Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.     

Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity

The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.     

Retinal Cell Death Induced by TRPV1 Activation Involves NMDA Signaling and Upregulation of Nitric Oxide Synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.     

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.     

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.     

Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region

Pannexin 1 (PANX1) channels mediate release of ATP, a "find-me" signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1.     

Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.     

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F–C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys³⁰ and Cys²⁷²) engineered into the EC3-N terminus (Ser³⁰ and Ser²⁷²) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser²⁷² on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.     

Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels

KCNH1 (EAG1) is a member of the Kv family of voltage-gated potassium channels. However, KCNH1 channels also show some amino-acid sequence similarity to cyclic-nucleotide-regulated channels: they harbor an N-terminal PAS domain, a C-terminal cyclic nucleotide binding homology domain (cNBHD), and N- and C-terminal binding sites for calmodulin. Another notable feature is the channels' high sensitivity toward oxidative modification. Using human KCNH1 expressed in Xenopus oocytes and HEK 293 cells we investigated how oxidative modification alters channel function. Intracellular application of H₂O₂ or cysteine-specific modifiers potently inhibited KCNH1 channels in two phases. Our systematic cysteine mutagenesis study showed that the rapid and dominant phase was attributed to a right-shift in the voltage dependence of activation, caused by chemical modification of residues C145 and C214. The slow component depended on the C-terminal residues C532 and C562. The cysteine pairs are situated at structural elements linking the transmembrane S1 segment with the PAS domain (N-linker) and the transmembrane channel gate S6 with the cNBH domain (C-linker), respectively. The functional state of KCNH1 channels is determined by the oxidative status of these linkers that provide an additional dimension of channel regulation.     

Potentiation of the Transient Receptor Potential Vanilloid 1 Channel Contributes to Pruritogenesis in a Rat Model of Liver Disease

Persistent pruritus is a common disabling dermatologic symptom associated with different etiologic factors. These include primary skin conditions, as well as neuropathic, psychogenic, or systemic disorders like chronic liver disease. Defective clearance of potential pruritogenic substances that activate itch-specific neurons innervating the skin is thought to contribute to cholestatic pruritus. However, because the underlying disease-specific pruritogens and itch-specific neuronal pathways and mechanism(s) are unknown, symptomatic therapeutic intervention often leads to no or only limited success. In the current study, we aimed to first validate rats with bile duct ligation (BDL) as a model for hepatic pruritus and then to evaluate the contribution of inflammation, peripheral neuronal sensitization, and specific signaling pathways and subpopulations of itch-responsive neurons to scratching behavior and thermal hypersensitivity. Chronic BDL rats displayed enhanced scratching behavior and thermal hyperalgesia indicative of peripheral neuroinflammation. BDL-induced itch and hypersensitivity involved a minor contribution of histaminergic/serotonergic receptors, but significant activation of protein-activated receptor 2 (PAR₂) receptors, prostaglandin PGE₂ formation, and potentiation of transient receptor potential vanilloid 1 (TRPV1) channel activity. The sensitization of dorsal root ganglion nociceptors in BDL rats was associated with increased surface expression of PAR₂ and TRPV1 proteins and an increase in the number of PAR₂- and TRPV1-expressing peptidergic neurons together with a shift of TRPV1 receptor expression to medium sized dorsal root ganglion neurons. These results suggest that pruritus and hyperalgesia in chronic cholestatic BDL rats are associated with neuroinflammation and involve PAR₂-induced TRPV1 sensitization. Thus, pharmacological modulation of PAR₂ and/or TRPV1 may be a valuable therapeutic approach for patients with chronic liver pruritus refractory to conventional treatments.     

Nitric oxide block of outward-rectifying K+ channels indicates direct control by protein nitrosylation in guard cells

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant pathogen defense and are a prerequisite for drought and abscisic acid responses in Arabidopsis (Arabidopsis thaliana) and Vicia faba guard cells. Nonetheless, its mechanism(s) of action has not been well defined. NO regulates inward-rectifying K+ channels of Vicia guard cells through its action on Ca2+ release from intercellular Ca2+ stores, but alternative pathways are indicated for its action on the outward-rectifying K+ channels (I(K,out)), which are Ca2+ insensitive. We report here that NO affects I(K,out) when NO is elevated above approximately 10 to 20 nm. NO action on I(K,out) was consistent with oxidative stress and was suppressed by several reducing agents, the most effective being British anti-Lewisite (2,3-dimercapto-1-propanol). The effect of NO on the K+ channel was mimicked by phenylarsine oxide, an oxidizing agent that cross-links vicinal thiols. Neither intracellular pH buffering nor the phosphotyrosine kinase antagonist genistein affected NO action on I(K,out), indicating that changes in cytosolic pH and tyrosine phosphorylation are unlikely to contribute to NO or phenylarsine oxide action in this instance. Instead, our results strongly suggest that NO directly modifies the K+ channel or a closely associated regulatory protein, probably by nitrosylation of cysteine sulfhydryl groups.     

Citral Sensing by TRANSient Receptor Potential Channels in Dorsal Root Ganglion Neurons

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1–3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral''s actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral''s stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral''s actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral''s broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.     

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues—I696 and W697—with Ala markedly affects TRPV1’s response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.     

