ASSOCIATE EDITOR: ARTHUR CHRISTOPOULOS The G Protein ...pharmrev.aspetjournals.org/content/pharmrev/67/1/36.full.pdf · stimuli (hyperalgesia), the perception of pain from normally

1521-0081/67/1/36–73$25.00 http://dx.doi.org/10.1124/pr.114.009555PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:36–73, January 2015Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: ARTHUR CHRISTOPOULOS

The G Protein–Coupled Receptor–Transient ReceptorPotential Channel Axis: Molecular Insights for

Targeting Disorders of Sensation and InflammationNicholas A. Veldhuis, Daniel P. Poole, Megan Grace, Peter McIntyre, and Nigel W. Bunnett

Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (N.A.V., D.P.P., N.W.B); Departments of Genetics (N.A.V.),Anatomy and Neuroscience (D.P.P.), and Pharmacology (N.W.B.), The University of Melbourne, Melbourne, Victoria, Australia; School ofMedical Sciences and Health Innovations Research Institute, RMIT University, Bundoora, Victoria, Australia (M.G., P.M.); and ARC Centre

of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria, Australia (N.W.B.)

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38II. The G Protein–Coupled Receptor–Transient Receptor Potential Axis in Pain Transmission . . . . 40

A. G Protein–Coupled Receptors and Transient Receptor Potential Channels as MolecularSensors of Nociceptive Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

B. The Evolutionarily Conserved G Protein–Coupled Receptor–Transient ReceptorPotential Ion Channel Coupling Paradigm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

C. G Protein–Coupled Receptors that Sense Noxious Stimuli and InitiateNeurogenic Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411. Algesic G Protein–Coupled Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

a. Protease-activated receptors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42b. Bradykinin receptors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

2. Analgesic G Protein–Coupled Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44D. Pain Transducing Transient Receptor Potential Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

1. Transient Receptor Potential Vanilloid 1.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462. Transient Receptor Potential Vanilloid 2.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463. Transient Receptor Potential Vanilloid 3.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464. Transient Receptor Potential Vanilloid 4.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475. Transient Receptor Potential Melastatin 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476. Transient Receptor Potential Melastatin 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477. Transient Receptor Potential Melastatin 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478. Transient Receptor Potential Ankyrin 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489. Transient Receptor Potential Canonical.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

III. Molecular Substates for the G Protein–Coupled Receptor–Transient Receptor Potential ChannelAxis: Regulation of Transient Receptor Potential Channels by Lipid and Protein Interactions . . . . 49A. Transient Receptor Potential Channel Structure: Improving Our Understanding

of Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49B. Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49C. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50D. Kinase Scaffold Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

1. A-Kinase Anchoring Protein and Receptor for Activated C-Kinase 1. . . . . . . . . . . . . . . . . . . . 512. b-Arrestins as Adaptors for G Protein–Coupled Receptors and Transient Receptor

Potential Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51IV. The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis in Visceral Pathways. . . . 52

Research in the authors’ laboratories is supported by the National Health & Medical Research Council; The Australian Research Council;RMIT University; and Monash University.

Address correspondence to: Dr. Nigel Bunnett, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, VIC 3052,Australia. E-mail: [email protected]

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A. Transient Receptor Potential Channels in Inflammatory Bowel Disease and FunctionalBowel Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521. Sensitization of Transient Receptor Potential Channels: Mechanisms and Role

in Colonic Hypersensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522. Protease-Dependent Sensitization of Transient Receptor Potential Channels in

Inflammatory Bowel Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53B. Transient Receptor Potential Channels, Pancreatitis, and Pancreatic Pain . . . . . . . . . . . . . . . . 54

1. Transient Receptor Potential Channels and Pancreatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542. Sensitization and Activation of Transient Receptor Potential Channels

in Pancreatitis and Pancreatic Pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54V. Not-So-Painful: Contributions of the G Protein–Coupled Receptor–Transient Receptor

Potential Channel Axis for Irritant Sensation and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55A. The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis in Pruritus . . . 55

1. Transient Receptor Potential Vanilloid 1.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552. Transient Receptor Potential Ankyrin 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563. Transient Receptor Potential Vanilloid 3.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574. Bile Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

B. The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis inAirway Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581. Transient Receptor Potential Vanilloid 1.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582. Transient Receptor Potential Ankyrin 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583. Transient Receptor Potential Vanilloid 4.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594. Transient Receptor Potential Melastatin 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595. G Protein–Coupled Receptor–Dependent Sensitization of Transient Receptor

Potential Channels in Airway Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60VI. Targeting G Protein–Coupled Receptors, Transient Receptor Potential Channels, and the

G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis . . . . . . . . . . . . . . . . . . . . . 60A. The Need for Alternative Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60B. Lessons from Analysis of Transient Receptor Potential Channel Expression in Sensory Neurons . . . 61

1. Improved Techniques for Expression Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612. Upregulation of Transient Receptor Potential Channels in Pain Conditions. . . . . . . . . . . . . 61

C. Current State of Transient Receptor Potential Agonists and Antagonists . . . . . . . . . . . . . . . . . . 611. Transient Receptor Potential Vanilloid 1 Agonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622. Other Transient Receptor Potential Agonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623. Transient Receptor Potential Vanilloid 1 Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624. Other Transient Receptor Potential Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

D. Therapeutic Targeting of G Protein–Coupled Receptor–Transient ReceptorPotential Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

VII. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Abstract——Sensory nerves are equippedwith recep-tors and ion channels that allow them to detect andrespond to diverse chemical, mechanical, and thermalstimuli. These sensory proteins include G protein–coupledreceptors (GPCRs) and transient receptor potential (TRP)

ion channels. A subclass of peptidergic sensory nervesexpress GPCRs and TRP channels that detect noxious,irritant, and inflammatory stimuli. Activation of thesenerves triggers protective mechanisms that lead towithdrawal from danger (pain), removal of irritants

ABBREVIATIONS: AA, arachidonic acid; AKAP, A-kinase anchoring protein; BCTC, N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide; BK, bradykinin; CaM, Ca2+-binding calmodulin protein; CB, cannabinoid receptor; CGRP, calcitonin gene-related peptide; COPD, chronicobstructive pulmonary disease; DAG, diacylglycerol; DRG, dorsal root ganglion; EET, epoxyeicosatrienoic acid; EP R, prostaglandin E2 receptor 1; ERK1/2,extracellular signal-related kinases (also known as MAPK1/2 or p44/p42); Ga, heterotrimeric GTP-binding protein a subunit; Gbg, heterotrimericG protein subunits b and g; GPCR, G protein–coupled receptor; HC-030031, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropyl-phenyl)acetamide; HNE, 4-hydroxynonenal; 5-HT, 5-hydroxytryptamine; IB4+, isolectin B4+; IBD, inflammatory bowel disease; IBS, irritable bowelsyndrome; IP3, inositol-1,4,5-trisphosphate; Mrgpr, Mas-related G protein–coupled receptor; NGF, nerve growth factor; PAR, protease-activated receptor;PDE, phosphodiesterase; PG, prostaglandin; PKA, cAMP-dependent protein kinase A; PKC, protein kinase C; PKD, protein kinase D (atypical PKCm); PIP2,phosphatidylinositol-4,5-bisphosphate; Pirt, PIP-binding regulator of TRP channels; PLA, phospholipase A; PLC, phospholipase C; SP, substance P; TG,trigeminal ganglion; TGR5, bile acid receptor (GPBA receptor); TLSP, thymic stromal lymphopoietin; TRPA, transient receptor potential ankyrin; TRPC,transient receptor potential canonical; TRPM, transient receptor potential melastatin; TRPV, transient receptor potential vanilloid.

The GPCR-TRP Axis 37

(itch, cough), and resolution of infection (neurogenicinflammation). The GPCR-TRP axis is central to thesemechanisms. Signals that emanate from the GPCRsuperfamily converge on the small TRP family, leading tochannel sensitization and activation, which amplify pain,itch, cough, and neurogenic inflammation. Herein wediscuss how GPCRs and TRP channels function indepen-dently and synergistically to excite sensory nerves thatmediate noxious and irritant responses and inflammation

in the skin and the gastrointestinal and respiratory sys-tems. We discuss the signaling mechanisms that underliethe GPCR-TRP axis and evaluate how new informa-tion about the structure of GPCRs and TRP channelsprovides insights into their functional interactions. Wepropose that a deeper understanding of the GPCR-TRPaxis may facilitate the development of more selective andeffective therapies to treat dysregulated processes thatunderlie chronic pain, itch, cough, and inflammation.

I. Introduction

Sensory nerves that innervate the skin and the majorvisceral organ systems play a vital role in protection.The cell bodies of these neurons in dorsal root ganglia(DRG) or trigeminal ganglia (TG) project nerve fibers toperipheral tissues and to the dorsal horn of the spinalcord. Their peripheral projections are equipped withmany receptors and ion channels that allow them todetect and respond to diverse chemical, mechanical, andthermal stimuli. A subclass of these neurons expressesreceptors and channels that are specialized for thedetection of noxious, irritant, and inflammatory stimuli.The activation of these nerves triggers acute protectiveprocesses that lead to withdrawal from danger (pain),removal of irritants (itch, cough), and resolution of infection(neurogenic inflammation). These physiologic processesare essential for survival and are normally under tightcontrol. However, dysregulation and disease can lead tochronic pain, pruritus, cough, and inflammation, whichare poorly understood, difficult to treat, and are a majorcause of suffering. A deeper understanding of thesenormal protective processes and of how they becomedysregulated during diseases is required to developmore selective and effective therapies for these chronicconditions.Dysregulated sensory responses to noxious, irritant,

and inflammatory stimuli underlie diseases of globalrelevance and can be caused by chronic inflammatoryand neuronal diseases. Inflammatory and neuropathicpain is characterized by heightened responses to painfulstimuli (hyperalgesia), the perception of pain fromnormally non-noxious stimuli (allodynia), and by lossof inhibitory mechanisms that prevent central paintransmission (reviewed in Scholz and Woolf, 2007;Patapoutian et al., 2009). Pain is a major societal burden.It is estimated to afflict 1 in 5 people at some pointduring their lives, and pain is the leading public healthproblem in the United States, where 100 million peopleseek medical attention each year because of acute orchronic pain, with an enormous economic impact (Gaskinand Richard, 2012). The acute and protective process ofitch can also become dysregulated and chronic (seereviews by Ikoma et al., 2006, and Bautista et al., 2014).Acute itch, such as that caused by an insect sting, istransient and can be readily treated with histaminereceptor antagonists. However, dermal and metabolicdiseases can lead to chronic pruritus that is resistant

to antihistamine drugs and is poorly understood anddifficult to treat. Neuropathic itch is caused by manyneurologic disorders (Binder et al., 2008). For example,the pruritus of patients with cholestatic liver disease canbe so profound and intractable that it is an indicationfor liver transplantation (European Association for theStudy of Liver, 2009). Defects in the normally protectiveprocesses of acute inflammation can lead to the chronicinflammatory diseases that affect all organ systems, andwhich can also lead to chronic pain (see reviews by Galliet al., 2008, and Grace et al., 2014b).

G protein–coupled receptors (GPCRs) and transientreceptor potential (TRP) ion channels are cell surfaceproteins of sensory nerves functioning at the “front line”for sensing noxious, irritating, and inflammatory stimu-lants (Fig. 1). With;850 members in mammals, GPCRsare the largest family of signaling proteins. They arereceptors for structurally diverse endogenous and exog-enous ligands, participate in all pathophysiological pro-cesses, and are established therapeutic targets, with 30%of current drugs targeting GPCRs (Overington et al.,2006). More than 40 GPCR families contribute to painprocessing and function as sensors on peripheral nervesto detect noxious, irritant, and inflammatory stimuli,including proteases, peptides, amines, and lipids (Stoneand Molliver, 2009). Drug-screening efforts targetingnociceptive GPCRs, such as neurokinin or bradykininreceptors, have resulted in the identification of manyantagonists that are effective in preclinical studies ofdisease and led to great expectations for new effectivepain treatments. However, with the exception of histaminereceptor antagonists, these drugs have generally beendisappointing in clinical trials (Steinhoff et al., 2014). TRPchannels comprise a small family of nonselective cationchannels (28 members in mammals) that also participatein widespread pathophysiologic processes and are anemerging therapeutic target (Patapoutian et al., 2009;Brederson et al., 2013). TRP channels of sensory nervesdirectly sense endogenous and exogenous chemical,mechanical, and thermal stimuli. TRP channels arealso major downstream effectors of GPCR signaling.This GPCR-TRP axis is vitally important for pain, itch,cough, and neurogenic inflammation (Basbaum et al.,2009; Bautista et al., 2014; Grace et al., 2014b). Signalingpathways that emanate from the GPCR superfamilyconverge on the small TRP family, leading to alteredchannel activity or expression. GPCR signaling events

38 Veldhuis et al.

can generate mediators that stimulate TRP channels(i.e., channel activation) or that enhance their responsesto TRP agonists (i.e., TRP sensitization). The net resultis that TRP channels can amplify that effects of GPCRsand mediate their contributions to transmission of pain,itch, cough, and neurogenic inflammation (Clapham,2003).The discovery of functional cross-talk between GPCRs

and TRP channels occurred concurrently with thecharacterization of the first TRP channels in Drosophilamelanogaster. Known as receptor-operated channel

theory, this mechanism describes how GPCRs stimulatecell signaling and lipid metabolism pathways, whichconverge on TRP channels to lower their activationthreshold and initiate rapid ion flux across the cell surfaceor internal membranes (Montell, 2005b). In mammals, theGPCR-TRP axis is enriched by the coexpression of GPCRsand TRPs in select cell types (e.g., subpopulations ofsensory neurons) and by an abundance of signalingintermediates (e.g., adaptor proteins, kinases, lipidmetabolites) that functionally link GPCRs to TRPs(Peth}o and Reeh, 2012). These intermediates afford

Fig. 1. Classification of GPCR-TRP channel interactions involved in major pain and sensory conditions. Acute activation of nociceptive or pruriceptivesensory neurons can trigger protective behavioral responses, such as avoidance behavior and irritant removal, including coughing and scratching.Dysregulation of these mechanisms can be caused by nerve damage, infection, or inflammation leading to heightened, chronic sensory, or pain states,including nociceptive pain, inflammatory pain, neuropathic pain, and itch. The associated disease states and their clinical settings, as well as theunderlying mediators, are described. These states are stimulated by different cellular processes (e.g., infection) or exogenous agents (e.g., insect sting)located centrally or in tissues of the periphery, such as the skin and peripheral nerve terminals. Numerous TRP channels contribute to inflammatorystates and can be sensitized by a host of GPCRs to reduce their activation threshold. This enhances normal nociceptive responses, leading toheightened pain (hyperalgesia) or allodynia. Associated references for GPCR-TRP channel sensitization can be found in Tables 1 and 2. CNS, centralnervous system; PNS, peripheral nervous system (adapted with permission from Patapoutian et al., 2009).


a high degree of specificity within the GPCR-TRPsignaling axis and explain how multiple GPCRs canconverge upon a limited number of TRP channels topromote different physiologic outcomes (e.g., pain versusitch). These signaling molecules may also provide newtargets for treatment of chronic pain, itch, cough, andinflammatory conditions. Furthermore, they may alsoincrease the usefulness of established GPCR and TRPchannel antagonists that are currently compromised byon-target side effects related to the widespread distri-bution of GPCRs and TRPs and their participation indiverse physiologic processes. A deeper understandingof the high-resolution structures of GPCRs and TRPchannels, including the identification of protein andlipid binding sites and of key regulatory domains, mayfacilitate the development of drugs that target theGPCR-TRP axis in a cell type–specific manner, leadingto improved treatments for disorders of sensation andinflammation.Herein we discuss the importance of the GPCR-TRP

axis in the detection of noxious, irritant, and in-flammatory stimuli in the skin and gastrointestinaland respiratory systems and summarize the relevanceof the axis to chronic pain, itch, cough, and inflammation.We discuss the signaling mechanisms that underlie theGPCR-TRP axis and evaluate how new informationabout the structure of GPCRs and TRP channels providesinsights into functional interactions. We review the utilityof antagonists of GPCRs and TRP channels for the treat-ment of chronic sensory and inflammatory disorders andassess whether targeting the GPCR-TRP axis representsa viable option for the treatment of chronic pain, itch,cough, and inflammation.

II. The G Protein–Coupled Receptor–TransientReceptor Potential Axis in Pain Transmission

A. G Protein–Coupled Receptors and TransientReceptor Potential Channels as Molecular Sensors ofNociceptive Neurons

Subsets of primary afferent neurons are the firstcells in the pathway that sense noxious stimuli. Theseare first-order pseudo-unipolar neurons, with a singleaxon branch that projects both peripherally to targetorgans and centrally to second-order neurons in thedorsal horn of the spinal cord. Neurons within the DRGproject to visceral organs and the periphery; afferentswithin the TG project to the head and face; vagalafferents of the nodose and jugular ganglia facilitatesensation in the head and visceral organs such as thelung and gastrointestinal tract. The peripheral projec-tions of these neurons express multiple receptors andion channels that allow them to detect and respond todiverse stimuli, providing critical sensory feedbackupon exposure to innocuous or noxious substances andenvironments. There are two classes of high-thresholdnociceptors that can centrally transmit information

about noxious stimuli: the moderate-conducting, thinlymyelinated Ad fibers and the slow-conducting smalldiameter, unmyelinated C-fibers. C-fibers are the pre-dominant form in the periphery and possess high noxiousthresholds to generate pain responses of greater intensityand duration (Stucky et al., 2001). These nociceptors arecommonly differentiated into two groups, those that are“nonpeptidergic” and bind the plant isolectin B4+ (IB4+)or the “peptidergic” neurons that express the nervegrowth factor (NGF) TrkA tyrosine kinase receptor andthe pronociceptive and proinflammatory neuropeptidessubstance P (SP), neurokinin A, and calcitonin gene-related peptide (CGRP). In addition to afferent fiber sizeand nerve conductance velocity, the receptor/ion channelexpression profile determines their sensory functionand defines subtypes of sensory neurons as “polymodalnociceptors” that can respond to more than one type ofstimulus (e.g., chemical, mechanical, thermal). For exam-ple, a subset of TrkA-positive C-fiber afferents expresstransient receptor potential ankyrin 1 (TRPA1) andtransient receptor potential vanilloid 1 (TRPV1) chan-nels to facilitate responses to noxious heat (.43°C) andnoxious chemicals (Story et al., 2003; Kobayashi et al., 2005;Wilson et al., 2011). However, not all C-fiber excitationleads to nociception, because polymodal C-fibers canalso express transient receptor potential melastatin 8(TRPM8) for response to nonpainful cooling stimuli(Basbaum et al., 2009).

