Dissecting TRPV1: lessons to be learned?

The transient receptor potential channel TRPV1 is a polymodal nociceptor. It is primarily expressed in dorsal root ganglia and peripheral sensory nerve endings, and to a much lesser extent, in the central nervous system. It has also been implicated in the functional properties of e.g. urinary and bronchial epithelia. TRPV1 has long been under intensive investigation by the pharmaceutical industry as a candidate drug target especially for pain conditions. This review summarizes the current knowledge of the molecular determinants of TRPV1 channel activation by heat, protons and capsaicin. Newly discovered heat and proton activation sites within the pore domain are discussed as well as potential consequences for drug discovery. Polymodal TRPV1 antagonists were found to cause hyperthermia in a species-dependent manner in-vivo, hence the discovery of euthermic compounds with an appropriate modality selectivity profile will be crucial for TRPV1's future as a drug target.     

Dissecting TRPV1: Lessons to be learned?

The transient receptor potential channel TRPV1 is a polymodal nociceptor. It is primarily expressed in dorsal root ganglia and peripheral sensory nerve endings, and to a much lesser extent, in the central nervous system. It has also been implicated in the functional properties of e.g. urinary and bronchial epithelia. TRPV1 has long been under intensive investigation by the pharmaceutical industry as a candidate drug target especially for pain conditions. This review summarizes the current knowledge of the molecular determinants of TRPV1 channel activation by heat, protons and capsaicin. Newly discovered heat and proton activation sites within the pore domain are discussed as well as potential consequences for drug discovery. Polymodal TRPV1 antagonists were found to cause hyperthermia in a species-dependent manner in-vivo, hence the discovery of euthermic compounds with an appropriate modality selectivity profile will be crucial for TRPV1's future as a drug target.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.     

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.     

The biophysical and molecular basis of TRPV1 proton gating

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.     

4-Aminophenyl acetamides and propanamides as potent transient receptor potential vanilloid 1 (TRPV1) ligands

A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.     

TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca²⁺ influx. Interestingly, nifedipine, a specific L-type Ca²⁺ channel blocker, attenuated capsaicin cytotoxicity, even when applied 2-4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca²⁺ channel opening, Ca²⁺ influx, ERK phosphorylation, and reactive oxygen species production.     

Functional analysis of thermo-sensitive TRPV1 in an aquatic vertebrate,masu salmon (Oncorhynchus masou ishikawae)

Transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in nociceptive primary sensory neurons and acts as a sensor for heat and capsaicin. The functional properties of TRPV1 have been reported to vary among species and, in some cases, the species difference in its thermal sensitivity is likely to be associated with thermal habitat conditions. To clarify the functional properties and physiological roles of TRPV1 in aquatic vertebrates, we examined the temperature and chemical sensitivities of TRPV1 in masu salmon (Oncorhynchus masou ishikawae, Om) belonging to a family of salmonids that generally prefer cool environments. First, behavioral experiments were conducted using a video tracking system. Application of capsaicin, a TRPV1 agonist, induced locomotor activities in juvenile Om. Increasing the ambient temperature also elicited locomotor activity potentiated by capsaicin. RT-PCR revealed TRPV1 expression in gills as well as spinal cord. Next, electrophysiological analyses of OmTRPV1 were performed using a two-electrode voltage-clamp technique with a Xenopus oocyte expression system. Heat stimulation evoked an inward current in heterologously expressed OmTRPV1. In addition, capsaicin produced current responses in OmTRPV1-expressing oocytes, but higher concentrations were needed for its activation compared to the mammalian orthologues. These results indicate that Om senses environmental stimuli (heat and capsaicin) through the activation of TRPV1, and this channel may play important roles in avoiding environments disadvantageous for survival in aquatic vertebrates.     

Heteromeric heat-sensitive transient receptor potential channels exhibit distinct temperature and chemical response

TRPV1 and TRPV3 are two heat-sensitive ion channels activated at distinct temperature ranges perceived by human as hot and warm, respectively. Compounds eliciting human sensations of heat or warmth can also potently activate these channels. In rodents, TRPV3 is expressed predominantly in skin keratinocytes, whereas in humans TRPV1 and TRPV3 are co-expressed in sensory neurons of dorsal root ganglia and trigeminal ganglion and are known to form heteromeric channels with distinct single channel conductances as well as sensitivities to TRPV1 activator capsaicin and inhibitor capsazepine. However, how heteromeric TRPV1/TRPV3 channels respond to heat and other stimuli remains unknown. In this study, we examined the behavior of heteromeric TRPV1/TRPV3 channels activated by heat, capsaicin, and voltage. Our results demonstrate that the heteromeric channels exhibit distinct temperature sensitivity, activation threshold, and heat-induced sensitization. Changes in gating properties apparently originate from interactions between TRPV1 and TRPV3 subunits. Our results suggest that heteromeric TRPV1/TRPV3 channels are unique heat sensors that may contribute to the fine-tuning of sensitivity to sensory inputs.     

Oxidative stress-induced posttranslational modification of TRPV1 expressed in esophageal epithelial cells

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca(2+)-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.     

