TRPV1: on the road to pain relief

Abstract

Historically, drug research targeted to pain treatment has focused on trying to prevent the propagation of action potentials in the periphery from reaching the brain rather than pinpointing the molecular basis underlying the initial detection of the nociceptive stimulus: the receptor itself. This has now changed, given that many receptors of nociceptive stimuli have been identified and/or cloned. Transient Receptor Potential (TRP) channels have been implicated in several physiological processes such as mechanical, chemical and thermal stimuli detection. Ten years after the cloning of TRPV1, compelling data has been gathered on the role of this channel in inflammatory and neuropathic states. TRPV1 activation in nociceptive neurons, where it is normally expressed, triggers the release of neuropeptides and transmitters resulting in the generation of action potentials that will be sent to higher CNS areas where they will often be perceived as pain. Its activation also will evoke the peripheral release of pro-inflammatory compounds that may sensitize other neurons to physical, thermal or chemical stimuli. For these reasons as well as because its continuous activation causes analgesia, TRPV1 has become a viable drug target for clinical use in the management of pain. This review will provide a general picture of the physiological and pathophysiological roles of the TRPV1 channel and of its structural, pharmacological and biophysical properties. Finally, it will provide the reader with an overall view of the status of the discovery of potential therapeutic agents for the management of chronic and neuropathic pain.

Figure 1. TRPV1 residues involved in ligand- and modulator interactions

Some of the residues responsible for the actions of some TRPV1 agonists or modulators have been identified. TRPV1 has multiple phosphorylation sites. cAMP-dependent protein kinase (PKA) directly phosphorylates the TRPV1 channel at several sites. Residues Ser 116 and Thr 370 are phosphorylated by PKA and have been implicated in desensitization, while residues Thr 144, Thr 370, and Ser 502 have been implicated in sensitization of heat-evoked TRPV1 responses when phosphorylated by PKA. Ser 502 is also the target for protein kinase C (PKC) and calcium-calmodulin dependent kinase II (CaMKII). PKC can phosphorylate the channel at a second residue, Ser 800, while CaMKII phosphorylates Thr 704. The region formed by amino acids 777–820 and both charged aminoacids R701 and K710 of TRPV1 are responsible for some of the actions of PIP₂ on the channel. Cysteine 157, at the N-terminus, reacts to cysteine-modifying agents such as allicin, a pungent compound present in garlic and onion, which cause TRPV1 channel activation. Several residues responsible for the actions of capsaicin and other vanilloids have been identified. Glu761 in the C-terminus and Arg 114 at the N-terminus have been postulated as agonist recognition sites. Ser 512 is important for capsaicin-mediated activation of the channel, while Thr 550 and Tyr 511 are necessary for maintaining capsaicin sensitivity. Met 547 is responsible for RXT binding and sensitivity, and is also involved in some of the vanilloid’s actions. Glu 600 serves as an important regulator site for proton potentiation of TRPV1 activity, while Glu 648 seems to be involved in direct proton-evoked activation of TRPV1. Both Glu 648 and Glu 600 are responsible for the Gd³⁺ activating effects on the channel. Additionally Glu648, together with Asp646, are responsible for polyamine actions on TRPV1. The three pore-cysteines, but especially C621, are important for TRPV1 modulation by extracellular reducing agents. Two calmodulin binding sites in the TRPV1 channel have been identified, one in the N-terminus and one in the C-terminus of the protein. Calmodulin bound to calcium in the N-terminus of the channel causes desensitization. Glu 636 is a key molecular determinant of the TRPV1 pore region, and is responsible for TRPV1 channel block by ruthenium red. Asp 604 is a site for N-glycosylation.

Figure 2. TRPV1 regulation by intracellular signaling pathways

The TRPV1 channel is coupled to many intracellular signaling cascades related to inflammatory processes that regulate channel activity. Multiple G-protein coupled receptors (GPCRs) are activated by pro-inflammatory agents including histamine and prostaglandins. The activation of receptors coupled to G_s proteins leads to adenylate-cyclase (AC) stimulation, cAMP production and concomitant cAMP-dependent protein kinase (PKA) activation. PKA directly phosphorylates the TRPV1 channel modulating its activity. Activation of GPCRs coupled to G_q proteins leads to phospholipase-C (PLC) stimulation, which degrades plasma membrane-associated PIP₂ into 1,2-diacylglycerol (DAG) and (1,4,5)-inositol triphosphate (IP₃). Increases in IP₃ lead to Ca²⁺ release from intracellular stores such as the endoplasmic reticulum (ER). Both Ca²⁺ and DAG activate protein kinase C (PKC), which also phosphorylates the TRPV1 channel and regulates its function. PIP₂ regulates the TRPV1 channel in a complex manner, with both positive and negative effects depending on the state of the receptor. TRK receptor activation by extracellular signals such as nerve growth factor (NGF) also leads to PLC activation. Intracellular calcium elevation through TRPV1 or voltage-dependent calcium channel (VDCC) activation or through calcium release from intracellular calcium stores due to pro-inflammatory mediators also regulates channel function: calcium binds to calmodulin which is associated with TRPV1 and promotes channel desensitization. Calcium-calmodulin dependent kinase II (CaMKII) directly phosphorylates the channel. Mechanical stimuli lead to integrin-dependent src-Protein tyrosine kinase (srcPTK) activation and direct action on the TRPV1 channel. Additionally, cell depolarization due to voltage-dependent calcium or sodium (VDSC) channel activation directly gates the channel. All regulators depicted in the figure are positive regulators of the channel except for the bimodal action of PIP₂.

Figure 3

Chemical structures of capsaicin and selected TRPV1 antagonists. Antagonists such as A-425619 (1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea) [175,241,242], SB-705498 (N-(2-bromophenyl)-N′-[((R)-1-(5-trifluromethyl-2-pyridyl)pyrrolidin-3-yl)]urea)[178,243], BCTC (N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide)[244], quinazolinone compound 26 [245] and AMG 9810 ([(E)-3-(4-t-Butylphenyl)-N-(2,3-dihydrobenzo[b] dioxin-6-yl)acrylamide) [246] (Fig. (3), compounds (3), (4), (6), (7) and (8), respectively) have implicated TRPV1 as an important mediator of nociceptive responses to mechanical stimuli under inflammatory conditions [17].