TRP channels are sensors of the environment andintegrators of receptor signaling in a variety of celltypes. In primary neurons, relative to the ion currentsof their voltage-gated cousins (L-type calcium, sodium,and potassium ion channels), TRP channels exhibit slowerkinetics and initiate pain transmission via sustainedion flux, resulting in depolarizing membrane potentialsof sufficient duration and magnitude to evoke actionpotentials. To ensure that nociceptors generate robustand frequent action potentials only in response toharmful stimuli, TRP channels possess high thresholdsof activation, whereby ion conductance occurs in re-sponse to substantial changes in voltage, temperature,exogenous ligands, and intracellular lipids. Further-more, the likelihood of a nociceptive TRP channel tobecome active is determined by its open probability andcell surface expression, which can be markedly influencedby channel agonists and sensitizing agents (Studer andMcNaughton, 2010). GPCRs regulate kinases and thesynthesis of lipid second messengers, both of which cancontrol TRP activity. In this manner GPCRs influencemultiple pathways to lower the TRP channel activationthreshold (Ramsey et al., 2006). Once activated, TRPchannel-mediated pain transmission occurs via con-ductance of ions on nerve terminals to cause localizedmembrane depolarization and stimulation of ion-sensitive and voltage-sensitive channels to producegenerator potentials and elicit downstream nerve ex-citation. TRP channels also stimulate cation-sensitive

40 Veldhuis et al.

signaling pathways (e.g., Ca2+-stimulated proteinkinase C [PKC] activity) and transcriptional changes topromote expression and release of painful or proin-flammatory peptides, such as SP and CGRP, frompeptidergic neurons.

B. The Evolutionarily Conserved G Protein–CoupledReceptor–Transient Receptor Potential Ion ChannelCoupling Paradigm

The seminal discovery of a D. melanogaster mutantphenotype that rapidly loses sensitivity to sustainedlight led to the identification of the founding TRPchannel family members (Hardie and Minke, 1992;Phillips et al., 1992). Characterization of the signalingpathways in photoreceptors has contributed enormouslyto our understanding of GPCR signaling in mammaliansystems, forming the basis of a central paradigm forreceptor-operated TRP channel gating across species(Minke and Cook, 2002). In the plasma membrane ofDrosophila photoreceptor cells, the receptor-operatedTRP channel pathway requires GPCR stimulation andrelease of a heterotrimeric GTP-bound G protein com-plex, phospholipase C (PLC) activity, and metabolism ofphospholipids and phosphatidylinositol-4,5-bisphosphate(PIP2) to generate lipid second messengers, such asinositol-1,4,5-trisphosphate (IP3), membrane-associateddiacylglycerol (DAG) and arachidonic acid (AA). IP3 bindsits receptor to release Ca2+ from intracellular stores, andDAG stimulates kinase activity. Although the mecha-nism is still not entirely clear, the activation threshold ofTRP channels is lowered (i.e., sensitized) by the presenceof Ca2+ ions and fatty acid second messengers to permitchannel opening and produce a light-induced current.This process is desensitized by DAG-sensitive kinasephosphorylation of the TRP channel and the Ca2+-sensitivecalmodulin protein (CaM), which binds to the channel toprevent further ionic permeability (Fig. 2A) (Hardie andRaghu, 2001; Montell, 2005a). The GPCR-TRP functionalrelationship is also evident in the nematodeCaenorhabditiselegans, where GPCR and TRP channel homologs arecritical for survival by conferring the ability to detect andrespond to changes in temperature, noxious substances,pheromones, and osmotic conditions (Clapham et al., 2001;de Bono and Maricq, 2005; Xiao and Xu, 2009).The regulatory components of vertebrate GPCR-TRP

signaling axis are largely conserved in pain transmissionpathways within mammalian sensory neurons.Phospholipid metabolism generates lipid precursors,such as DAG, that can activate protein kinases (e.g.,DAG stimulates PKC), which phosphorylate TRPs.Moreover, phospholipids can be converted into poly-unsaturated fatty acids, which are TRP channel ligands(Minke and Cook, 2002). TRP channels also exhibitindependent sensory functions and can also regulateGPCRs, which is suggestive of functional cooperativitybetween these two classes of membrane proteins (Xiaoand Xu, 2009). GPCRs and TRPs can be recruited to

dynamic “supramolecular” complexes that facilitate func-tional interactions (Sheng and Sala, 2001). The discoveryof multiple algesic or analgesic GPCRs and nociceptiveTRP channels in sensory neurons and other cell typesreveals a complex regulatory system that controls neuronalexcitability and nonneuronal cell function to mediatenociceptive and inflammatory responses (Fig. 2B).

C. G Protein–Coupled Receptors that Sense NoxiousStimuli and Initiate Neurogenic Inflammation

GPCRs are structurally characterized by seven trans-membrane domains, an extracellular N terminus, anintracellular C terminus, and three extracellular andthree intracellular loops. They are receptors for diverseexogenous and endogenous ligands and are criticallyimportant for sensation (e.g., vision, taste, smell, pain,and itch) and pathophysiologic control. The peripheralprojections of nociceptive neurons express a suite ofGPCRs that allow them to “sample” the extracellularenvironment for noxious stimuli. These stimuli includeproteases (e.g., mast cell tryptase), peptides (e.g., bradykininfrom circulation), purines (e.g., ATP), cytokines/chemokines(e.g., interleukins), and lipids (e.g., prostaglandins), resultingin the activation of different classes of GPCRs. Eachreceptor is exquisitely selective for particular ligandsand ligand binding results in diverse, cell- and tissue-dependent outcomes that can ultimately cause pain orinflammatory processes to occur (Nygaard et al., 2013).TRP channels amplify or sustain these painful processes,and GPCRs transduce signals to activate or sensitize TRPchannels through several mechanisms. GPCR-stimulatedsecond messenger kinases (e.g., cAMP-dependent pro-tein kinase A [PKA], PKC) phosphorylate TRPs toreduce activation thresholds in response to endogenouschannel agonists (Peth}o and Reeh, 2012). GPCRs stimu-late phospholipase activity to release tonic inhibitoryeffects of PIP2 on ion channels (Huang et al., 2010).Metabolic generation of lipids such as anandamide(N-arachidonoylethanolamine) or epoxyeicosatrienoic acids(EETs) are endogenous TRP channel ligands (Watanabeet al., 2003). Receptor tyrosine kinase receptors canpromote TRP channel expression (Brederson et al., 2013)and plasma membrane insertion (Zhang et al., 2005) toenhance neuronal responsiveness to TRP stimuli. Togetherthese processes stimulate the release of neuropeptides suchas SP and CGRP from peripheral nociceptive endings ofnociceptors to promote arteriolar vasodilation, plasmaextravasation, and granulocyte infiltration in postcapillaryvenules (i.e., neurogenic inflammation). The stimulation ofTRP channels of the peripheral nerve terminals also has anexcitatory effect, resulting in the activation of voltage-sensitive, Ca2+-activated and ATP-sensitive channels,which induce localized membrane depolarization andformation of generator potentials that are capable ofpropagating action potentials (Bourinet et al., 2014).Hence, GPCRs are entirely dependent upon ion chan-nels for the central transduction of painful signals from


the periphery and viscera. Several key “algesic” and“analgesic” GPCRs that control TRP channels aredescribed below.1. Algesic G Protein–Coupled Receptors. The gener-

ation and release of painful and inflammatory media-tors from immune cells, epithelial tissue, and thecirculation activate multiple different classes of GPCRs,and these are identified according to their activatingligand or stimulatory mechanism. Classes that mediateproinflammatory, nociceptive, or pruritic events includethose stimulated by peptides (e.g., bradykinin andneurokinin receptors), lipids (e.g., prostaglandin receptors),amines (e.g., 5-hydroxytryptamine [5-HT] and histaminereceptors), nucleotides (e.g., purinergic and adenosinereceptors), and proteases (e.g., protease-activated recep-tors). Evidence indicates that each of these GPCRclasses functionally interacts with TRP channels toinduce painful, inflammatory, or pruritic cellular out-comes and an extensive summary of these pathways isprovided (Tables 1 and 2). Key sensitizing receptors,such as the tyrosine kinase receptor TrkA, 5-HT,purinergic, and the prostaglandin-activated EP receptors,have been reviewed (Peth}o and Reeh, 2012; McKelveyet al., 2013; Bannwarth and Kostine, 2014; Bautistaet al., 2014). The best studied sensory GPCR-TRP channelregulatory pathways include the protease-activated receptorand bradykinin receptor families, and these are discussed ingreater detail below. Intriguingly, the versatility of thesereceptors to respond to injury, inflammation, itch, andneuropathic disorders requires stimulation of multiple TRPchannels through multiple signaling pathways.a. Protease-activated receptors. The pathophysiologic

roles of protease-activated receptors (PARs) havebeen extensively reviewed (Macfarlane et al., 2001;Noorbakhsh et al., 2003; Ossovskaya and Bunnett, 2004;Ramachandran et al., 2012). PARs are a family of 4 GPCRs(PAR1–4) that are widely expressed in the peripheral and

central nervous systems as well as many other cell types,where they participate in hemostasis, inflammation,pain, and repair mechanisms. In contrast to most GPCRsthat interact with soluble extracellular ligands, PARligands reside within the receptors themselves. Proteasescleave specific sites within the extracellular N-terminaldomains of PAR1, PAR2, and PAR4 to reveal tetheredligand domains that bind to and activate cleavedreceptors. PAR1, PAR2, and PAR4 are expressed bynociceptive neurons (Vellani et al., 2010), and PAR2

in particular can sensitize or excite sensory neuronsto promote neurogenic inflammation, pain, and itch(Ramachandran et al., 2012). PARs couple to the majorGa subunits (Gaq/11, Gas, Gai/o, Ga12/13) to stimulatePLC and phospholipase A2 (PLA2) and activate PKA,PKC, and protein kinase D (atypical PKCm). These,in turn, sensitize or activate TRP channels, includingTRPV1, TRPA1, and TRPV4, to initiate pain andinflammation through peripheral and central mecha-nisms (Veldhuis and Bunnett, 2013). PARs can becleaved by multiple proteases at different locations toexpose different N-terminal sequences and stabilizedistinct “biased” GPCR active conformations. Neutro-phil elastase and the proinflammatory cysteine proteasecathepsin S have shown PAR2 cleavage specificity atsites distinct from the canonical trypsin/tryptase site,resulting in activation of alternative G protein signalingpathways (Ramachandran et al., 2009; Zhao et al.,2014). Multiple PAR-activated pathways, including trypsin-stimulated Gaq protein, PLA2 activity, and tyrosine kinasesignaling or cathepsin S–stimulated Gas and PKAsignaling may therefore converge on a single TRP channeleffector (e.g., TRPV4) to cause inflammatory pain (Pooleet al., 2013; Zhao et al., 2014). Furthermore, cleavage ofthe canonical PAR2 cleavage site induces rapid receptorphosphorylation and internalization, whereas cathepsin Scleavage does not, potentially influencing the duration and

Fig. 2. Conserved mechanisms demonstrate fundamental concepts in TRP channel sensitization. GPCR-TRP interactions in vision (Drosophila sp.)and in pain are depicted in (A) and (B), respectively. GPCR stimulation and PLC activity generates lipid precursors (DAG) to stimulate kinases (PKC),release intracellular Ca2+ stores (IP3), or directly activate TRP channel Ca2+ conductance (AA and polyunsaturated fatty acids [PUFAs]). Elevatedintracellular Ca2+ leads to CaM binding and channel desensitization. AC, adenylyl cyclase; COX, cyclooxygenase; LOX, lipoxygenase; RTK, receptortyrosine kinase. Adapted from Minke and Cook, 2002.

42 Veldhuis et al.

TABLE 1Nociceptive, inflammatory, and pruritic pathways caused by direct stimulation

PIP2 metabolism is considered to relieve inhibition of most TRP channels that regulate nociceptive responses and is therefore omitted, unlessindicated to increase activity (Rohacs, 2007).

Direct Mechanismsof Activation Reference

TRPV1 ThermalNoxious heat .43°C Tominaga et al., 1998

ChemicalProtons pH ,5.9 Tominaga et al., 19984-HNE DelloStritto et al., 2014Arachidonic acid/AEA Smart et al., 2000; Sousa-Valente et al., 2014ATP Moriyama et al., 2003; Lishko et al., 2007;

Ma et al., 2012Leukotriene B4 Vigna et al., 2011Oleoylethanolamide Suardíaz et al., 200720-HETE Wen et al., 20125-(S)-HETE , 12-(S)-HETE Hwang et al., 20005-(S)-HPETE, 12-(S)-HPETE Hwang et al., 2000PGE2/ PGI2 Moriyama et al., 2005LPA Nieto-Posadas et al., 2012Capsaicin Caterina et al., 1997Resiniferatoxin Szallasi et al., 1999Piperine McNamara et al., 2005Tarantula toxin (DxTx) Bohlen et al., 2010

TRPV2 ThermalNoxious heat .52°C Hu et al., 2004

Chemical2-APB Monet et al., 2009Arachidonic acid Hu et al., 2005Lysophospholipids Stokes et al., 2004

TRPV3 ThermalWarm .32°C Peier et al., 2002b

ChemicalCarvacrol, eugenol, thymol Xu et al., 20062-APB Hu et al., 2006Arachidonic acid Hu et al., 2006Monoterpenoids (camphor) Vogt-Eisele et al., 20071,8-Cineole Takaishi et al., 2012

TRPV4 ThermalWarm .28°C Güler et al., 2002; Todaka et al., 2004

MechanicalMembrane stretch/swelling Alessandri-Haber et al., 2003; Mochizuki et al., 2009Cilia beating Lorenzo et al., 2008

ChemicalArachidonic acid, AEA, 59,69-EET, 89,99-EET Watanabe et al., 2003

TRPM2 ChemicalProtons ,pH 6.7 Du et al., 2009H2O2 Fonfria et al., 2004; Naziroglu et al., 2011Adenine dinucleotides Kraft et al., 2004; Grubisha et al., 2006; Du et al., 2009

TRPM3 ThermalHeat .37°C Vriens et al., 2011

ChemicalPregnenolone sulfate Wagner et al., 2008D-Erythro-sphingosine Grimm et al., 2005Clotrimazole Vriens et al., 2014

TRPM8 ThermalCold 2–28°C McKemy et al., 2002; Peier et al., 2002a

ChemicalPIP2 Rohács et al., 2005Lysophospholipids Andersson et al., 2007Menthol, icilin Peier et al., 2002a1,8-Cineole Takaishi et al., 2012Camphor Selescu et al., 2013

TRPA1 ThermalNoxious cold ,17°C

ChemicalCannabinoids Andersson et al., 20114-HNE Trevisani et al., 2007; Taylor-Clark et al., 2008a59,69-EET Sisignano et al., 2012

(continued )


intensity of subsequent cell signaling events (DeFea et al.,2000; Zhao et al., 2014). The specificity for initiatingdistinct signaling and lipid metabolic pathways may bea requirement to promote distinct physiologic outcomessuch as inflammatory pain and wound healing.b. Bradykinin receptors. The contributions of bradykinin

(BK) receptors (B1R and B2R) to pain, inflammation, andTRP channel activation has been the subject of multiplereviews (Peth}o and Reeh, 2012; Regoli et al., 2012; Blaesand Girolami, 2013). Proteases of the kallikrein-kininogensystem cleave propeptides to produce painful ligands ofthe kinin family. Blood-derived BK is a key mediator ofnociception and can be differentially processed to evokea variety of physiologic responses via B1R and B2R.These receptors contribute to chronic diseases of pain,inflammation, neurodegeneration, and cardiovascularsystems and are closely associated with the renin-angiotensin system.BK-dependent pain transmission is predominantly

attributed to the B2R, which is coexpressed by peptidergicprimary afferents with TRPV1, TRPA1, TRPM8, andTRPV4. B2R couples to Ga proteins (Gaq, Gas, GaI, andGa12/13) on DRG nerve terminals to stimulate PKA, PKC,cyclooxygenases 1/2, lipoxygenase (e.g., 5- or 12-lipoxygenase)and phospholipases (PLC and PLA2) (Liebmannet al., 1996; Wang et al., 2008; Peth}o and Reeh, 2012).This leads to increased production of inflammatoryprostanoids and eicosanoids and rapid stimulationof ion channels to evoke action potentials resulting inpain, inflammation, or itch. TRPV1 and TRPA1 knock-out mice show diminished nocifensive behaviors whenstimulated with moderate BK doses (Katanosaka et al.,2008). This is supported by neuronal studies demon-strating that BK-dependent intracellular Ca2+ tran-sients or action potentials are partially mediated byTRPV1 or TRPA1 (Bandell et al., 2004; Bautista et al.,2005). Normal nociceptor excitability is observed whentrpv12/2 mice are exposed to higher BK concentrations,which may also suggest that TRPV1 and TRPA1 aredifferentially regulated by BK receptors in the samecell (Kwan et al., 2006). BK can also regulate TRP-dependent itch sensory responses, and this is discussedin section V.2. Analgesic G Protein–Coupled Receptors. Some of

the most effective strategies for counteracting nociception

may be derived from endogenous analgesic systemswithin the body. Tissue insult and recruitment ofimmune cells counteract peripheral and central paintransmission via production of endogenous opioid andsomatostatin peptides and the fatty acid cannabinoids.Activation of opioid receptors on peripheral sensoryneurons reduces nerve excitability and suppresses neuro-transmitter or neuropeptide release. There is also evidencefor the analgesic opioid receptors (m-, k-, d-ORs) andcannabinoid (CB1 and CB2) receptors to function syner-gistically in the inhibition of inflammatory or nociceptivepain (Anand et al., 2009). The opioid, cannabinoid, andsomatostatin receptors stimulate Gai/o to inhibit adenylylcyclase–dependent cAMP production, resulting in down-regulation of PKA-dependent TRP channel activity andrelease of bg trimeric subunits to inhibit N-, T-, andP/Q-type Ca channels (Williams et al., 2013). Together,such processes reduce nerve excitability and presynap-tic neurotransmitter release (Pinter et al., 2006; Steinand Machelska, 2011; Williams et al., 2013)

The Mas-related G protein–coupled receptor familymembers (Mrg receptors) also contribute to analgesiain animal models of protracted pathologic pain. Thesereceptors are selectively expressed on small diameterneurons and contribute to processing of pain and itchresponses (Dong et al., 2001; Han et al., 2013). Severallines of evidence from wild-type or Mrg knockout miceindicate that these receptors are upregulated in responseto sciatic nerve injury and provide an endogenousmechanism for the inhibition of mechanical, thermal,and inflammatory pain hypersensitivity (Guan et al.,2010; He et al., 2014). MrgC receptor–dependent mecha-nisms of analgesia are not entirely clear but may enhancem-opioid receptor Gai/o signaling (Wang et al., 2013).