Effect of increasing temperature on TRPV1-mediated responses in isolated rat pulmonary sensory neurons

Hyperthermia has been shown to sensitize vagal pulmonary C-fibers in anesthetized rats. However, it was not clear whether the effect was due to a direct action of hyperthermia on these sensory neurons. To answer this question, we carried out this study to determine the effect of increasing temperature on the responses to various chemical stimuli in isolated nodose and jugular ganglion neurons innervating the rat lungs. In the whole cell perforated patch-clamp study, when the temperature was increased from normal (approximately 36 degrees C) to hyperthermic (approximately 40.6 degrees C) level of the rat body temperature, the inward currents evoked by capsaicin, a selective activator of the transient receptor potential vanilloid type 1 (TRPV1), and 2-aminoethoxydiphenyl borate (2-APB), a nonselective activator of TRPV1-3 receptors, were both significantly increased. This potentiating effect was clearly present even at a moderate level of hyperthermia (approximately 39 degrees C). However, only the slow, sustained component of acid-evoked current mediated through the TRPV1 receptor was potentiated by hyperthermia, whereas the rapid, transient component was inhibited. In contrast, the currents evoked by adenosine 5'-triphosphate and acetylcholine, neither of which is known to activate the TRPV1 channel, did not increase when the same temperature elevation was applied. Furthermore, the hyperthermia-induced potentiation of the cell response to 2-APB was significantly attenuated by either capsazepine or AMG 9810, selective TRPV1 antagonists. In conclusion, increasing temperature within the physiological range exerts a potentiating effect on the response to TRPV1 activators in these neurons, which is probably mediated through a positive interaction between hyperthermia and these chemical activators at the TRPV1 channel.     

Heat attenuates sensitivity of mammalian cells to capsaicin

The transient receptor potential (TRP) channels are thermo‐sensors, and transient receptor potential vanilloid (TRPV)1 and V4 are widely expressed in primary afferent neurons and nonneuronal cells. Although heat acclimation is considered as changes of thermoregulatory responses by thermo‐effectors to heat, functional changes of TRP channels in heat acclimation has not been fully elucidated. Here, we investigated whether heat acclimation induces capsaicin tolerance. NIH3T3 cells were incubated at 39.5°C. We determined the expression level of TRPV1 and TRPV4 messenger RNA (mRNA), performed cellular staining of TRPV1 and TRPV4, and investigated actin assembly and activation of the extracellular signal‐regulated kinase (ERK). Exposure to moderate heat decreased the levels of TRPV1 but not TRPV4 mRNA. It also induced stress fiber formation and the intensity of TRPV1 seemed to be decreased by chronic heat stimuli. In addition, heat acclimation attenuated the capsaicin‐induced activation of ERK. Heat acclimation may induce capsaicin tolerance via the downregulation of TRPV1.     

Discovery of small molecule antagonists of TRPV1

Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca(2+) based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.     

Discovery of SB-705498: a potent, selective and orally bioavailable TRPV1 antagonist suitable for clinical development

Small molecule antagonists of the vanilloid receptor TRPV1 (also known as VR1) are disclosed. Pyrrolidinyl ureas such as 8 and 15 (SB-705498) emerged as lead compounds following optimisation of the previously described urea SB-452533. Pharmacological studies using electrophysiological and FLIPR-Ca2+-based assays showed that compounds such as 8 and 15 were potent antagonists versus the multiple chemical and physical modes of TRPV1 activation (namely capsaicin, acid and noxious heat). Furthermore, 15 possessed suitable developability properties to enable progression of this compound into in vivo studies and subsequently clinical development.     

Tunable calcium current through TRPV1 receptor channels

TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore.     

Alpha-methylation at benzylic fragment of N-aryl-N'-benzyl ureas provides TRPV1 antagonists with better pharmacokinetic properties and higher efficacy in inflammatory pain model

SAR studies for N-aryl-N'-benzyl urea class of TRPV1 antagonists have been extended to cover alpha-benzyl alkylation. Alkylated compounds showed weaker in vitro potencies in blocking capsaicin activation of TRPV1 receptor, but possessed improved pharmacokinetic properties. Further structural manipulations that included replacement of isoquinoline core with indazole and isolation of single enantiomer led to TRPV1 antagonists like (R)-16a with superior pharmacokinetic properties and greater potency in animal model of inflammatory pain.     

TRPV1通道的功能、门控机制及其调节剂在药物研发中的应用

TRPV1 （transient receptor potential vanilloid-1）是配体门控的非选择性阳离子通道，属于瞬时受体电位通道家族，能够被多种物理和化学刺激激活。TRPV1是药物研发的重要靶点之一，其异常刺激和表达与多种疾病的发病机制有关。一直以来，TRPV1因其调节剂优异的镇痛效果而备受关注。2021年诺贝尔生理学奖对温度和触觉感受器研究工作的认可，使TRPV1再一次成为关注的焦点。TRPV1已有20多年的研究基础，但是其门控机制和药物研发仍然是研究的难点。本文从TRPV1的生理功能、门控机制和药物发现的角度出发，综述了TRPV1的表达分布、功能特点和结构特征，重点阐述了3种门控机制及TRPV1调节剂在药物发现上的进展，并对未来的TRPV1药物进行展望。     

Discovery of 2-(3,5-difluoro-4-methylsulfonaminophenyl)propanamides as potent TRPV1 antagonists

A series of A-region analogues of 2-(3-fluoro-4-methylsufonamidophenyl) propanamide 1 were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that a fluoro group at the 3- (or/and) 5-position and a methylsulfonamido group at the 4-position were optimal for antagonism of TRPV1 activation by capsaicin. The most potent antagonist 6 not only exhibited potent antagonism of activation of hTRPV1 by capsaicin, low pH and elevated temperature but also displayed highly potent antagonism of activation of rTRPV1 by capsaicin. Further studies demonstrated that antagonist 6 blocked the hypothermic effect of capsaicin in vivo, consistent with its in vitro mechanism, and it showed promising analgesic activity in the formalin animal model.     

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.     

Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1

The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in [Ca(2+)](i) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca(2+)](i) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca(2+)](i) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca(2+)](i) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.