Human Sensory Neuron-specific Mas-related G Protein-coupled Receptors-X1 Sensitize and Directly Activate Transient Receptor Potential Cation Channel V1 via Distinct Signaling Pathways

Sensory neuron-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are primate-specific proteins that are exclusively expressed in primary sensory neurons and provoke pain in humans. Hence, MRGPR-X1 represent promising targets for future pain therapy, but signaling pathways activated by MRGPR-X1 are poorly understood. The transient receptor potential cation channel V1 (TRPV1) is also expressed in primary sensory neurons and detects painful stimuli such as protons and heat. G_q-promoted signaling has been shown to sensitize TRPV1 via protein kinase C (PKC)-dependent phosphorylation. In addition, recent studies suggested TRPV1 activation via a G_q-mediated mechanism involving diacylglycerol (DAG) or phosphatidylinositol-4,5-bisphosphate (PIP₂). However, it is not clear if DAG-promoted TRPV1 activation occurs independently from classic TRPV1 activation modes induced by heat and protons. Herein, we analyzed putative functional interactions between MRGPR-X1 and TRPV1 in a previously reported F11 cell line stably over-expressing MRGPR-X1. First, we found that MRGPR-X1 sensitized TRPV1 to heat and protons in a PKC-dependent manner. Second, we observed direct MRGPR-X1-mediated TRPV1 activation independent of MRGPR-X1-induced Ca²⁺-release and PKC activity or other TRPV1 affecting enzymes such as lipoxygenase, extracellular signal-regulated kinases-1/2, sarcoma, or phosphoinositide 3-kinase. Investigating several TRPV1 mutants, we observed that removal of the TRPV1 binding site for DAG and of the putative PIP₂ sensor decreased MRGPR-X1-induced TRPV1 activation by 71 and 43%, respectively. Therefore, we demonstrate dual functional interactions between MRGPR-X1 and TRPV1, resulting in PKC-dependent TRPV1 sensitization and DAG/PIP₂-mediated activation. The molecular discrimination between TRPV1 sensitization and activation may help improve the specificity of current pain therapies.     

Regulation of the Temperature-dependent Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) by Phospholipids in Planar Lipid Bilayers

TRPV1 (transient receptor potential vanilloid 1) proteins are heat-activated nonselective cation channels. TRPV1 channels are polymodal in their function and exhibit multifaceted regulation with various molecular compounds. In this regard, phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, are important channel regulators. However, their effects on TRPV1 channel activity have not been conclusively determined. To characterize temperature-induced activation of TRPV1 in the presence of different phospholipids, we purified the TRPV1 protein from HEK-293 cells and incorporated it into planar lipid bilayers. In the presence of 2.5 μm phosphatidylinositol 4,5-bisphosphate, TRPV1 channels demonstrated rapid activation at 33–39 °C and achieved full channel opening at 42 °C. At this temperature range, TRPV1 heat activation exhibited steep temperature dependence (temperature coefficient (Q₁₀) of 18), and the channel openings were accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. At a similar temperature range, another phosphoinositide, phosphatidylinositol 4-phosphate, also potentiated heat activation of TRPV1, but with much lower efficiency. Negatively charged phosphatidylglycerol could also induce heat activation of TRPV1 channels, although with a small-conductance state. Our data demonstrate that phospholipids, specifically phosphoinositides, are important regulators of TRPV1 and are required for heat-induced channel activity.     

Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.     

Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways

The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells resulting in high levels of expression at the plasma membrane. Channel activation was assessed from changes in calcium influx (fura-2 fluorescence measurements) or whole cell currents (patch clamp analysis). At room temperature (22-24 degrees C), exposure of TRPV4-transfected cells to hypotonic medium (225 mOsm/liter) or a non-protein kinase C (PKC)-activating phorbol ester derivative, 4alpha-phorbol 12,13-decanoate (100 nm), induces modest channel activation, whereas phorbol 12-myristate 13-acetate (100 nm), a PKC-activating phorbol ester, and shear stress (3-20 dyne/cm2) had minimal or no effect on channel activation. In contrast, at elevated temperatures (37 degrees C) the channel was rapidly activated by all stimuli. Inhibition of PKC by calphostin C (50 nm) or staurosporine (500 nm) abolished phorbol 12-myristate 13-acetate-induced activation of the channel without affecting the response to other stimuli. Ruthenium red (1 microm) effectively blocked the channel activity by all stimuli. It is concluded that temperature is a critical modulator of TRPV4 channel gating, leading to activation of the channel by a diverse range of microenvironmental chemical and physical signals utilizing a least two transduction pathways, one PKC-dependent and one PKC-independent. The convergence of multiple signals and transduction pathways on the same channel indicate that the channel functions as a molecular integrator of microenvironmental chemical and physical signals.     

Agonist- and Ca2+-dependent desensitization of TRPV1 channel targets the receptor to lysosomes for degradation

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca(2+) cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood. Here, we report that prolonged exposure of TRPV1 to agonists induces rapid receptor endocytosis and lysosomal degradation in both sensory neurons and recombinant systems. Agonist-induced receptor internalization followed a clathrin- and dynamin-independent endocytic route, triggered by TRPV1 channel activation and Ca(2+) influx through the receptor. This process appears strongly modulated by PKA-dependent phosphorylation. Taken together, these findings indicate that TRPV1 agonists induce long-term receptor down-regulation by modulating the expression level of the channel through a mechanism that promotes receptor endocytosis and degradation and lend support to the notion that cAMP signaling sensitizes nociceptors through several mechanisms.