D. Pain Transducing Transient ReceptorPotential Channels

The discovery of TRP channels, their expression profiles,and biophysical properties have been reviewed in detail(Ramsey et al., 2006; Hardie, 2007; Nilius et al., 2007; Wuet al., 2010). Themammalian TRP channels are tetrameric,nonselective cation permeable pores that are associatedwith intracellular and plasma membranes. There are 28TRP genes in mammals that are divided into six TRPchannel protein families: canonical (TRPC), melastatin

TABLE 1—Continued

Direct Mechanismsof Activation Reference

PGA1, PGA2, 15-dPGJ2 Cruz-Orengo et al., 2008; Materazzi et al., 2008;Taylor-Clark et al., 2008b

Allyl isothiocyanate Macpherson et al., 2007Lipopolysaccharide Meseguer et al., 2014Hydrogen sulfide Andersson et al., 2012Cinnemaldehyde Macpherson et al., 2007Allicin, diallyl disulphide, Taylor-Clark et al., 2009Nitrooleic acidChloroquine Than et al., 2013

44 Veldhuis et al.

TABLE 2Nociceptive, inflammatory, and pruritic pathways caused by receptor-mediated TRP channel stimulation

Receptor Signaling-Mediated Activation

Receptor (Ligand) Signaling Mechanism Reference

TRPV1 B1, B2 (bradykinin) PKC, 12-LOX Cesare and McNaughton, 1996; Vellani et al.,2001; Shin et al., 2002; Katanosakaet al., 2008

H1R, H2R (histamine) PLA2/ 12-LOX, PLC/PKC Kim et al., 2004; Shim et al., 2007; Imamachiet al., 2009; Kajihara et al., 2010

PAR2 (trypsin, PAR2-activating peptide) PLC, PKA, PKC, PKD Dai et al., 2004; Amadesi et al., 2006, 2009;Vellani et al., 2010

NPR-C (natriuretic peptide C) bg G proteins/ PLCb/PKC Loo et al., 2012Prokineticin PK1, PK2 (Bv8) PKC Negri et al., 2006; Vellani et al., 2006TrkA (NGF) PKC, PI3K, p38, B2 Ji et al., 2002; Lee et al., 2002b; Bonnington and

McNaughton, 2003; Stein et al., 2006m-Opioid receptor (morphine, DAMGO) b-Arrestin Zhang et al., 2005; Forster et al., 2009;

Rowan et al., 2014MrgA3 (chloroquine) PLCb, PKC Than et al., 2013EP1 EP2, & EP4 (PGE2) PKC, PKA, and AKAP Gu et al., 2003; Moriyama et al., 2005;

Zhang et al., 2008IP receptor (PGI2) PKA Moriyama et al., 20055-HT receptor (5-HT2, 5-HT4, 5-HT7) PKC, PKA, and AKAP Sugiuar et al., 2004; Ohta et al., 2006Signaling with unknown receptor or

intermediatesProlactin receptor (prolactin/estradiol) — Diogenes et al., 2006; Patil et al., 2013TrkA (NGF), Ret/GFRa (GDNF),

Mrg receptor— Luo et al., 2007; Ciobanu et al., 2009

TRPV2 Fc«RI (IgE) Acyl CoA/PKA Stokes et al., 2004Signaling with unknown receptor or

intermediates— Lysophospholipids/ Gaq/ PI3K Monet et al., 2009

TRPV3 H1R (histamine) Gq/11, PLCb Xu et al., 2006B2 (bradykinin) PKC Hu et al., 2004Muscarinic M1 (carbachol) PLC Xu et al., 2006; Doerner et al., 2011

TRPV4 B2 (Bradykinin) PLC, PKC Fan et al., 2009PAR2 (proteases) PLC, PLA2, PKA, PKC, PKD,

MAPK, and 59,69-EETGrant et al., 2007; Cenac et al., 2008; Sipe et al.,

2008; Li et al., 2011; Poole et al., 20135-HT receptor PLC, PLA2, PKC, and MAPK Cenac et al., 2010Signaling with unknown receptor or

intermediatesH1R (histamine) — Cenac et al., 2010Muscarinic M1 (carbachol) — Sonkusare et al., 2012

Src Kinase Wegierski et al., 2009; Poole et al., 2013PGE2 Alessandri-Haber et al., 2003

TRPM3 Muscarinic M1 (Carbachol) — Lee et al., 2003

TRPM8 EP1 (PGE2) PKA Linte et al., 20075-HT1B (sumatriptan, CP 94253) Phospholipase D1, PIP5-kinase Vinuela-Fernandez et al., 2014Signaling with unknown receptor or

intermediatesTrkA (NGF) Ret/GFRa

(GDNF, artemin), Mrgpr— Luo et al., 2007; Ciobanu et al., 2009;

Lippoldt et al., 2013Prolactin receptor (prolactin/estradiol) — Patil et al., 2013

PLA2 Gentry et al., 2010Gaq (inhibition) Zhang et al., 2012

TRPA1 B2 (bradykinin) PLC/PKA/TRPV1 Ca2+ flux Bautista et al., 2006; Katanosaka et al., 2008;Wang et al., 2008; Schmidt et al., 2009

H1R (histamine) Mrgpr signaling Wilson et al., 2011PAR2 (proteases) PLC Bautista et al., 2005; Dai et al., 2007;

Cattaruzza et al., 2010MrgC11 (Bam8-22) PLC, PKC Wilson et al., 2011MrgA3 (chloroquine) bg G proteins Wilson et al., 2011; Than et al., 2013Signaling with unknown receptor or

intermediatesTrkA (NGF) Ret/GFRa (GDNF),

Mrg receptor— Diogenes et al., 2007; Luo et al., 2007;

Ciobanu et al., 2009Prolactin receptor (prolactin/estradiol) — Patil et al., 2013m-Opioid receptor (morphine) — Forster et al., 2009

PI3K, phosphoinositide 3-kinase; PKD, protein kinase D.


(TRPM), vanilloid (TRPV), polycystin (TRPP), mucolipin(TRPML), and ankyrin (TRPA). With the exception ofthe pore and transmembrane regions, these familiesshare low-level sequence identity and possess distinctmolecular architectures and varied expression profiles(Montell, 2001). Many of these channels have beenidentified as therapeutic targets for pain and inflam-mation (Wu et al., 2010), and this has been confirmed byTRP antagonism or genetic deletion in inflammatory,puriritic, and neuropathic pain models.TRP channels are ligand gated and voltage sensitive,

and most family members are inward rectifying channelsthat nonselectively conduct cations (Ca2+, Na+), leadingto rapid changes in intracellular cation concentrations.On sensory neurons, sustained ion flux regulatesCa2+-sensitive proteins and cellular events and thestimulation of other voltage-gated channels to initiategenerator potentials, neuronal excitation, and centraltransmission of sensory information. They possess severalcommon features: 6 transmembrane domains, a pore-forming channel gating region between the 5th and 6thtransmembrane domains, and intracellular N and C ter-mini (Fig. 3). In the TRPV, TRPM, and TRPC families, theintracellular end of the 6th transmembrane domain par-ticipates in formation of the “lower gate” of the pore andcontains a highly conserved helical sequence known asthe “TRP domain,” which has been proposed to regulatechannel tetramerization and interactions with phospholi-pids (Clapham, 2003). Highly structured ankyrin repeatsat the N terminus participate in ligand binding, protein-protein interactions, and tetramerization (Lishko et al.,2007). Recently, the structure of TRPV1 was solved to3.4 Å by electron cryomicroscopy and confirms these priorassessments (Liao et al., 2013) (see secton III.A).TRP channels can bind many endogenous lipids and

exogenous natural or synthetic compounds. They aretargets for kinases, signaling adaptors, such as theA-kinase anchoring protein (AKAP; described below),and trafficking proteins. The capacity of TRP channelsto sense endogenous and environmental stimuli and toregulate ion fluxes illustrates their important roles ascellular sensors and signaling effectors. An extensivelist of the thermal, mechanical, chemical, and signalingstimuli that can control TRP channel activity is providedin Table 1. Key features of proinflammatory, pruritogenic,or nociceptive TRP channels are summarized below.1. Transient Receptor Potential Vanilloid 1. The

vanilloid receptor subtype 1 or TRPV1 was the firstmammalian TRP vanilloid ion channel to be cloned(Caterina et al., 1997). Small diameter, unmyelinatedC-fibers and lightly myelinated Ad fibers are majorcellular sites of TRPV1 expression (Cavanaugh et al.,2008). Trpv12/2 mice respond poorly to noxious heat,acid, and vanilloid-evoked pain, and lack the ability topromote tissue swelling, neuropeptide release, andcytokine release after inflammation, illustrating thekey role of this channel in nociception and neurogenic

inflammation (Caterina et al., 2000; Keeble et al., 2005).TRPV1 also contributes to painful disorders, includingdiabetic-induced neuropathic pain, cancer pain, andinflammatory pain (Alawi and Keeble, 2010). Lipidderivatives that can potentiate or stimulate TRPV1activity include prostaglandin E2 (PGE2) (Moriyamaet al., 2005) and numerous polyunsaturated fattyacids (Matta et al., 2007). The arachidonic fatty acidderivative and CB1 receptor agonist anandamide is awell studied example that functionally inhibits neu-ronal activation at low concentrations and activatesTRPV1 at higher concentrations (Tognetto et al., 2001).Complementary studies on capsaicin-induced releaseof the painful neuropeptide substance P into rat spi-nal cords reveal that similar effects can be observedwith pharmacological inhibition of CB1 (Lever andMalcangio, 2002). Many GPCRs sensitize or activateTRPV1, including receptors for proteases, serotonin,histamine, tachykinins, and bradykinins (see Tables 1and 2). However, not all GPCRs signal through the samecascades and the presence of auxiliary TRPV1-interactingscaffolding proteins, such as AKAP150 and arrestins, canpotentially influence tissue-specific outcomes (see sectionIII.D) (Schnizler et al., 2008; Jeske et al., 2009; Zhanget al., 2008).

2. Transient Receptor Potential Vanilloid 2. TRPV2shares 50% sequence homology with TRPV1, is activatedby high heat thresholds (52°C), and is expressed onsmall, medium, or large diameter primary afferentneurons, motor neurons of the spinal cord, and inosmosensing regions of the hypothalamus (Caterinaet al., 1999; Lewinter et al., 2008; Nedungadi et al.,2012). TRPV2 responds to stretch, lysophospholipids,and cannabinoids, with the most potent being thenonpsychotropic compound cannabidiol. Activation ofTRPV2 stimulates CGRP release from DRG sensoryneurons, indicating a pronociceptive and proinflam-matory role (Qin et al., 2008). More recently TRPV2has also been implicated in nitric oxide production forthe control of intestinal motility (Mihara et al., 2013).

3. Transient Receptor Potential Vanilloid 3. TRPV3 is awarm-sensing channel that is expressed by TRPV1-positiveafferent neurons and in the skin, brain, colon, andepithelia of the nose, tongue, and cornea, where itcontributes to sensory, nociceptive, inflammatory, andpruritic pathways (Xu et al., 2002; Moussaieff et al.,2008; Ueda et al., 2009; Nilius et al., 2014). A TRPV3-dependent Ca2+ flux is essential for skin barrier formation,the release of interleukin and PGE2, and nitric oxideproduction. Uniquely, TRPV3 is potentiated by repeatedexposure to activating stimuli like warm temperatures(Peier et al., 2002b). Sensitization of TRPV3 by GPCR-mediated signaling has been demonstrated by stimulationof M1 acetylcholine receptor (in a heterologous ex-pression system) and reveals the sensitization ofTRPV3 activity occurs through PIP2 depletion andCa2+ sensitivity (Doerner et al., 2011) and inflammatory

46 Veldhuis et al.

fatty acids, such as AA, but not through lipoxygenaseand epoxygenase-derived products of AA metabolism(Hu et al., 2006). PGE2 can also sensitize TRPV3 responsesin the skin to cause peripheral pain (Huang et al., 2008).TRPV3 is involved in cutaneous thermosensation andcan convey sensory information from keratinocytesto sensory neurons via an ATP-release mechanism(Moqrich et al., 2005; Mandadi et al., 2009). TRPV3also contributes to pruritus, atopic dermatitis, andloss of wound healing in dysregulated systems and isinvolved in epidermal growth factor receptor signalingand transglutaminase activity, which are essential forepidermal barrier integrity and defense (Asakawa et al.,2006; Xu et al., 2006; Imura et al., 2007; Cheng et al.,2010; Miyamoto et al., 2011; Nilius and Biro, 2013).4. Transient Receptor Potential Vanilloid 4. TRPV4 is

expressed by Ad- and C-fibers and by nonneuronalcells (e.g., epithelia, endothelia, and skin) and issensitive to membrane stretch or hypotonic conditions(Alessandri-Haber et al., 2003; Suzuki et al., 2003b;Hartmannsgruber et al., 2007). Studies using selectiveTRPV4 agonists/antagonists and of trpv42/2 mice havedemonstrated an important role for TRPV4 in thermaland mechanical hyperalgesia and in chronic inflamma-tory conditions, such as colitis, through transcriptionalregulation and the release of cytokines, SP, and CGRP(Brierley et al., 2008; D’Aldebert et al., 2011). TRPV4also regulates vasodilatation, ciliary beating frequency,wound healing, skin barrier formation, and bladdervoiding (Liedtke and Friedman, 2003; Todaka et al.,2004; Alessandri-Haber et al., 2006; Lorenzo et al., 2008;Earley et al., 2009). TRPV4 activity is modulated by manyGPCRs, including those for proteases, histamine, seroto-nin, and acetylcholine (Tables 1 and 2). GPCR-dependentphosphorylation is an essential mechanism for TRPV4sensitization, yet TRPV4 is also highly sensitive toendogenous inflammatory lipid mediators, includinganandamide and AA metabolites (e.g., 59,69-EET or119,129-EET) (Watanabe et al., 2003; Earley et al.,2005). TRPV4 is also a key integrator of many signalingmolecules. It can associate with PKA and PKC, whichrequires the scaffold A-kinase anchoring protein AKAP79(Fan et al., 2009), and interacts with Src kinase,integrins, cytoskeletal proteins, tight junction, and adap-tor proteins (Suzuki et al., 2003a; Wegierski et al., 2006;Alessandri-Haber et al., 2008; Becker et al., 2009;Fan et al., 2009; Akazawa et al., 2013). Members ofthe PKC and CKII substrate in neurons adaptorprotein family (PACSIN; also known as Syndapin 1)bind to an N-terminal proline-rich region of TRPV4to regulate channel internalization and responses tohypotonicity (Fig. 3D) (Cuajungco et al., 2006; D’Hoedtet al., 2008). TRPV4 activity contributes to pain andproinflammatory processes through upregulation ofedema (Vergnolle et al., 2010); epidermal, epithelialand endothelial cell permeability (Reiter et al., 2006;Willette et al., 2008; Sokabe et al., 2010); and chemokine/

cytokine production (D’Aldebert et al., 2011). Thus, cell-specific auxiliary proteins that regulate the intracellularlocalization and function of TRPV4 are recognized aspotential therapeutic targets for the regulation of TRPV4in its capacity as a proinflammatory effector molecule(Garcia-Elias et al., 2013).

5. Transient Receptor Potential Melastatin 2. The eightmembers of the melastatin subfamily have low-levelsequence homology. They are grouped according to theirlack of N-terminal ankyrin repeats, and three of themembers possess an extended C-terminal tail that con-tains an active kinase domain (TRPM6 and TRPM7) orADP ribose-binding phosphohydrolase homology domains(TRPM2). These channels have diverse roles in coldsensation, neuronal development, taste reception, T-cellregulation, glucose-induced insulin release, and regula-tion of mast and glial cells (Wu et al., 2010). TRPM2,TRPM3, and TRPM8 contribute to pain processing.TRPM2 is expressed in central and peripheral neurons,microglia, and neutrophils and is sensitive to oxidativeor nitrosative stress, intracellular Ca2+, and acidity(Kraft et al., 2004; Lange et al., 2008; Du et al., 2009;Nazıro�glu et al., 2011). Trpm22/2 mice have normalthermal or mechanical thresholds yet possess reducednociceptive responses in inflammatory or neuropathicpain, most likely via TRPM2-dependent chemokinerelease from infiltrating peripheral macrophages, neu-trophils, and spinal microglia (Yamamoto et al., 2008;Haraguchi et al., 2012; Isami et al., 2013).

