 12:73–85. their epigenetic changes may be involved in the development of Aizawa N, Wyndaele JJ, Homma Y, and Igawa Y (2012) Effects of TRPV4 cation chronic pain. In mice, the temperature sensitivity of the TRPA1 channel activation on the primary bladder afferent activities of the rat. Neurourol channel protein (cold vs. warm) is apparently determined by only three Urodyn 31:148–155. Akazawa Y, Yuki T, Yoshida H, Sugiyama Y, and Inoue S (2013) Activation of TRPV4 amino acid residues (Jabba et al., 2014); however, the native channel strengthens the tight-junction barrier in human epidermal keratinocytes. Skin is not involved in autonomic thermoregulation (de Oliveira et al., Pharmacol Physiol 26:15–21. 2014). It is noteworthy that TRPA1 blockade exerted an anxiolytic-like Akbar A, Yiangou Y, Facer P, Walters JR, Anand P, and Ghosh S (2008) Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome action in mice (de Moura et al., 2014). If this observation holds true in and their correlation with abdominal pain. Gut 57:923–929. humans, TRPA1 antagonists (currently investigated in clinical trials Akiyama T, Carstens MI, and Carstens E (2010) Enhanced scratching evoked by as and cough-relieving drugs) may have an added clinical PAR-2 agonist and 5-HT but not histamine in a mouse model of chronic dry skin – benefit. itch. Pain 151:378 383. Akiyama T, Tominaga M, Carstens MI, and Carstens EE (2012) Site-dependent and Another recently discovered TRP channel involved in anxiety is state-dependent inhibition of pruritogen-responsive spinal neurons by scratching. TRPC4 (Riccio et al., 2014). Of note, Trpc4 KO mice also show Eur J Neurosci 36:2311–2316. reduced self-administration (Rasmus et al., 2013), implying Akopian AN, Ruparel NB, Jeske NA, Patwardhan A, and Hargreaves KM (2009) Role of ionotropic cannabinoid receptors in peripheral antinociception and anti- a role for addictive behavior. Because TRPC4 blockade restores hyperalgesia. Trends Pharmacol Sci 30:79–84. erectile function in diabetic rats (Sung et al., 2014), drugs targeting Akopian AN, Ruparel NB, Patwardhan A, and Hargreaves KM (2008) Cannabinoids this channel seem to have a number of attractive indications, ranging desensitize capsaicin and mustard oil responses in sensory neurons via TRPA1 activation. J Neurosci 28:1064–1075. from behavioral health to erectile dysfunction. Al-Shawaf E, Naylor J, Taylor H, Riches K, Milligan CJ, O’Regan D, Porter KE, Li J, The small-molecule dual TRPC3 and TRPC6 antagonists and Beech DJ (2010) Short-term stimulation of calcium-permeable transient re- GSK2332255A and GSK2833503A prevented the development of ceptor potential canonical 5-containing channels by oxidized phospholipids. Arte- rioscler Thromb Vasc Biol 30:1453–1459. cardiac hypertrophy in rodents subjected to pressure overload, AL-Shawaf E, Tumova S, Naylor J, Majeed Y, Li J, and Beech DJ (2011) GVI although genetic deletion of Trpc3 or Trpc6 alone was not protective phospholipase A2 role in the stimulatory effect of sphingosine-1-phosphate on in the same model (Seo et al., 2014). This finding supports the notion TRPC5 cationic channels. Cell Calcium 50:343–350. that nonselective TRP channel blockers may be superior to selective Albert AP, Saleh SN, and Large WA (2009) Identification of canonical transient receptor potential (TRPC) channel proteins in native vascular smooth muscle cells. ones in pharmacotherapy. Curr Med Chem 16:1158–1165. Lastly, TRPC5 expression was identified as an adverse prognostic Alenmyr L, Greiff L, Andersson M, Sterner O, Zygmunt PM, and Högestätt ED (2012) marker in breast cancer, predicting resistance to chemotherapy and Effect of mucosal TRPV1 inhibition in allergic rhinitis. Basic Clin Pharmacol Toxicol 110:264–268. poor survival (Ma et al., 2014). Alenmyr L, Uller L, Greiff L, Högestätt ED, and Zygmunt PM (2014) TRPV4-mediated calcium influx and ciliary activity in human native airway epithelial cells. Basic Clin Pharmacol Toxicol 114:210–216. Acknowledgments Alessandri-Haber N, Dina OA, Chen X, and Levine JD (2009) TRPC1 and TRPC6 TheauthorsthankDrs.GiovanniAppendino,DottieCimino- channels cooperate with TRPV4 to mediate mechanical hyperalgesia and noci- ceptor sensitization. J Neurosci 29:6217–6228. 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