6. Transient Receptor Potential Melastatin 3.TRPM3 has diverse roles in sensation and insulinsecretion and is expressed in the kidney, testes, brain,and spinal cord (Lee et al., 2003). TRPM3 is alsoexpressed on sensory neurons of DRG and TG, andtrpm32/2 mice show deficiencies in heat sensation anddevelopment of inflammatory heat hyperalgesia (Vrienset al., 2011). Intriguingly, the cation permeationpathway in TRPM3 may function independently of thecanonical central ion pore, allowing Na+ influx andsensory neuronal excitation even after pharmacologicalinhibition of the central pore (Vriens et al., 2014). Itremains to be determined if other TRP channels possessa similar secondary ion permeation pathway and howpassage of both Ca2+ and Na+ may influence neuronalexcitatory responses and other channels in a neuropathicpain setting. The potential for TRPM3 to be sensitized byGPCR signaling has been demonstrated with carbachol-induced muscarinic receptor signaling and sensitivity toCa2+ store depletion by thapsigargin (Lee et al., 2003).TRPM3 has also been observed to interact withCa2+-sensitive proteins CaM and S100b, which areproposed to compete for a PIP2 binding site in the Nterminus, yet the functional consequences of this remainunclear (Holendova et al., 2012).

7. Transient Receptor Potential Melastatin 8.TRPM8 is a well known thermosensitive ion channelfor cold temperatures, responding within 8–28°C, and


also to cooling agents, such as menthol and icilin (Bautistaet al., 2007; Colburn et al., 2007; Dhaka et al., 2007; Gavvaet al., 2008). Initially shown to be expressed in small-diameter TrkA-positive sensory neurons (Peier et al.,2002a), tissue analyses in mice engineered to express agreen fluorescent protein reporter from the TRPM8 locusreveal channel expression in a unique population ofneurons that do not express typical nociceptive markerssuch as IB4 or TRPV1 (Dhaka et al., 2008). This is incontrast to ;30–50% of acutely dissociated or culturedTRPM8-positive neurons that also functionally expressTRPV1, suggesting species differences or artifacts relatedto neuronal isolation and culturing (Babes et al., 2004;Okazawa et al., 2004). Assessment of trpm82/2 mice anduse of pharmacologic agents indicate that TRPM8 hasdiverse sensory roles and contributes to cold-inducedallodynia and analgesia, pruritus, and inflammatoryprocesses (Namer et al., 2008; Caspani et al., 2009; Suet al., 2011; Calvo et al., 2012; Lippoldt et al., 2013;Ramachandran et al., 2013; Shapovalov et al., 2013;Than et al., 2013). The use of selective antagonists haveconfirmed the involvement of TRPM8 in thermoregula-tion and augmented nerve excitability through voltage-gated Na+ channels (Gavva et al., 2012; Sarria et al.,2012). Furthermore, TRPM8 is an integrator of cellsignaling pathways: it can bind G proteins and there-fore play a role in initiating signaling pathwaysthrough mechanisms that are yet to be completely un-derstood (Klasen et al., 2012). The regulation of TRPM8by G protein signaling pathways involves typicalPIP2/phospholipase activity and noncanonical pathways.The PIP2-binding protein Pirt (PIP-binding regulator ofTRP channels) interacts with TRPM8 (and can also bindTRPV1), and the expression of Pirt enhances thermo-sensitivity (Tang et al., 2013). Several lines of evidencesuggest that TRPM8 functions as an anti-inflammatoryTRP channel. TRPM8 exerts an anti-inflammatory activityin the mouse colon via inhibition of neuropeptide release(Ramachandran et al., 2013), and bradykinin or histamine-induced inflammatory signaling can also lead to directTRPM8 interactions with the Gaq subunit, resulting inTRPM8 inhibition (Zhang et al., 2012; Li and Zhang, 2013).8. Transient Receptor Potential Ankyrin 1. TRPA1 pos-

sesses 17 N-terminal ankyrin repeats (in human) andis sensitive to a wide range of noxious stimuli (Coreyet al., 2004). It is expressed by TrkA- and TRPV1-positiveC-fibers, and although TRPA1 sensitivity to noxious coldand mechanical stress has been debated (McKemy, 2005),functional studies and a familial TRPA1 gain-of-functionmutation has associated TRPA1 currents with cold andchemical nociception (e.g., ,16°C) or environmental/endogenous irritants (Kwan et al., 2006; Kremeyeret al., 2010). The N terminus contains key regulatoryregions in the linker region between the ankyrin repeatsand first transmembrane domain, including conservedN-terminal cysteine residues (Doerner et al., 2007). Thesesites are essential for TRPA1 to “sense” and covalently

bind painful molecules that possess electrophilic chemicalgroups, such as noxious irritants in airways (cigarettesmoke, tear gas), pungent chemicals in the digestivesystem (allicin from garlic, allyl isothiocyanate frommustard oil, cinnamaldehyde from cinnamon) and re-active oxygen species released by damaged tissue (Tables1 and 2) (Bautista et al., 2006; Macpherson et al., 2007;Andre et al., 2008; Brone et al., 2008; Bessac et al., 2009;Taylor-Clark et al., 2009; Cordero-Morales et al., 2011).TRPA1 is highly sensitive to inflammatory fatty acidsand highly reactive thiol-reactive species, such as4-hydroxynonenol (4-HNE), prostaglandin metabolites[15-dPGJ(2), PGA(2), and PGA(1)], and hydrogen peroxide(Trevisani et al., 2007; Andersson et al., 2008; Materazziet al., 2008). The role of TRPA1 in inflammatory pain wasrecently discussed in detail (Bautista et al., 2013). It isa key integrator of receptor signaling by all major painfuland inflammatory GPCRs (proteases, bradykinin, prosta-glandins, ATP, or histamine) to promote pain, plasmaextravasation and edema, itch, and vasodilatation, andalthough multiple kinases sensitize TRPA1 channelgating, the phosphorylation status of TRPA1 is notwell characterized (Bandell et al., 2004; Chen et al.,2011; Wilson et al., 2011; Brederson et al., 2013).Animal studies have linked TRPA1 activity to mechan-ical hyperalgesia, visceral pain, inflammatory boweldisease, and respiratory disease (Engel et al., 2011;Sisignano et al., 2012; Hughes et al., 2013; Kondo et al.,2013; Lin et al., 2013).

9. Transient Receptor Potential Canonical. TheTRP canonical (TRPC) subfamily is divided into threesubgroups by sequence homology and functional similar-ities: C1/C4/C5 and C3/C6/C7, with the rat pheromonesignaling receptor TRPC2 being classed as a pseudogenein humans (Clapham, 2003). Analogous to theDrosophilaphototransduction pathway, TRPC channels are receptor-operated channels that respond to GPCR signaling, re-ceptor tyrosine kinases, and intracellular Ca2+

(Clapham, 2003). The Gaq/PLC-dependent signalingand generation of DAG and IP3 are proposed to bethe major mechanism for activation of TRPC gating(Schaefer et al., 2000). Several TRPC members areimplicated in neurogenic pain and inflammatory pro-cesses. TRPC1 and TRPC6 are “stretch-activated ionchannels” that frequently share expression with TRPV4on small and medium diameter sensory neurons(Alessandri-Haber et al., 2009). Both channels are pro-posed to function synergistically with TRPV4 to contributeto inflammation-induced mechanical hyperalgesia in mice.TRPC6 is also associated with wound healing and edemacaused by dysfunction of endothelial cells of the lung(Davis et al., 2012; Weissmann et al., 2012). The stretch-activated ion channel TRPC5 is highly sensitive tointracellular Ca2+ ion concentrations and temperaturesbelow 37°C and may contribute to cold sensitivity orallodynia in the temperature range not processed byTRPM8 or TRPA1 (Zimmermann et al., 2011). Stimulation

48 Veldhuis et al.

of Gai/o-coupled signaling through M2 muscarinic, 5-HT1A

serotonin, andm-opioid receptors leads to direct activation ofTRPC4 or TRPC5 channels (Miller et al., 2011; Jeon et al.,2012). Further studies may elucidate important functionalroles for TRPC channels in itch sensation and analgesia.

III. Molecular Substates for the G Protein–Coupled Receptor–Transient Receptor PotentialChannel Axis: Regulation of Transient Receptor

Potential Channels by Lipid andProtein Interactions

The interaction between TRP channels, signalingproteins, and second messengers determines channelgating or cellular localization and can influence paintransmission. Hence, the intracellular N- and C-terminalregions of TRP channels are key regulatory domains thatare essential for function (Phelps and Gaudet, 2007).Although some of these interactions are tissue or TRPspecific, conservation of regions in the C terminus of mostTRP channel family members favors interactions withPIP2 and CaM. However, new protein binding partners(e.g., AKAP) and lipid metabolites (e.g., anionic longchain acyl CoA esters) continue to be discovered (Jeskeet al., 2009; Yu et al., 2014). The characterization of theselipid and protein interactions provides information aboutkey domains that are important for TRP channel gatingand may therefore be therapeutically “tuned” to avoidnoxious sensory nerve excitation and inflammation indysregulated systems. Recent advances in our under-standing of TRP structure have provided informationabout these regulatory domains and given insight intotetrameric subunit assembly and channel gating. Theseprocesses are essential for understanding how GPCRmodulation of TRP channels may occur.

A. Transient Receptor Potential Channel Structure:Improving Our Understanding of Protein Interactions

Cryoelectron microscopy has provided greater un-derstanding of the molecular architecture of the TRPhomotetramers, such as TRPV4, and has revealed thatcytoplasmic regions of a TRP channel form a largebasket-like domain, which is proposed to influenceassembly, channel gating, and protein-protein interactions(Shigematsu et al., 2010). Advanced electron cryomicroscopyhas provided the high-resolution (3.4 Å) structure of TRPV1(Fig. 3) (Cao et al., 2013b; Liao et al., 2013). These dataare consistent with X-ray crystallography studies on theisolated vanilloid TRPV1 ankyrin domain, which en-abled characterization of interactions with CaM andidentified ATP as a novel TRP channel ligand (Jin et al.,2006; Lishko et al., 2007; Phelps and Gaudet, 2007; Inadaet al., 2012). The latest structural studies of TRPV1provide insights into subunit assembly and reveal thata b-sheet is formed between C- and N-terminal domains(Liao et al., 2013). Specifically, the linker region be-tween the N-terminal ankyrin domain and the first

transmembrane domain associates with a singleb-strand within the C terminus (Fig. 3B). The linkerregion is in close proximity to the ankyrin repeats,which enhances association between N and C terminiand provides contacts for tetrameric subunit assembly(Liao et al., 2013). This interaction may explain bio-chemical studies in which PIP2 or protein interactionsare observed to occur on both intracellular tails of a TRPsubunit (Lau et al., 2012; Garcia-Elias et al., 2013). TheTRPV1 structure is further supported by the firststructure of TRPV1 bound to a peptide ligand (extracel-lular spider toxin) or vanilloid-based ligand (ultrapotentresiniferatoxin) to demonstrate the adaptation of TRPV1to ligand-specific conformations and may provide cluesfor ion permeability through the channel pore (Caoet al., 2013b). These data also provide an enhanced3-dimensional prototype for understanding how pro-teins and other ligands interact with all TRP channels.TRPV1 may be used as a structural template to builda TRP channel “interactome,” to provide new perspec-tives on TRP channel regulation. Figure 3 highlightsdistinct and overlapping binding sites and key reg-ulatory regions of TRPV1, TRPV4, TRPA1, and TRPM8.Furthermore, structural information provides us with anunderstanding of phosphorylation and how proinflamma-tory mediators, pruritogens, and receptor signaling mayinfluence these processes.

B. Calmodulin

CaM participates in TRP channel desensitizationby binding to N-terminal ankyrin repeats and theC-terminal tail of TRPs. With respect to pain-relatedpathways, Ca2+-dependent CaM interacts with TRPV1-4,TRPM2, and TRPM8, predominantly to facilitate chan-nel desensitization (Numazaki et al., 2003; Rosenbaumet al., 2004; Lishko et al., 2007; Sarria et al., 2011).The role of Ca2+ and CaM in TRP channel regulationhas been reviewed (Zhu, 2005) and demonstrates howall GPCRs that signal though PLC pathways have thepotential to influence TRP channel gating.

Ca2+-CaM and intracellular ATP compete for a com-mon binding site to influence channel-gating proper-ties. Although the mechanism remains unknown, ATPbinding is proposed to prevent Ca2+-CaM desensitiza-tion (Phelps et al., 2010). The high-resolution crystalstructure Ca2+-CaM bound to a C-terminal fragment ofTRPV1 reveals how the two Ca2+-bound lobes of CaMclasp the helical TRPV1 peptide to form a tight in-teraction. In contrast, interactions between CaM andthe N-terminal ankyrin repeats are predicted to occurwith lower affinity yet promote greater desensitizationof TRPV1 (Lau et al., 2012). The high-affinity bindingwith the C terminus may be important for coordinatingTRP-CaM docking to facilitate interactions with theN-terminal ankyrin site for optimal TRP channel de-sensitization (Lau et al., 2012).


C. Phospholipids

Phosphoinositide lipids, such as PIP2, are essentialintermediates of the GPCR-TRP channel axis and areknown to modulate the activity of more than 30 channels(Suh and Hille, 2005). PIP2 is present in limitingquantities in the phospholipid bilayer and binds atdistinct sites within the cytoplasmic tail of TRPVchannels (Fig. 3, C and D). In TRPV1, these sites overlapwith binding regions for the Ca2+-sensitive CaM, whichcan exert inhibitory effects by competing with PIP2

(Fig. 3) (Brauchi et al., 2007; Garcia-Elias et al., 2013).For TRPV4, PIP2 interactions within the N-terminalankyrin region are proposed to “rearrange” cytoplasmicdomains to either facilitate channel gating or exposebinding sites for endogenous inflammatory lipids, such aseicosanoids (Garcia-Elias et al., 2013). Multiple signalingproteins contribute to somatosensation by modulatingPIP2-mediated TRP regulation. PLC-dependent break-down of PIP2 leads to increased intracellular Ca2+ andproduction of DAG, leading to PKC stimulation and TRPphosphorylation. Furthermore, the accessory protein Pirt

indirectly regulates PIP2-dependent TRPV1 activity, andphosphoinositide 3-kinase–dependent phosphorylation ofPIP2 produces PI(3,4,5)P3 to enhance TRPV1 cell surfaceexpression and extracellular signal-regulated kinase(ERK)–dependent thermal pain (Zhuang et al., 2004;Zhang et al., 2005; Stein et al., 2006; Zhu and Oxford,2007; Hernandez et al., 2008; Kim et al., 2008; Ufret-Vincenty et al., 2011).

Although ion channels appear to nonselectively bindPIP2 over other PI species, its precise regulatory rolecontinues to be debated (Chuang et al., 2001; Prescottand Julius, 2003; Hilgemann, 2012; Cao et al., 2013a).PIP2 interactions with TRP channels regulate theirfunction through PLC-mediated breakdown of PIP2,which relieves TRP channel inhibition, leading to heat/capsaicin-induced pain. Hence, TRP channel sensitizationoccurs as a consequence of PIP2 turnover and generationof phosphoinositide-derived second messengers, such asAA (Huang et al., 2010; Cao et al., 2013a). A new modelhas been proposed to explain how PLC isoforms mediatebradykinin-dependent TRPV1 sensitization: submaximal

Fig. 3. Structural information provides new insights for ligand binding and the identification of key regulatory regions. (A) Electron cryomicroscopy ofTRPV1 reveals how 4 homomeric subunits are assembled into a tetrameric structure. The ribbon representation shows a large intracellular region,consisting of C-terminal and N-terminal domains (ankyrin containing region) and the formation of a b-sheet, as indicated. (B) A single TRPV1 subunitshows the assembly of transmembrane domains and intracellular regions. A simplified cartoon structure that is color coded to match the ribbon diagram isshown in (C). (D) TRPV1 is used as a template to map the sites of ligand binding and interaction partners. These include PIP2, reactive irritants that bindTRPA1, TRPV4 protein partners, TRPM8–G protein interactions, and TRPV1/TRPV4 phosphorylation sites that may influence protein-proteininteractions. All of these interactions are discussed and cited in the text. Adapted with permission from Liao et al., 2013.

50 Veldhuis et al.

PLCb activity (and PKC phosphorylation) sensitizesTRPV1, whereas increased TRPV1 Ca2+ influx activatesCa2+-sensitive PLCd isoforms to decrease PIP2 levelsand serve as a negative feedback mechanism (Lukacset al., 2013).TRP channel interaction with PIP2 is one example of how

receptors, phospholipases, and lipids can influence neuronalexcitability. Anandamide (arachidonoylethanolamide), AA,and its metabolites constitute a large group of endog-enous TRP channel ligands that contribute to pain andinflammation (Zygmunt et al., 1999). The role of AA andother fatty acid derivatives, such as eicosanoids, in TRPchannel biology has been extensively studied (Hardie,2003; Watanabe et al., 2003; Kahn-Kirby et al., 2004;Hu et al., 2006; Matta et al., 2007; Meves, 2008; Peth}oand Reeh, 2012). Their roles in pathophysiologic settingsare also discussed below.

D. Kinase Scaffold Proteins

1. A-Kinase Anchoring Protein and Receptor forActivated C-Kinase 1. Receptor-dependent control ofTRP channel activity requires channel modification byphosphorylation and the coordinated activities of kinasesand phosphatases. AKAP proteins (AKAP79 in human/AKAP150 in rodents) have a central role in receptor-mediated TRP channel activity by facilitating associationsbetween kinases, phosphatases, and phosphodiesterasesto arrange functionally and spatially distinct pools of sig-naling molecules, including PKA, PKC, and cAMP(Houslay and Adams, 2003). The PGE2-sensitive EPreceptor stimulates Gas pathways (Malmberg et al.,1997), and AKAPs have been confirmed as crucialscaffolding intermediates for PGE2-stimulated TRPV1-mediated hyperalgesia (Rathee et al., 2002; Zhang et al.,2008). AKAP150 knockout mice and AKAP150 siRNAknockdown reveal how AKAP scaffolding participatesin receptor-mediated nociception by orientating PKA,PKC, and the phosphatase PP2B toward TRPV1. Thisprocess mediates PKA- and PKC-dependent sensitiza-tion of TRPV1 responses to heat, capsaicin, and GPCR-dependent bradykinin and PGE2 stimulation (Jeskeet al., 2008, 2009; Schnizler et al., 2008; Zhang et al.,2008). TRPV4 phosphorylation by PKA/PKC is alsoenhanced by AKAP79 expression to promote channelsensitization to hypotonic cell swelling and bradykinin(Fan et al., 2009).TRP channel interactions with these adaptor pro-

teins can also provide negative feedback mechanismsto facilitate TRP channel desensitization. Phosphodi-esterase PDE4D5 activity downregulates cAMP levels,and for the b adrenoreceptor, this regulates switchingbetween G protein signaling pathways (Baillie et al.,2003). The PKC scaffold receptor for activated C-kinase1 coordinates PKC and the PDE4D5 protein to regulateTRPC3 and TRPM6 activity by regulating PKC andPKA activity (Bandyopadhyay et al., 2008; Cao et al., 2008).

Interactions between receptor for activated C-kinase 1 andother nociceptive TRP channels are yet to be determined.

2. b-Arrestins as Adaptors for G Protein–CoupledReceptors and Transient Receptor Potential Channels.The four members of the arrestin protein family in-clude arrestin-1 and -4 of visual sensory tissue andthe nonvisual ubiquitously expressed arrestin-2 and-3, otherwise known as b-arrestin-1 and b-arrestin-2.Initially named for their capacity to prevent continuedsignaling of b-adrenergic receptors, b-arrestins are nowknown to regulate multiple GPCRs and TRP channelsin the pain pathway (Rowan et al., 2014). The associationof b-arrestins with GPCRs is facilitated by receptorphosphorylation, where agonist-occupied GPCRs arephosphorylated by a family of G protein–coupled re-ceptor kinases (GRKs), in addition to PKC and PKA,which enhances association with b-arrestins. b-Arrestinsuncouple GPCRs from heterotrimeric G proteins anddesensitize the cell surface component of signal trans-duction through recruitment of GPCRs to clathrin andother endocytic adaptor proteins, to mediate receptorendocytosis. Internalized GPCRs then either recycleto the cell surface for reactivation (e.g., neurokinin 1receptor [NK1R] in the spinal cord) or are degraded inlysosomes (e.g., PAR2 in sensory neurons; reviewed byMurphy et al., 2009, and Shenoy and Lefkowitz, 2011).Rapid and specific receptor phosphorylation eventspromote different outcomes of G protein signaling,b-arrestin recruitment, and GPCR trafficking. Highlyphosphorylated receptors, such as PAR2 andNK1R, stronglyassociate with b-arrestin-1 and -2, resulting in rapidand robust internalization and sequestration into endo-somal pools (Cattaruzza et al., 2013; Pal et al., 2013).

The contribution of GPCR phosphorylation and traf-ficking to complex pathophysiologic processes, such asinflammatory pain, are poorly understood. However, it isestablished that endosomes are not merely a sortingstation for delivery of receptors to recycling or degrada-tive pathways but exist as platforms for receptors tosignal from intracellular locations (Murphy et al., 2009).Arrestins are crucial in this process. In the case of NK1Rtrafficking, b-arrestin affords NK1R an extended resi-dence in the endosomes and serves as a scaffold forERK1/2 and Src kinases, which may promote pain-transmitting signaling cascades.

Although most studies of b-arrestins have examinedtheir interactions with GPCRs, b-arrestins can alsoassociate with TRP channels to directly regulate channelactivity. At the cell surface of sensory neurons, phos-phorylation within the TRPV1 N terminus (Ser116 andThr370) enhances channel activity and reduces pharma-cological desensitization. Two additional PKA phosphor-ylation sites (Thr370 and Thr382) in the linker region(immediately downstream of the N-terminal ankyrinrepeat domain) are proposed to increase electrostaticinteractions between TRPV1 and b-arrestin-2, providinga scaffold for PDE4D5, resulting in reduced cAMP and


desensitization of TRPV1 (Por et al., 2012, 2013). Thisprocess may also contribute to endocannabinoid- andopioid-dependent downregulation of TRPV1-dependentpain (Patwardhan et al., 2006). Conversely, chronic opioid-induced hyperalgesia was recently investigated, andevidence suggests that stimulation of the m-opioid receptorwith agonists that cause robust receptor internalizationenhance TRPV1 activity due to a loss of TRPV1-arrestininteractions (Rowan et al., 2014). Similarly, angiotensin1R signaling in HEK-293 and vascular smooth musclecells promotes TRPV4 internalization through an arrestin-dependent mechanism (Shukla et al., 2010).In summary, GPCR-TRP channel cross-talk requires

lipid second messengers and signaling proteins. Theseintermediates function in different ways to modulateTRP channel activity, phosphorylation state, and cellsurface expression. Furthermore, new structural in-formation has revealed locations of lipid or proteininteractions, and together these data provide valuableinsights into the regulation of TRP channels at themolecule level.

IV. The G Protein–Coupled Receptor–TransientReceptor Potential Channel Axis in

Visceral Pathways

GPCRs and TRP channels are essential for all gas-trointestinal functions. GPCRs mediate the actions ofmany gastrointestinal hormones and neurotransmitters,and GPCRs and TRP channels participate in chemo-sensation, regulate motility, secretion, and homeostasisand contribute to visceral sensation. Dysregulation ofthese functions, for example, with respect to the ac-tivation properties and expression of TRP channels invisceral afferents, contribute to visceral pain and in-flammation. In addition to activation by environmentalstimuli and endogenous mediators, a wide variety ofpungent substances commonly associated with spices arealso potent TRP channel agonists, and traditional treat-ments for gut-related disorders often contain TRPchannel activators as active ingredients (Vriens et al.,2008; reviewed by Holzer, 2011). This section provides anoverview of the sensitization and function of neuronalTRP channels in the etiology of digestive disease, with aparticular emphasis on pain and neurogenic inflammation.

A. Transient Receptor Potential Channels inInflammatory Bowel Disease and FunctionalBowel Disorders

Visceral pain caused by inflammation, obstruction,or functional disorders is the most common form ofdisease-derived pain and is often poorly managedtherapeutically. The extrinsic innervation of viscera,particularly the gastrointestinal tract, has been thesubject of several recent reviews (Robinson and Gebhart,2008; Blackshaw et al., 2010; Brookes et al., 2013).

The expression, activation, and sensitization of TRPchannels expressed by visceral afferents are perhapsbest characterized for extrinsic sensory neurons in-nervating the colon. Studies on knockout mice or theuse of TRP-selective agents have demonstrated thatTRP channels play integral roles in both the de-velopment and maintenance of colitis, as well as beingmajor mediators of colonic afferent hypersensitivity.TRP channels are also upregulated in colonic afferentsof animals with inflammatory bowel disease (IBD).Most clinical studies have focused on TRPV1, andthere is an increased density of TRPV1-positive fibersinnervating the colonic mucosa in both IBD andfunctional bowel disorders, such as irritable bowelsyndrome (IBS). These changes have been positivelycorrelated to visceral pain scores.

1. Sensitization of Transient Receptor PotentialChannels: Mechanisms andRole in Colonic Hypersensitivity.The factors that lead to abdominal pain, such as occursin IBS and IBD, are poorly understood. IBS-relatedpain is often associated with a past infection andinflammation, and sensory afferents may exhibit hy-perexcitability long after the initial inflammation isresolved (Collins, 2001; Beyak et al., 2004; Ibeakanmaet al., 2009). In visceral afferents of the colon, sen-sitization of TRPV1, TRPV4, and TRPA1 by GPCRagonists, inflammatory changes, and exposure of nerveterminals to acid has been reported. These pathwaysare illustrated in Fig. 4. The sensitization of visceralafferents, particularly after exposure to GPCR agonists,is a major mechanism through which postinflammatoryvisceral pain is initiated and maintained. Evidence forfunctional expression of TRPV1, TRPV4, TRPA1, andTRPM8 by colonic afferent neurons is provided bystudies demonstrating mRNA and protein expressionand by Ca2+ imaging and electrophysiologic studies thatshow responsiveness by these neurons to establishedTRP channel activators (Christianson et al., 2007, 2010;Brierley et al., 2008, 2009; De Schepper et al., 2008; Tanet al., 2008; Harrington et al., 2011).

TRP channel sensitization through activation ofGPCRs has been demonstrated by a number of studies.Peripheral sensitization of colonic afferents to stretchstimulation occurs after exposure to an acidic in-flammatory soup containing bradykinin, 5-HT, hista-mine, and PGE2 (Jones et al., 2005). Diminished afferenthypersensitivity in trpv12/2 mice demonstrates thatthis sensitization is partly mediated via TRPV1 and isconsistent with the use of an H2 histamine receptorantagonist to reduce capsaicin-sensitive neurogenicinflammation of the colon (Okayama et al., 2004).Activation of TRPV1 by capsaicin, heat, or by protons isalso augmented after pre-exposure of colonic sensoryneurons to 5-HT and involves a G protein–dependentPKA/AKAP mechanism (Sugiuar et al., 2004). 5-HT2

and 5-HT4 reduce the temperature threshold for neuronalfiring, an effect that is lost in neurons from trpv12/2 mice

52 Veldhuis et al.

and requires PKC activity. Pre-exposure to the proin-flammatory mediators 5-HT or histamine enhances in-tracellular Ca2+ responses of colonic afferent neurons tothe TRPV4 agonist 4a-PDD, a response that is attenuatedby intrathecal delivery of TRPV4 siRNA (Cenac et al.,2010). The underlying sensitization mechanism involvesPLC, PLA2, PKC, and mitogen-activated kinase, andimaging studies revealed that 5-HT and histaminetreatment promoted increased cell surface expressionof TRPV4 in neurons. In an analogous system, micelacking TRPA1 show deficiencies in early responses tobradykinin-induced colonic afferent mechanosensitivity(Brierley et al., 2005, 2009; Wang et al., 2008).Growth factors, including NGF and glial-derived

neurotrophic factor, are also upregulated in IBD and inanimal models of colitis (Christianson et al., 2010).Acute exposure of colonic afferent neurons to NGFpotentiates TRPV1- and TRPA1-evoked Ca2+ signalingin colonic afferents, suggesting an important role forTrkA receptor signaling in TRP channel activity, poten-tially due to increased TRP channel surface expression(Zhang et al., 2005). Similarly, glial-derived neurotrophicfactor–mediated sensitization of TRPA1 has been in-vestigated in cutaneous afferents and shows no sensitiza-tion, thus highlighting the functional importance ofpeptidergic neurons in neurogenic inflammation (Malinet al., 2011).2. Protease-Dependent Sensitization of Transient

Receptor Potential Channels in Inflammatory BowelDisease. Proteases are major mediators of neurogenic

inflammation and hyperalgesia through specific activa-tion of PARs and subsequent sensitization of TRPchannels (Amadesi et al., 2004; Ossovskaya and Bunnett,2004; Dai et al., 2007; Grant et al., 2007). Cellularsources of proteases in the intestine under normal andpathologic conditions include digestive enzymes, inflam-matory cells (e.g., mast cells, macrophages), epithelialcells, and gut-associated pathogens, including bacteria(Barbara et al., 2004; Ossovskaya and Bunnett, 2004;Barbara, 2007). These proteases are present in biopsysupernatants and can activate PARs (particularly PAR2),leading to the sensitization of mouse sensory neuronsand colonic afferents (Cenac et al., 2007). PAR2-inducedmechanical hyperalgesia to colorectal distension isTRPV4 dependent, as PAR2-evoked action potentialsare effectively prevented by TRPV4 deletion. Prior ex-posure of colonic sensory neurons to PAR2 activatingpeptide (PAR2-AP; SLIGRL-NH2) augments cellularresponses to the TRPV4 activators 5,6-EET and 4aPDD,consistent with the PAR2-dependent sensitization ofTRPV4 in colonic afferents (Sipe et al., 2008) and DRGs(Poole et al., 2013). Similar PAR2-TRPA1 studies havebeen performed and show that intracolonic delivery ofPAR2-AP increase visceromotor responses to colorectaldistension in wild-type, but not trpa12/2, mice (Cattaruzzaet al., 2010). However, a functional interaction betweenPAR2 and TRPA1 is not apparent in studies of colonicmechanosensory transduction (Brierley et al., 2009) andmay suggest off-target effects for PAR2-AP, as observed inother systems (Betts et al., 2012).

Fig. 4. TRP channels are major mediators of inflammatory bowel disease and visceral pain. The expression of TRP channels within the colon and bycolonic extrinsic primary afferent neurons is significantly increased in both IBD and IBS. The activation of TRP channels expressed by primaryafferents leads to the peripheral release of proinflammatory neuropeptides (SP, CGRP), resulting in neurogenic inflammation. The release of theseneuropeptides at the level of the spinal dorsal horn results in pronociceptive signaling. Agonists of cell surface receptors, including GPCRs, are releasedduring tissue damage or inflammation. The activation of these receptors leads to the sensitization or recruitment of TRP channels (see examplesshown). This effectively augments TRP channel activity in these neurons, resulting in enhanced neuropeptide release and to greater inflammation andpain. Modified from Furness et al., 2013.


PAR4 activation by selective activating peptides orby cathepsin G reduces responses to colorectal distension(Annahazi et al., 2009) through presumed activation ofPAR4 expressed on nerve terminals (Annahazi et al.,2012). Prestimulation with PAR4 activating peptidecan significantly reduce allodynia and hyperalgesiain response to PAR2 and TRPV4 agonists and caneffectively inhibit elevated intracellular Ca2+ levels inprimary afferent neurons in response to these ago-nists (Auge et al., 2009). Intracolonic PAR1 activationsimilarly reduces behavioral responses, indicative ofantinociceptive regulation of visceral pain (Kawaoet al., 2004).

B. Transient Receptor Potential Channels,Pancreatitis, and Pancreatic Pain

The pancreas is innervated by both vagal and spinalafferents, with cell bodies located within nodose anddorsal root ganglia, respectively, and sensitization byinflammatory mediators likely contributes to pancreatitispain. Several lines of evidence support the importance ofneurogenic inflammation in the etiology of pancreatitis,including increased pancreatic innervation, elevatedneurotransmitter content in pancreatic afferents, andreduction in pancreatitis severity by destruction ofprimary afferent neurons (Nathan et al., 2002;Hartel et al., 2006; Noble et al., 2006; Schwartzet al., 2013).1. Transient Receptor Potential Channels and

Pancreatitis. The involvement of TRPA1, TRPV1, andTRPV4 in pancreatitis and pancreatic pain has beeninvestigated, and observations support a role for TRPV1and TRPA1 in neurogenic inflammation of the pancreas.TRPV4-positive nerve fibers innervate the pancreas, andpancreatic DRG afferents express functional TRPV4,although no functional changes in TRPV4 have beenassociated with acute pancreatic inflammation (Ceppaet al., 2010). Nerve fibers positive for both TRPV1 andCGRP are also in close association with pancreatic acinarcells and predominantly originate from spinal afferentDRG neurons (Fasanella et al., 2008). TRPV1mRNA andprotein are upregulated in animals with pancreatitis,in line with enhanced responses by pancreatic afferentneurons to capsaicin (Xu et al., 2007; Schwartz et al.,2011, 2013; Zhu et al., 2011). Consistent with thesestudies, capsaicin-treated animals are less susceptible topancreatitis and treatment with the TRPV1 antagonistcapsazepine or resiniferatoxin-induced denervation pro-tects against caerulein-induced pancreatitis (Nathanet al., 2001; Noble et al., 2006). TRPA1-positive afferentnerve fibers innervate the pancreas and respond to theTRPA1 activators allyl isothiocyanate and endogenousagonists produced in pancreatic inflammation (15dPGJ2and 4-HNE), and responses are attenuated by TRPA1inhibitors or in trpa12/2mice (Ceppa et al., 2010; Schwartzet al., 2011, 2013; Li et al., 2013). The administrationof TRPV1 and TRPA1 agonists into the pancreatic duct

induces c-fos expression and NK1R internalization inspinal cord and causes nocifensive behavior, whichtogether suggest activation of pain pathways. Theseeffects are diminished by intrathecal delivery of NK1Rand CLR antagonists, TRPV1/TRPA1 antagonists, orgenetic deletion of TRPA1 (Wick et al., 2006; Ceppaet al., 2010; Schwartz et al., 2013).

2. Sensitization and Activation of Transient ReceptorPotential Channels in Pancreatitis and PancreaticPain. The inflamed pancreas is an ideal environmentin which TRP channels may be directly activated orindirectly sensitized. The initial tissue injury associatedwith pancreatitis leads to premature activation of trypsin,an established agonist of PAR2. PAR2 has proinflamma-tory and protective roles in pancreatitis, depending on themodel used (Kawabata et al., 2006; Laukkarinen et al.,2008). PAR2 activation by intraductal delivery of PAR2-APor trypsin results in c-fos induction in spinal neuronsthat is attenuated by capsazepine (Nishimura et al.,2010). PAR2-dependent activation of spinal ERK isalso blocked by capsazepine and is thus mediatedthrough PAR2-TRPV1 interaction (Fukushima et al.,2010). PAR2 sensitization of other TRP channelshas not been examined with respect to pancreaticpain.

NGF promotes upregulation of TRPV1 gene expres-sion, sensitization of TRPV1 currents, and increasedfunctional TRPV1 at the surface of nociceptive neurons(Bron et al., 2003; Zhuang et al., 2004; Zhang et al., 2005).Blockade of NGF signaling using NGF neutralizingantibodies reduces pancreatic hyperalgesia in an intra-ductal model of chronic pancreatitis, induced with 2,4,6-trinitrobenzenesulfonic acid (Zhu et al., 2011). The AAderivative leukotriene B4 induces pancreatic inflamma-tory damage, which is diminished by capsazepine orinhibition of the leukotriene B4 biosynthesis (Vigna et al.,2011). Other potential TRPV1 activators are producedin pancreatitis, including protons, anandamide, andendovanilloids, although their specific roles are notwell defined.

In conclusion, considerable evidence supports anintegral role for TRP channels in the control of normalvisceral function and sensation. Changes in TRPchannel expression and sensitivity are associated withinflammatory disease and visceral hypersensitivity inanimal models, and these changes may also occur inhuman disease. Although the functional interactionbetween GPCRs and TRP channels may underlievisceral hypersensitivity, the mechanisms of theseinteractions, particularly in functionally relevant cells,such as visceral afferent neurons, remain unclear.GPCR-TRP signaling pathways require greater char-acterization and may be supported by further studieson human tissue or innovative approaches, such as theassessment of visceral diseases in mice lacking keysignaling proteins, such as the AKAP scaffold (Zhanget al., 2011).

54 Veldhuis et al.

V. Not-So-Painful: Contributions of theG Protein–Coupled Receptor–Transient

Receptor Potential Channel Axis for IrritantSensation and Inflammation

The first line of defense against noxious substancesand invasive organisms is formed by epithelial layerssuch as the epidermis (skin), the mucosal lining of theairways, and the gastrointestinal tract. These barriersare highly innervated for detection of potentially harm-ful stimuli, including noxious temperatures, chemicalirritants, and microorganisms. Exposure to noxiousstimuli triggers pain, which leads to withdrawal fromor avoidance of damaging agents. However, noxiousstimuli also activate reflexes that remove the stimulus(e.g., cough, scratching) and initiate protective measures(e.g., mucus secretion, activation of an inflammatoryimmune response). As is the case in pain, GPCRs andTRP channels play a major role in these protectivemechanisms. Thus, sensory processes stimulated byGPCRs and TRP channels are integral to the body’sdefense mechanisms, but are not always painful. More-over, abnormalities of the GPCR-TRP axis may contributeto dysregulation and chronic disease, such as chroniccough, pruritus, and inflammation.

A. The G Protein–Coupled Receptor–TransientReceptor Potential Axis in Pruritus

Itch (pruritus) is a defense mechanism that has beendefined as an “unpleasant skin sensation that elicitsthe desire or reflex to scratch” (Rothman, 1941). Whenperipheral nerve terminals in the skin and mucosalsurfaces detect pruritogenic stimuli they elicit a scratch-ing response that protects by removing the associatedirritant. By contrast, chronic itch is a common clinicalproblem that exacerbates cutaneous conditions and leadsto further injury (reviewed in LaMotte et al., 2014). Themost well established pruritogen, histamine, is secretedby dermal mast cells and excites nearby sensory fibersby acting on histamine H1 receptors (Liu et al., 2009).However, chronic pruritus is insensitive to antihistaminetreatment. Chronic itch is commonly associated withdermal diseases and metabolic disorders and can beclassified according to the source or cause of the pruriticsymptoms: dermal or peripheral itch (e.g., dermatitis,psoriasis), neurogenic itch (e.g., chronic renal failure,cholestatic liver disease), and neuropathic itch (e.g.,multiple sclerosis, diabetes) (Binder et al., 2008).In contrast to the classic theory that itch as a “sub-

threshold pain” evoked by weak activation of nociceptors,the “specificity theory of itch” describes the differentia-tion of pain and itch sensory pathways (Han et al., 2013;Tóth and Bíró, 2013). The sensations of itch and pain aredistinct; however, they are both protective mechanismsand involve a similar neuronal pathway of transmission.Like pain, itch is mediated by subpopulations of un-myelinated small diameter C-fiber sensory neurons with

cell bodies in the DRG and TG and is transmitted centrallyto second-order neurons of the spinal cord (Caterina andJulius, 1999; Paus et al., 2006; Shim and Oh, 2008; Hanet al., 2013). Moreover, there are important functionalinteractions between pain and itch pathways that areexemplified by the phenomenon that painful scratchingcan inhibit itch, and intrathecal analgesics can cause itch.These interactions depend on regulatory mechanisms,such as itch-inhibitory interneurons, that are stimulatedto reduce the postsynaptic transmission of itch whennociceptive neurons are activated. For example, theadministration of opiates can cause itch by both inhibitingsynaptic transmission of the pain signal and preventingactivation of the itch-inhibitory interneurons (Xiao andPatapoutian, 2011; Baraniuk, 2012). In addition, MrgprA3is a pruritogen-responsive GPCR expressed in neuronsthat innervate the epidermis. Genetic ablation ofMrgprA3-positive neurons in mice substantially reducedscratching behavior to a range of stimuli (histamine,chloroquine, dry skin, allergic inflammation) withoutaffecting pain sensitivity (Han et al., 2013). Interestingly,in mice engineered to exclusively express TRPV1 inMrgprA3-positive neurons, scratching behavior in re-sponse to capsaicin remains intact, whereas painresponses are absent (Han et al., 2013). There is evidencefor multiple nociceptive TRP channels to contribute topruritus, and Han et al. (2013) hypothesize that ex-pression of specific GPCRs within a neuronal subpopu-lation may be a key determinant that influences theability of a TRP channel to contribute to either nociceptiveor pruritic processes.

The circuitry controlling itch is complex and involvesmany pruritogenic substances (histamine, tryptase,serotonin, kinins, chemokines) released from neuronsand cutaneous cells, including keratinocytes, mast,endothelial, and immune cells (Tóth and Bíró, 2013).TRPA1, TRPV1, TRPV3, and TRPV4 are expressed onall of these cell types and contribute to acute andchronic itch processes either by facilitating pruritogenrelease or sensory itch responses in neurons (Ikomaet al., 2006). A more comprehensive understanding ofthe fundamental molecular basis of itch, including theGPCR-TRP axis, is required to improve the treatmentof chronic itch conditions. GPCRs expressed in theperiphery contribute to the processing of itch andinclude those receptors sensitive to histamine (H1R,H4R), proteases (PAR2), bradykinin (B1R, B2R), bileacids (TGR5), prostanoids (EP1R), SP (NK1R), and theMas-related GPCRs (MrgprA3, MrgprC11). Their rolesin pruritus and regulation of TRP channels have beenreviewed (Ikoma et al., 2006; Bautista et al., 2014).GPCR-TRP sensitization pathways that have beenclearly defined to promote acute or chronic itch aresummarized in Fig. 5.

1. Transient Receptor Potential Vanilloid 1. TRPV1has been implicated in multiple itch pathways, andalthough TRPV1 can directly induce itch by capsaicin, it


primarily functions downstream of receptors that arestimulated by pruritogens (Imamachi et al., 2009;Sikand et al., 2009). Histamine is released by dermalmast cells during inflammation or in response to allergensand stimulates H1 and H4 GPCRs on unmyelinatedC-fibers to cause itch (Bell et al., 2004; Dunford et al.,2007; Shim et al., 2007). Two major intracellular signalingpathways have been postulated to mediate H1 receptorresponses. In CHO cells, H1 causes the release of in-tracellular calcium stores mediated by the PLC/IP3

pathway (Leurs et al., 1994). In rat DRGs, H1 receptoractivation stimulates PLA2 and lipoxygenase activity toincrease gating of TRPV1 (Kim et al., 2004). The DRGmodel is supported by reduced histamine-evoked scratch-ing in mice exposed to inhibitors of PLA2, lipoxygenase,H1, and TRPV1, or in trpv12/2mice. However, these micestill exhibit scratching behavior, which suggests thatalternate histamine-dependent pruritic pathways exist(Shim et al., 2007). Histamine receptor signaling canstimulate kinase activity and the production of sensi-tizing agents, such as prostaglandin PGE2, suggestingthe involvement of other TRP-sensitizing pathways(Belghiti et al., 2013).Neurogenic inflammation can lead to pruritus through

the local release of NGF and neuropeptides, such astachykinins and CGRP (Holzer, 1998; Costa et al., 2008).PAR2-dependent sensitization of TRPV1 on sensoryafferents is also proposed to be pruritogenic (Belghitiet al., 2013). However, itch induced by the PAR2

activating peptide SLIGRL is potentially mediated bytheMrgprC11, rather than PAR2. In contrast, a truncatedpeptide SLIGR activates PAR2 to cause pain but does not

induce MrgprC11-dependent scratching (Liu et al., 2011).Although the signaling mechanisms by which MrgprC11regulates TRPV1 are not fully understood, MrgprC11activates TRPA1 via Gaq/11-PLC signaling (Wilsonet al., 2011). Alternatively, MrgprC11 may cause mastcell degranulation, neurogenic inflammation, and therelease of a multitude of mediators, including pro-teases and kinins, all of which contribute to itch andTRPV1 activation (Liu et al., 2011; Wilson et al.,2011).

Pirt regulates PIP2-dependent activation of TRPV1and TRPM8 (Kim et al., 2008) and participates inhistamine-dependent and -independent (chloroquine,5-HT, endothelin-1) itch, as well as TRPV1-dependentand -independent responses (Patel et al., 2011). How-ever, Pirt does not contribute directly to the activation ofother TRP channels, such as TRPA1, and it is thereforehypothesized that Pirt may modulate specific pruritogen-induced GPCR signaling cascades, such as PLCb3 activity(Patel et al., 2011).

2. Transient Receptor Potential Ankyrin 1. TRPA1 isa major target of GPCR-dependent itch processes and isa key mediator of chronic itch, either directly or viaclinically relevant histamine-independent pruritogens in-cluding BAM8-22 and chloroquine, which stimulate Mrgreceptors MrgprC11 and MrgprA3, respectively (Liu et al.,2009). MrgprA3- and MrgprC11-induced itch is absent intrpa12/2 mice, and both receptors can activate TRPA1 invitro (Wilson et al., 2011). These two GPCRs activateTRPA1 via distinct signaling mechanisms; pharmacologicexperiments suggest that MrgprC11 is functionally linkedto TRPA1 by stimulating PLC activity, whereas MrgprA3

Fig. 5. Sensory and inflammatory pathways leading to sensitization or activation of TRP channels in the skin. Exposure of the skin to pruritogensresults in activation of sensory neurons, resulting in the generation of itch responses. In addition, endogenous pruritogens are released from variouscell subtypes, such as keratinocytes, mast cells, and endothelial cells. Several itch pathways are now known to exist, and TRP channels are integralcomponents of these. TRPV1 is thought to largely mediate histamine-dependent itch downstream of H1 receptors via PLA2 and lipoxygenase.Nonhistaminergic itch involves the activation of TRPA1. Key intracellular mechanisms of GPCR-TRP channel interaction are outlined.

56 Veldhuis et al.

couples to TRPA1 via the Gbg subunits, independently ofPLC activation (Wilson et al., 2011). Related studies havealso demonstrated that chloroquine-induced sensitizationof TRPV1, TRPA1, and TRPM8 in DRG neurons occursvia similar PLC-independent pathways (Than et al.,2013). This intracellular signaling diversity highlightsthe complexity of itch sensation yet may help to explainthe selectivity of the distinct transduction pathways.Several animal models of chronic itch have been used

to understand human processes of itch (Bautista et al.,2014). Comprehensive gene expression-profiling analysesin mouse models of chronic itch (alcohol-ether-water dryskin) reveal that TRPA1 activity is a key mediator ofchronic itch phenotypes. TRPA1 activity induces ex-pression changes in many genes contributing to scratchbehavior, dermatitis, dry skin, and other sensory, in-flammatory, or immune responses (Wilson et al., 2013a).This study also highlighted a potential role for TRPA1in itch hypersensitivity incurred through increases in theexpression of itch receptors (e.g., B2R) and innervationby itch-detecting primary afferent nerve fibers, as pre-viously observed in dry skin mouse models (Tominagaet al., 2007).Conditions of chronic pruritus, such as atopic derma-

titis, psoriasis, cholestasis, and diabetes, are associatedwith high levels of oxidative stress. TRPA1 is anestablished mediator of oxidant-induced pain (Anderssonet al., 2008; Bessac and Jordt, 2008), and, unsurprisingly,TRPA1 has also been implicated in itch associated withoxidative-stress induced by hydrogen peroxide andtert-butylhydroperoxide. In contrast, this itch is notinfluenced by blockade of histamine receptors. Thisevidence reinforces the concept that TRPA1 is a media-tor of nonhistaminergic pruritus (Wilson et al., 2011).More recently neuronal TRPA1 has also been linked tothe actions of the cytokine thymic stromal lymphopoietin(TLSP) in atopic dermatitis-associated itch (Wilsonet al., 2013b). A relationship between proteases, PAR2

signaling, and TLSP has been reported for modelsof dermatitis (Briot et al., 2009, 2010). The study byWilson et al. (2013b) extends these findings to show thatprotease-dependent stimulation of PAR2 in keratino-cytes results in intracellular Ca2+ release, stimulation ofendoplasmic reticulum STIM-1 protein, and the cellsurface ORAI1 Ca2+ channel to initiate expression andsecretion of TLSP. The activation of TLSP receptorson adjacent sensory neurons subsequently stimulatesTRPA1 activity. Together, this mechanism revealspruritogenic processes that require communicationbetween cells and between GPCRs and ion channelsto generate itch responses in chronic skin conditions(Wilson et al., 2013b).3. Transient Receptor Potential Vanilloid 3. A role

for TRPV3 in itch sensation has been demonstratedusing an experimentally induced dry skin pruritusmodel, where trpv32/2 mice show decreased scratchingin response to acetone-ether-water (Yamamoto-Kasai

et al., 2012). Moreover, an increase in the density ofinvasive nerve fibers is observed in this model. Thisobservation is consistent with atopic dermatitis patients,where increased epidermal C-fiber innervation is thoughtto aggravate the disease (Paus et al., 2006).

A genetic basis for itch disorders involving TRPV3hyperactivity has been identified, where a gain-of-function mutation in the trpv3 gene (Gly573Ser) causesspontaneous dermatitis and itch in rodents (Asakawaet al., 2006). Gain-of-function missense mutations inTRPV3 (Gly573Ser, Gly573Cys, and Trp692Gly) have beendescribed in Olmstead syndrome, a rare congenital skincondition that causes severe itching. Mutant TRPV3channels expressed in HEK cells or in keratinocytes ofOlmstead Syndrome patients show increased inwardlyrectifying currents and apoptotic cell death (Lin et al.,2012). TRPV3 can be sensitized via the PKC signalingpathway (Hu et al., 2006) and could contribute to itchdownstream of GPCR activation; however, this has notbeen reported to date.

4. Bile Acids. Bile acids are essential for the digestionand absorption of dietary fat and can also activate thebile acid receptor TGR5 (GPBA), a GPCR that couples toGas, adenylyl cyclase, and cAMP production (Kawamataet al., 2003; Rajagopal et al., 2013). Bile acids are markedlyincreased in the circulation and tissues of patients withcholestatic liver disease and may contribute to the chronicand intractable pruritus that is a common feature of thiscondition (Hayashi and Majima, 1999). TGR5 is expressedin peptidergic small diameter primary sensory neuronsinnervating the skin, and activation by bile acids causesa TGR5-dependent hyperexcitability and stimulates thesecretion of the itch-selective transmitter gastrin releasingpeptide (Alemi et al., 2013). The intradermal injection ofbile acids and TGR5-selective agonists also caused robustscratching in wild-type but not tgr52/2mice, whereas gain-of-function transgenic mice exhibit a spontaneous pruritus.Evidence suggests that TGR5 agonists promote pruritusvia a TRPA1-dependent process (Lieu et al., 2014). Bileacids can also evoke scratching by alternative mechanisms.The intradermal administration of deoxycholic acid to rats,for example, activates kallikreins to generate bradykininand stimulate B2R signaling (Hayashi and Majima, 1999).The role of B1R and B2R activity in PAR2-dependentpruritus has also been investigated and suggests that kininreceptors function downstream of protease-induced pruriticresponses (Costa et al., 2010). Although the bile acid-bradykinin system has not been clearly defined, TRPV1sensitization was recently demonstrated to contribute toliver disease-induced itch and is predicted to play a majorrole, potentially via receptors for bradykinin, histamine,serotonin, or PAR2 (Belghiti et al., 2013). In addition,TRPV1 and TRPA1 are expressed in distinct andoverlapping populations of peptidergic and IB4+ neu-rons (Bhattacharya et al., 2008; Kim et al., 2010), andcoexpression of TRPA1 and TRPV1 on peptidergicC-fibers is hypothesized to result in Ca2+-dependent


channel cross-talk (Bautista et al., 2006). AlthoughTRPA1 is a major downstream target of Gs-stimulatedTGR5 signaling in the presence of elevated endogenouspruritogens (Lieu et al., 2014), it is tempting to speculatethat TRPV1-induced Ca2+-dependent upregulation ofTRPA1 may contribute to scratching behavior via aregulatory pathway similar to that observed forbradykinin-induced inflammation via a TRPV1-TRPA1and Ca2+-dependent pathways (Bautista et al., 2006).

B. The G Protein–Coupled Receptor–TransientReceptor Potential Axis in Airway Diseases

The lungs are constantly exposed to the externalenvironment, and the respiratory epithelium providesthe first point of contact with the majority of inhaledchemicals, pathogens, allergens, and pollutants. Theimmune system plays a crucial role in maintaininglung health by detecting and removing potentiallyharmful agents. Inflammatory processes are driven by anorchestrated interplay between the respiratory epithelium,the innate immune system, and the adaptive immunesystem. Nonspecific protection also includes anatomicbarriers, mucus, saliva, cough, and the recruitmentof inflammatory cells. The cough reflex is a protectivemechanism that helps to clear foreign material from thelungs and aids in immune defense. However, chroniccough can lead to additional injury, airway inflamma-tion, and exacerbation of disease (Ford et al., 2006). Themechanisms driving cough have only recently started tobe elucidated, and TRP ion channels appear to play afundamental role in both the healthy reflex andexacerbated inflammatory cough pathologies (Graceet al., 2013).The prevalence of asthma, chronic obstructive pulmo-

nary disease (COPD), and other respiratory diseases isincreasing on a global scale, and the etiology of asthmaand COPD has been extensively reviewed (Rabe et al.,2007; Mathers et al., 2008; Barnes, 2011, 2013). Briefly,asthma pathology is characterized by chronic airwayinflammation, variable airflow obstruction, airway hyper-responsiveness, and symptoms, such as cough, dyspnea,wheezing, and chest tightness. COPD encompasses aheterogeneous group of airway pathologies, includingchronic bronchitis, bronchiectasis, and emphysema(Bateman et al., 2008). In contrast to asthma, COPDis characterized by airflow limitation that is not fullyreversible and is usually progressive because of persis-tent inflammation and typical functional changes in theairways resulting from repeated tissue injury and repair(Di Stefano et al., 1998).The potential for TRP channels to contribute to early

airway responses is evident after inhaled constituentsof food products that contain noxious or pungent TRPV1and TRPA1 agonists (e.g., capsaicin, mustard, cinnamon,wasabi). Environmental particles, such as cigarettesmoke, wood smoke, or air pollution, also contain TRPA1agonists (e.g., acrolein, crotonaldehyde), which cause

reflex cough or bronchoconstriction (Grace and Belvisi,2011). In contrast, substances such as menthol (TRPM8)are reported to inhibit cough and elicit bronchodilation(Materazzi et al., 2009). Much of what we know aboutTRP channels in the lung has been inferred from studiesinvestigating the pain pathway. However, in some in-stances, the airways show important differences to the restof the body. For example, PGE2 is generally consideredto be a proinflammatory mediator in the periphery,which causes pain and is implicated in diseases, such asrheumatoid arthritis and ultraviolet B-induced cutane-ous inflammation (Materazzi et al., 2008). However, inthe lungs PGE2 has both beneficial (e.g., bronchodilatoryand anti-inflammatory) as well as unwanted (e.g.,protussive) effects (Pavord et al., 1993; Gauvreau et al.,1999). The following discussion focuses on the impor-tance of TRP channels in the airways and our currentunderstanding of how GPCRs contribute to TRP channelfunction in this context. These airway GPCR-TRP sensiti-zation pathways are summarized in Fig. 6.

1. Transient Receptor Potential Vanilloid 1. Indiseases such as asthma and COPD, activators andsensitizers of TRPV1 are present at increased concen-trations in the lung. This can induce tonic activation ofTRPV1 and is proposed to contribute to underlyingairway disease pathology (Profita et al., 2003; Gattiet al., 2006). The biologic response to TRPV1 stimula-tion is consistent with respiratory disease symptomsand processes, including cough, bronchoconstriction,and inflammation. Moreover, cigarette smoke expo-sure, allergy, and virus cause hypersensitivity of thecough reflex to TRPV1 agonist inhalation, which can beprevented by TRPV1 antagonists (reviewed by Adcock,2009, and Banner et al., 2011). Increased expression ofTRPV1 in airway nerves and smooth muscle is alsocorrelated with sensitization of the cough reflex inhumans (Groneberg et al., 2004; Mitchell et al., 2005;Butler et al., 2010). In contrast to cough, a direct rolefor TRPV1 in the etiology of other airway diseases isstill unclear. For example, in animals, TRPV1 agonistscause tracheal smooth muscle contraction in vitro andbronchoconstriction in vivo via neurogenic pathways(Belvisi et al., 1992; Lalloo et al., 1995). Conversely,capsaicin has been observed to cause bronchodilation inhumans (Ichinose et al., 1988).

2. Transient Receptor Potential Ankyrin 1. TRPA1 isexpressed by bronchial epithelia and TRPV1-positive vagalafferent fibers innervating the lung (Nassenstein et al.,2008; Mukhopadhyay et al., 2011). TRPA1 is highlysensitive to respiratory irritant exposure, includingpungent cysteine-reactive chemicals, or irritants, suchas cigarette smoke, to cause TRPA1-dependent protussiveevents in human and animal models and is therefore akey therapeutic target for respiratory disorders (Trevisaniet al., 2007; Birrell et al., 2009; Preti et al., 2012). Thepathophysiologic consequences of this include neuro-genic release of the proinflammatory neuropeptide SP.

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In a positive feedback cycle, SP released from sensorynerve terminals increases NK1R-dependent generationof reactive species, including the lipid peroxidation product4-HNE, which can further increase TRPA1 activity(Rahman et al., 2002; Boldogh et al., 2005; Taylor-Clarket al., 2008a). Together, an initial TRPA1 response torespiratory irritants, followed by secondary responses toinflammatory mediators, implicates TRPA1 in sensoryprocessing and augmented dysregulation in airway dis-eases due to enduring inflammation and lung remodeling(Springer et al., 2007; Li et al., 2008). TRPA1 is also likelyto mediate allergen-induced airway inflammation in pa-tients with asthma, as demonstrated using trpa12/2 miceor by treatment with the selective TRPA1 inhibitorHC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide], whichreduces allergen-induced leukocyte infiltration, cytokineand mucus production, and airway hyper-responsiveness(Caceres et al., 2009; Raemdonck et al., 2012). Animalmodels also show that TRPA1 plays an important rolein the early phase of bronchial inflammation to cigarettesmoke, causing release of interleukin-8, which is achemoattractant for neutrophils, T cells, and monocytes(Mukhopadhyay et al., 2011). Contraction of bronchialrings to cigarette smoke extract, acrolein, andcrotonaldehyde are also reduced by inhibition of TRPA1but not by TRPV1 inhibition (Andre et al., 2008).TRPA1 can also mediate the irritant effects of certainvolatile anesthetics in the airways, where activationtriggers the release of neuropeptides that can causebronchospasm and neurogenic inflammation (Mattaet al., 2008; Eilers et al., 2010).3. Transient Receptor Potential Vanilloid 4. Al-

though a loss-of-function mutation in TRPV4 does not

show significant association to asthma (Cantero-Recasenset al., 2010), diesel exhaust particles can activatePAR2 expressed by isolated human bronchial epithe-lial cells, leading to activation of TRPV4 and secre-tion of matrix metalloproteinase-1, which plays a rolein tissue remodeling and contributes to COPD andemphysema pathogenesis (Li et al., 2011). Moreover,bronchial smooth muscle cells and nerve endings maybecome exposed to hypotonic bronchial fluid in patientswith asthma because of airway remodeling (Liedtke andSimon, 2004). A hypotonic solution such as this is pro-posed to elicit TRPV4-dependent smooth muscle contrac-tion in isolated human and rodent airways (Jia et al.,2004). A genome-wide association study has also revealedmultiple TRPV4 single-nucleotide polymorphisms asso-ciated with susceptibility to COPD (Zhu et al., 2009).Loss of TRPV4 Ca2+ permeability has also been linked tocystic fibrosis pathology, rendering the epithelia unableto respond to hypotonic stress (Arniges et al., 2004).

In murine ciliated tracheal cells, functional TRPV4regulates ciliary beat frequency associated with mildtemperature and ATP stimulation, implicating it inmucociliary clearance of the lungs (Lorenzo et al.,2008). Moreover, osmotic stress can cause ATP release(and activation of inflammatory purinergic receptors)from human bronchial epithelial cells via TRPV4 andthe RhoA pathway (Eltom et al., 2011; Seminario-Vidalet al., 2011). It is possible that genetic mutations inTRPV4 could contribute to disease pathogenesis throughinability of cilia to respond to environmental stimuli andhampered mucus clearance associated with respiratorydiseases such as COPD (Zhu et al., 2009).

4. Transient Receptor Potential Melastatin 8. A lackof selective pharmacologic tools and the fact that many

Fig. 6. Sensory and inflammatory pathways leading to sensitization or activation of TRP channels in the airways. High concentrations of endogenousinflammatory mediators that can indirectly activate or sensitize TRP channels are found in the blood and lungs of patients with inflammatory airwaydisease and are thought to play an important role in disease pathogenesis. The airways are also routinely exposed to exogenous irritants (e.g., cigarettesmoke) that also activate TRPs. Major sources of TRP channel activators and sites of expression and functional roles of TRPA1, TRPV1, and TRPV4 inthe respiratory tract are as outlined.


TRPM8 agonists can also modulate TRPA1 have led toan ongoing debate about the functional importance ofTRPM8 in airway processes. Breathing cold air caninduce respiratory autonomic responses that includecough, airway constriction, plasma protein extravasation,and mucosal secretion (Yoshihara et al., 1996; Carlsenand Carlsen, 2002). Conversely, TRPM8 has been shownto inhibit the cough reflex, although data are contradic-tory. Although menthol efficacy is not greater thanplacebo in clinical trials, menthol remains a key in-gredient in many over-the-counter antitussive therapies(Kenia et al., 2008).The modestly selective TRPM8 antagonist BCTC

[N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide; also described as a TRPV1 antagonist]and siRNA knockdown can inhibit TRPM8-inducedinflammatory cytokine production and suggests thatantagonism of TRPM8 could be beneficial for patientswith cold-induced asthma or related respiratory disease(Sabnis et al., 2008; Banner et al., 2011). Moreover,menthol has been claimed as an adjuvant agent and isassociated with relief of dyspnea in diseases such asCOPD (Eccles, 2003). Indeed, menthol is regularly usedin nasal decongestant therapies. However, there is noeffect of menthol on nasal airflow, although mentholdoes increase the perception of nasal patency (Keniaet al., 2008).5. G Protein–Coupled Receptor–Dependent Sensitiza-

tion of Transient Receptor Potential Channels in AirwayDiseases. The activation of TRP channels by GPCR-stimulated intracellular signaling is important in respi-ratory disease and congruent with pain transmissionmechanisms. High concentrations of inflammatory medi-ators that indirectly activate or sensitize TRPV1 andTRPA1 (e.g., PGE2, bradykinin, lipoxygenase metab-olites, proteases) are found in the blood and lungs ofpatients with inflammatory airway disease (Profitaet al., 2003; Birring et al., 2004; Gatti et al., 2006;Grace et al., 2012). Several of the pathways initiated bythese mediators activate Gas and Gaq-coupled pathways,which converge on PKA, PKC, or PLC (Bessac and Jordt,2008) (Fig. 6).Vagal bronchopulmonary C-fibers play an important

role in inducing bronchoconstriction, hypersecretion ofmucus, and the cough reflex (Lee et al., 2002a). Inparticular, the tussive effects of PGE2 have been linkedto EP3 receptor-dependent activation of TRPV1 andTRPA1 (Maher et al., 2009; Grace et al., 2012). In otherpathways, PGE2 sensitizes TRPV1 responses in cul-tured vagal neurons by activation of adenylyl cyclaseand through PKA-mediated TRPV1 phosphorylation,likely via the EP2 receptor (Kwong and Lee, 2002; Guet al., 2003).Kinins are released in response to tissue injury and

inflammation, and expression of B1R and B2R by pul-monary cells is upregulated under inflammatory condi-tions (Newton et al., 2002). Several pathways have been

suggested to mediate the effects of bradykinin on TRPV1and TRPA1. Activation of B2R stimulates Gas-coupledPKA signaling and PLC-dependent PKC activity andbradykinin-induced lipoxygenase products (Premkumarand Ahern, 2000; Ferreira et al., 2004; Grace et al., 2012;Gregus et al., 2012).

A balance between proteases that digest structuralproteins (e.g., elastin) and protease inhibitors thatprotect against digestion is important in maintainingthe integrity of the lungs. Inhaled irritants, includingcigarette smoke, cause the release of proteases from avariety of cells. Proteases activate PARs, digest connectivetissue in the lung parenchyma leading to emphysema, andalso stimulate mucus hypersecretion (Barnes, 2010).In addition, individuals with a1-antitrypsin deficiencycarry a greater risk of developing COPD (Brebner andStockley, 2013). Activation of PAR2, presumably onsensory afferents of the lung, potentiates TRPV1-induced cough responses through activation of PKC,PKA, and the release of prostanoids (Gatti et al., 2006).Therefore, endogenous release of proteases may contributeto the sensitized cough reflex to TRPV1 stimulationobserved in patients with asthma and COPD. Interestingly,the PAR2-induced activation of TRPV4 in airways requiresGai/o signaling and the activity of PLC and PI3K (Li et al.,2011). This may indicate cell type–specific regulatorymechanisms and a requirement of Gbg subunits.

Increased activation of TRPV4 by mechanical andosmotic stimuli in oviductal ciliated cells may dependon PLA2 activation and production of AA metabolitessuch as EET. Under conditions of low PLA2 activation,ATP (likely via the P2Y2 receptor) may contribute toTRPV4 gating by activating PLC, leading to IP3 genera-tion (Fernandes et al., 2008; Lorenzo et al., 2008).Importantly, alterations in the activity of PLA2 and AAlevels have been reported in cystic fibrosis airwayepithelia, which could explain the lack of TRV4 activa-tion by cell swelling (Arniges et al., 2004).

VI. Targeting G Protein–Coupled Receptors,Transient Receptor Potential Channels, and the

G Protein–Coupled Receptor–TransientReceptor Potential Channel Axis

A. The Need for Alternative Therapeutics

GPCRs and TRP ion channels are attractive targetsfor the treatment of pain and related disorders, such aschronic pruritus and inflammation. Current analgesicsinclude those that act locally (e.g., lidocaine) or attenuateneurotransmitter release through inhibition of ionchannels (voltage-sensitive Ca2+, Na+, or K+ channels)in central nerve pathways. Anticonvulsant and anes-thetic sodium channel blockers have long been used totreat acute or chronic pain, such as trigeminal neuralgia,by reducing the generation of action potentials and nerveexcitation (Bhattacharya et al., 2009). For peripheralpain, nonsteroidal anti-inflammatory drugs have mild

60 Veldhuis et al.

analgesic, anti-inflammatory, and antipyretic proper-ties that inhibit production of algesic prostaglandinsand are effective for inflammatory conditions such asarthritis (Andersson et al., 2011). Opioids reduce potassiumchannel activity and TRP channels to reduce presynapticneurotransmitter release and central nerve excitabilityand remain a first-line treatment of postoperative, acute,and chronic pain (Finnegan et al., 2006). Drawbacksassociated with these treatments include overdose,development of tolerance, seizures, confusion, seda-tion, and undesirable on-target effects (e.g., morphinecauses respiratory depression and constipation). Inaddition, the promise of key nociceptive receptors(e.g., bradykinin and neurokinin family members) hasled to the development of many selective, high-affinityantagonists that block nociceptive responses in ani-mal pain models yet have failed to yield successfuldrugs in the market. Hence, therapeutic interventionof peripheral sensory pathways targeted toward GPCRsor TRP channels at the source of pain transmissioncould be a better strategy (Patapoutian et al., 2009).Analgesics directed toward GPCRs and TRP chan-

nels within peripheral cells are currently providingpromise in the clinic (Brederson et al., 2013). However,this strategy may prove challenging, given the wide-spread expression of TRPs and their broad physiologicsignificance (Ramsey et al., 2006). As with so manytherapeutic targets, development of new treatments ispredominantly limited by our understanding of howthese receptors or ion channels function in differentcells or disease states. Species differences can also leadto different outcomes in human studies compared withanimal models (Steinhoff et al., 2014). To assess thepotential to generate novel therapeutics to alleviatechronic conditions while keeping basal physiologicfunctions intact is an enormous challenge and re-quires deeper understanding of all processes, includingthe mechanisms of GPCR and TRP channel cross-talk.

B. Lessons from Analysis of Transient ReceptorPotential Channel Expression in Sensory Neurons

1. Improved Techniques for Expression Analysis.TRP channels are expressed in primary sensory neuronsand are active targets of drug discovery efforts directedtoward the identification of novel ligands for paintherapies. Crucial to the outcomes of such drug discoveryprograms is the knowledge of the sites of TRP channelexpression. TRPV1, TRPV2, TRPV3, TRPV4, TRPA1,and TRPM8 are expressed by primary sensory neurons,which were once considered to be the sole site of ex-pression. However, these pain targets are also expressedin other cell types, where they can have distinct roles.Difficulties in detecting TRP channels include lack ofselective antibodies for anatomic studies and of agonistsand antagonists for functional studies. However, someof these challenges have been overcome by studies of

reporter mouse lines. For example, the availability ofTRPV1Cre/R26R-lacZ mice and TRPV1PLAP-nlacZ mice hasimproved the reliability of TRPV1 detection and hasrevealed new and unexpected information about TRPV1expression. Unexpected findings include the expressionof TRPV1 in a smaller subpopulation of sensory neuronsthan previously reported and the detection of TRPV1 inarterial smooth muscle, which may explain the cardio-protective effects induced by capsaicin or endogenousTRPV1 ligands (Peng and Li, 2009; Cavanaugh et al.,2011a,b). Clearly, a detailed understanding of the sites ofTRP channel expression in normal and diseased states isnecessary for the interpretation of clinical trials of TRPagonists or antagonists.

2. Upregulation of Transient Receptor PotentialChannels in Pain Conditions. There are marked changesin TRP expression in pain states that have importantimplications for therapeutic targeting. Damage to onebranch of the spinal root of a dorsal root ganglion leads toupregulation of TRPV1 in the adjacent undamaged nervethat can still transmit pain signals (Hudson et al., 2001).TRP channel expression is also altered in human tissuesduring chronic pain. A study of TRPV1, TRPV3, TRPV4,and TRPM8 expression revealed that TRPV1 is upregu-lated in the injured brachial plexus nerves and that TRPV1and TRPV3 are upregulated in hypersensitive skin afternerve repair, whereas TRPV4 is unchanged (Facer et al.,2007). In the same study, TRPV1-immunoreactive nerveswere present in injured dorsal spinal roots and dorsal hornof control spinal cord but not in ventral roots, whereasTRPV3 and TRPV4 were detected in spinal cord motorneurons. The upregulation of TRP channels in painfulconditions may contribute to neuronal sensitization andresultant hyperalgesia. Indeed, TRP channel antagonistsmay have more selective and beneficial therapeuticactions in diseases associated with a robust upregulationof TRPs in sensory neurons compared with other tissues(Basbaum et al., 2009).

C. Current State of Transient Receptor PotentialAgonists and Antagonists

TRP channels are attractive therapeutic targets: pre-clinical studies from multiple laboratories show thatTRP channels play a major role in pain, pruritus,and neurogenic inflammation and the channels are“drugable,” because selective agonists and antagonistshave been developed for many channels. However, thereare formidable challenges with targeting TRP channels.First, the preclinical models of chronic pain, itch, andinflammation in experimental animals may be poorrepresentations of the complexity of human diseases,and the translation of information from studies of laboratoryanimals to humans is always problematic. Second, there ismarked redundancy among TRP channels. Sensorynerves coexpress multiple TRP channels that participatein sensory transduction and neurogenic inflammation,often with overlapping functions. Third, TRP channels


are not confined to sensory nerves and participate inmany normal physiologic processes, such as thermoreg-ulation (e.g., TRPV1) and osmoregulation (e.g., TRPV4).The on-target disruption of these homeostatic mecha-nisms represents significant challenges to the targetingof TRP channels. Despite these concerns, several TRPagonists and antagonists have advanced to clinical trialsfor pain. In general, two different approaches have beenused to target TRPs for pain therapeutics: agonists havebeen screened to desensitize sensory neurons andantagonists have been generated as antihyperalgesicagents.1. Transient Receptor Potential Vanilloid 1 Agonists.

The burning sensation evoked by capsaicin eventuallydesensitizes or defunctionalizes primary nerve termi-nals and results in analgesia of the affected area. Thiswell known phenomenon has led to the topical use ofcapsaicin creams in the treatment of neuropathic andmusculoskeletal pain (Mason et al., 2004). Despite itswidespread use, capsaicin cream has moderate to poorclinical efficacy in treating chronic musculoskeletaland neuropathic pain, although it may be useful insome cases where other treatments are inadequate.The use of capsaicin is problematic mainly because ofthe slow time for the analgesic effect to become apparent(hours to weeks) and because of the risk of spreading thepainful substance to sensitive areas of skin and organs,such as wounds or mucus membranes containing TRPV1-positive C-fibers. An effective alternative for someneuropathic pain conditions includes the recently avail-able high-concentration (8% capsaicin) transdermal patchQutenza (Acorda Therapeutics Inc., Ardsley, NY). Thecontrolled release of capsaicin into the skin for 60 minutesin healthy patients results in loss of epidermal nervefibers for more than 12 weeks (Kennedy et al., 2010), andpreliminary studies of the high-dose patch on postherpeticneuralgia patients indicate that the patch is reasonablywell tolerated and improves analgesia compared withlow-dose topical creams (Martini et al., 2013).The highly potent TRPV1 agonist resiniferatoxin has

been used experimentally to treat intractable detrusorhyper-reflexia of the bladder (Evans, 2005; Apostolidis et al.,2006). Although the clinical efficacy of resiniferatoxin intreating pain is still unknown, it is currently beingtested for its ability to treat severe pain associatedwith advanced cancer in National Institute of Dentaland Craniofacial Research–sponsored phase I and phaseII trials (ClinicalTrials.gov identifier: NCT00804154).2. Other Transient Receptor Potential Agonists. Folk

remedies and over-the-counter liniments and rubs havebeen used to relieve muscle soreness for many years.Eucalyptus oil, ginger extract, and oil of wintergreencontain substances that are agonists of TRPV1 (gingerol)(Morera et al., 2012), TRPM8 (menthol) (Peier et al.,2002a), and TRPA1 (methyl salicylate, menthol, andgingerol) (McKemy et al., 2002; Bandell et al., 2004;Morera et al., 2012). However, when used topically,

there is little objective evidence that they act at sensorynerves and are analgesic.

Dietary agonists of TRPV1 (capsaicin, gingerol), TRPV3(thymol, eugenol, carvacrol), TRPM8 (menthol), andTRPA1 (cinnemaldehyde, mustard oil) stimulate Ca2+ fluxin the oral mucosa and gastrointestinal tract to regulatemucosal blood flow. Mediterranean diets rich in oregano(carvacrol) have been proposed to promote cardioprotec-tive effects via arterial dilatation, yet this has not beentherapeutically investigated (Xu et al., 2006; Earleyet al., 2010; Okumi et al., 2012). This may be attributedto the fact that in most cases the oral bioavailability ofthese compounds is extremely limited. Caution shouldalso be taken regarding modulation of TRPV3 becauseof observations of agonist-induced augmentation ofCa2+-dependent activity with repeated stimulation(Xiao et al., 2008). Constitutive TRPV3 activity is alsoattributed to chronic itch, dermatitis, hair loss, and cellproliferation (Asakawa et al., 2006; Yoshioka et al., 2009;Yamada et al., 2010).

3. Transient Receptor Potential Vanilloid 1 Antagonists.A detailed understanding of the pharmacologicalactions of capsaicin in the 1960s prompted the de-velopment TRPV1 antagonists before cloning of thecapsaicin receptor (Jancso et al., 1967; Caterina et al.,1997). Capsazepine, the first TRPV1 antagonist, reversespersistent nociceptive and neuropathic pain behavior inrats (Walker et al., 2003), and many new antagonists havesince reported effectiveness in animal models of pain(reviewed in Kort and Kym, 2012).

Although TRPV1 antagonists can provide therapeu-tic benefit for pain in humans, many compounds affectnormal thermoregulation and temperature perception(reviewed in Brederson et al., 2013). A phase I trial ofSB-705498 given orally to healthy volunteers showedeffectiveness in reversing heat-evoked pain and skinsensitization induced by capsaicin or ultraviolet Birradiation (Rami et al., 2006; Chizh et al., 2007). Onevolunteer experienced a modest increase (1.3°C) in corebody temperature, but no other adverse events werereported. A phase 1b trial of AMG517 to treat dentalpain was terminated because of marked hyperthermiain some subjects who experienced an increase in corebody temperature of over 4°C (Doherty et al., 2007;Gavva et al., 2008). After initial safety trials for theTRPV1 antagonist AZD-1386 on healthy volunteersand GERD patients (Krarup et al, 2011; ClinicalTrials.gov identifier: NCT00692146), a double-blind phase IItrial of dental pain from third molar extraction revealedthat AZD-1386 provided pain relief and was welltolerated, with an average 0.4°C increase in core bodytemperature (Quiding et al., 2013). MK-2295 markedlyblunted heat perception in healthy human subjectsmeasured using quantitative sensory testing of painevoked by immersion of the hand into heated water orsipping hot liquids without attenuation by repeated dosing(Eid, 2011; ClinicalTrials.gov Identifier: NCT00387140). A

62 Veldhuis et al.

multiple-dose, double-blind, placebo-controlled, randomizedtrial of healthy subjects showed that that ABT-102 causeda reversible change in oral and cutaneous sensitivity to heatbut not cold without adverse effects (Gomtsyan et al., 2008;Rowbotham et al., 2011). Thus, although TRPV1 antago-nists may blunt pain, there are variable effects on core bodytemperature and consistent effects on the perception of heat,where potentially noxious temperatures were perceived asinnocuous (Eid, 2011). The variability of effects of TRPV1antagonists on core body temperature are compoundselective and may thus depend on structure and could be“engineered out.” However, the uniform effects of TRPV1antagonists on the threshold of oral heat-pain are unaccept-able, because they introduce a risk of scalding injury fromhot drinks or food (Rowbotham et al., 2011). Given thatthis latter effect is likely an on-target action of TRPV1antagonists, it may represent a challenging problem fordevelopment of TRPV1 antagonists. Possibly allostericmodulators of TRPV1 activity will be more usefulanalgesics than full antagonists.4. Other Transient Receptor Potential Antagonists.

Research programs to identify and characterize novelantagonists selective for TRPA1, TRPV3, TRPV4, andTRPM8 to treat conditions of pain, itch, cough, andinflammation have also massively expanded in thepast 10 years, and the now abundance of literature ofpreclinical antagonist development is heavily supportedand scrutinized by the pharmaceutical industry (reviewedby Eid, 2011; Brederson et al., 2013). TRPA1, for example,is a tractable therapeutic target as demonstrated byreduced inflammatory or neuropathic responses inantagonist-treated or trpa12/2 mice. Selective TRPA1antagonists, such as HC-030031, have been identifiedas effective inhibitors of TRPA1 activity in primaryneurons when stimulated by irritants, bradykininreceptor signaling, and cold temperatures. NumerousTRPA1-selective compounds from Glenmark, Abbott,and Amgen followed, with varying degrees of oral bio-availability (Eid et al., 2008; Eid, 2011; Moran et al.,2011). Phase I and phase II human trials by Glenmark,Cubist, and Hydra are currently in progress, and thefindings are yet to be published. One could speculatethat topical application or inhalation of TRPA1 antag-onists may also be beneficial for the reduction orprevention of itch or chronic cough (Bautista et al.,2013). Although TRPA1 antagonist patent applicationshave been filed with intention to treat these disorders, itis yet to be investigated in detail. TRPV3 antagonistsalso have efficacy in models of inflammatory pain,formalin-induced flinching, thermal injury, spinal sen-sitization, and neuropathic pain (Brederson et al., 2013).These treatments may have additional benefit inpruritus and skin conditions, such as Olmsted Syn-drome (Lai-Cheong et al., 2012; Lin et al., 2012). TRPV4antagonists have wide potential for alleviating disordersof inflammation, central nervous system water reten-tion, metabolism, bladder distention, and circulation,

and several recent TRPV4 antagonist patent applica-tions have been filed (e.g., US20140135369 A1, 2014;WO2013/2013152109 A1, 2013; WO2013/2014089013A1, 2013). There is limited information on these com-pounds progressing beyond preclinical development,which may indicate complications in enhancing speci-ficity for target organs or tissues (Willette et al., 2008;Eid, 2011).

Toxins have also been the focus of drug screeningefforts to identify natural analgesics that target a rangeof proalgesic ion channels (Malmberg and Yaksh, 1995;Diochot et al., 2012). Recently, this has led to theidentification of ligands that selectively bind and regulateTRP ion channels, including two distinct Tarantulavenoms that antagonize TRPA1 (Gui et al., 2014) andstimulate TRPV1 (Bohlen et al., 2010). Continueddrug discovery efforts and development of structure-activity relationship studies on toxins as pharmacophoremodels may uncover new classes of therapeuticallyimportant analgesics (Lewis et al., 2012).

D. Therapeutic Targeting of G Protein–CoupledReceptor–Transient Receptor Potential Interactions

GPCRs play a major role in regulating TRP channelsin disease by stimulating intracellular signaling path-ways that can lower the activation thresholds of TRPchannels. These mechanisms are likely to be cell typespecific and require second messengers and auxiliaryproteins that may offer alternative targets for drugdiscovery. For example, mice lacking Pirt, which inter-acts with TRPV1 and TRPM8, exhibit decreased sensi-tivity to heat, itch, or cold in selective neurons (Kim et al.,2008; Patel et al., 2011; Tang et al., 2013). TRPV4 isa PAR2 receptor-operated ion channel and can contributeto neurogenic inflammation through a mechanism thatrequires generation of endogenous TRPV4 agonists andphosphorylation of a tyrosine residue 110 in TRPV4(Poole et al., 2013). Thus, in addition to direct targetingof GPCRs and TRP channels, therapies that target keykinases, lipids, or auxiliary proteins that mediate GPCR-TRP coupling offer an alternative therapeutic approachfor sensory disorders and neurogenic inflammation. Insupport of this concept, the tyrosine kinase inhibitorbafetinib was recently shown to block PAR2 couplingto TRPV4 in cell lines and attenuate PAR2-dependentmechanical hyperalgesia in mice (Grace et al., 2014a).Similarly, the novel PAR2 antagonist GB88 was recentlyshown to selectively block PAR2-induced Gaq signalingevents and inflammatory pain in mice (Suen et al., 2014)and demonstrates the potential for receptor-operated ionchannels to be selectively blocked via treatment withbiased antagonists.

More than 400 kinases are associated with humandiseases and they are a well studied therapeutic target(Zhang et al., 2009). Moreover, kinase-selective inhibitionis a successful treatment of inflammatory disorders(Patterson et al., 2014). Small-molecule inhibitors of


protein-protein interactions have had limited successowing to difficulty in disrupting the large interfacesbetween interacting proteins (Jin et al., 2014). How-ever, several approaches that have been explored todisrupt such interactions may allow for TRP channel-specific inhibition. Pepducins are small membrane-associated peptides that mimic intracellular loop 3 ofPARs and which disrupt the interactions between PARsand heterotrimeric G proteins and thereby inhibitinflammatory signaling (Sevigny et al., 2011). TRPducinsare palmitoylated peptide sequences from the C-terminal"TRP domain" of TRPs (Fig. 3B). TRPV1 TRPducinsselectively inhibits TRPV1 over other TRPV channelsand can block heat-, acid-, and voltage-evoked TRPV1currents. This process inhibits CGRP release from culturedneurons and lowers chemically- but not mechanically-induced pain (Valente et al., 2011). Similarly, small PKCsequence peptides fused to membrane-permeating TATsequences can selectively inhibit specific PKC isoformsand have been shown to reduce ischemic heart damage(Chen et al., 2001). Although these kinase inhibitorsmay selectively prevent inflammatory pathways (e.g.,PKC« is critical for PAR2 sensitization of TRPV1),penetrating peptides directed toward the b-strands,linker region, or ankyrins may similarly affect in-teraction sites in TRP channels (Amadesi et al., 2009).Finally, local anesthetics exert their analgesic effect

by inhibiting voltage-gated sodium channels by bind-ing to sites in the inner face of the plasma membrane.It has been observed that some TRP ion channelsundergo activation-dependent pore dilation, and con-sequently, large cations can be transported into sensorynerves. This pore dilation might be a means of producingTRP channel subtype–dependent inhibition. Specifically,coadministration of capsaicin with a local anestheticenhances TRPV1 pore dilation to facilitate the deliveryof membrane impermeant local anesthetics to voltage-dependent sodium channels, which inhibits action po-tentials in TRPV1-positive sensory neurons (Binshtoket al., 2007). This represents a novel means of exploitingion channels for drug delivery that may be used to increasethe specificity and efficacy of therapeutics (Roberson et al.,2011). This approach has also been used to investigatedistinct neuronal itch pathways and indicates the potentialto selectively inhibit pain-transmitting nerves where in-flammatory states have increased TRP channel expression(Roberson et al., 2013).

VII. Concluding Remarks

GPCRs and TRP channels are frontline partners forthe detection of noxious, irritant, and inflammatorystimuli. These integral membrane proteins are coex-pressed at the surface of sensory neurons and manyother cell types in most tissues. Although GPCRs andTRPs can independently detect diverse chemical, me-chanical, and thermal stimuli, the GPCR-TRP functional

axis is of vital importance. Through this axis, signals thatemanate from multiple GPCRs converge on a smallernumber of TRP channels, leading to channel activationand sensitization or altered channel expression andplasma membrane insertion. This convergence oftenamplifies the effects of GPCRs, which results inexacerbated responses to noxious, irritant, and in-flammatory stimuli. Accordingly, the GPCR-TRP axisis vitally important for the normal protective pro-cesses of pain, itch, cough, and neurogenic inflamma-tion and also participates in chronic conditions thatunderlie disease.

The realization that GPCRs and TRPs are keymediators of disease processes has provided theimpetus for efforts to develop antagonists of GPCRsand TRPs to treat chronic pain, itch, cough, andinflammation. Antagonists and agonists of GPCRsand TRP channels are effective treatments for manypreclinical models of sensory and inflammatory disor-ders in experimental animals and in some cases areuseful treatments for human diseases. However, thewidespread distribution of GPCRs and TRPs and theirinvolvement in diverse physiologic processes can lead toon-target side effects that can limit the effectiveness ofreceptor- and channel-directed drugs.

Whether targeting the GPCR-TRP axis is a moreselective, effective, and viable approach to treat chronicdisorders remains to be determined. A prerequisite forthe success of this approach is a detailed understand-ing of the molecular mechanisms that underlie theGPCR-TRP axis in functionally relevant cell types anddisease settings. Our current understanding of thisaxis mostly derives from studies of GPCRs and TRPsthat are overexpressed in model cell lines. Such studieshave identified multiple mechanisms, which are oftenoverlapping, by which GPCRs can regulate the activityof TRP channels. In many cases it is not known whetherthe same mechanisms also underlie the GPCR-TRPaxis in functionally relevant cells, such as primarynociceptive neurons. Furthermore, whether thesesame mechanisms also operate in disease settings orwhether defects in the GPCR-TRP axis are involved inthe etiology of chronic disorders of sensation andinflammation remains to be determined. However, therecent explosion of high-resolution information of GPCRand TRP channel structures has provided invaluableinsights into the mechanisms of GPCR and TRP activationand signaling. Whether such structural informationcan illuminate the mechanisms of the GPCR-TRP axisremains to be determined, but if it does it is likely toprovide valuable knowledge of potential therapeutictargets for conditions that are a major cause ofsuffering.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Veldhuis,Poole, Grace, McIntyre, Bunnett.

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ASSOCIATE EDITOR: ARTHUR CHRISTOPOULOS The G Protein ...pharmrev.aspetjournals.org/content/pharmrev/67/1/36.full.pdf · stimuli (hyperalgesia), the perception of pain from normally