Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR₂), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR₂ regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR₂ agonist stimulated a transient increase in [Ca²⁺]_i. TRPV4 expression led to a markedly sustained increase in [Ca²⁺]_i. Removal of extracellular Ca²⁺ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A₂ and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR₂ generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR₂-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR₂ was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR₂ signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR₂-stimulated neurogenic inflammation. Thus, PAR₂ activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR₂.     

Protease Signaling to G Protein-Coupled Receptors: Implications for Inflammation and Pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR₁, PAR₂, and PAR₄ either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR₂ agonists induce hyperalgesia and allodynia, and PAR₂ activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR₁ or PAR₄ increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

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.     

β1-Adrenergic Receptor Signaling Activates the Epithelial Calcium Channel,Transient Receptor Potential Vanilloid Type 5 (TRPV5), via the Protein Kinase A Pathway

Epinephrine and norepinephrine are present in the pro-urine. β-Adrenergic receptor (β-AR) blockers administered to counteract sympathetic overstimulation in patients with congestive heart failure have a negative inotropic effect, resulting in reduced cardiac contractility. Positive inotropes, β1-AR agonists, are used to improve cardiac functions. Active Ca²⁺ reabsorption in the late distal convoluted and connecting tubules (DCT2/CNT) is initiated by Ca²⁺ influx through the transient receptor potential vanilloid type 5 (TRPV5) Ca²⁺ channel. Although it was reported that β-ARs are present in the DCT2/CNT region, their role in active Ca²⁺ reabsorption remains elusive. Here we revealed that β1-AR, but not β2-AR, is localized with TRPV5 in DCT2/CNT. Subsequently, treatment of TRPV5-expressing mouse DCT2/CNT primary cell cultures with the β1-AR agonist dobutamine showed enhanced apical-to-basolateral transepithelial Ca²⁺ transport. In human embryonic kidney (HEK293) cells, dobutamine was shown to stimulate cAMP production, signifying functional β1-AR expression. Fura-2 experiments demonstrated increased activity of TRPV5 in response to dobutamine, which could be prevented by the PKA inhibitor H89. Moreover, nonphosphorylable T709A-TRPV5 and phosphorylation-mimicking T709D-TRPV5 mutants were unresponsive to dobutamine. Surface biotinylation showed that dobutamine did not affect plasma membrane abundance of TRPV5. In conclusion, activation of β1-AR stimulates active Ca²⁺ reabsorption in DCT2/CNT; an increase in TRPV5 activity via PKA phosphorylation of residue Thr-709 possibly plays an important role. These data explicate a calciotropic role in addition to the inotropic property of β1-AR.     

The protease-activated receptor-2-specific agonists 2-aminothiazol-4-yl-LIGRL-NH2 and 6-aminonicotinyl-LIGRL-NH2 stimulate multiple signaling pathways to induce physiological responses in vitro and in vivo

Protease-activated receptor-2 (PAR₂) is one of four protease-activated G-protein-coupled receptors. PAR₂ is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR₂ reveals a tethered ligand that activates PAR₂ and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca²⁺ signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR₂ (e.g. SLIGRL-NH₂ and SLIGKV-NH₂) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH₂ (2-f-LIGRLO-NH₂) underscores the use of peptidomimetic PAR₂ ligands as a mechanism to enhance pharmacological action at PAR₂, 2-f-LIGRLO-NH₂ has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH₂ and two recently described pentapeptidomimetic PAR₂-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH₂ (2-at-LIGRL-NH₂) and 6-aminonicotinyl-LIGRL-NH₂ (6-an-LIGRL-NH₂). All peptidomimetic agonists stimulated PAR₂-dependent in vitro physiological responses, MAPK signaling, and Ca²⁺ signaling with an overall rank order of potency of 2-f-LIGRLO-NH₂ ≈ 2-at-LIGRL-NH₂ > 6-an-LIGRL-NH₂ ≫ SLIGRL-NH₂. Because PAR₂ plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca²⁺ signaling in nociceptors in vitro, and both 2-at-LIGRL-NH₂ and 2-f-LIGRLO-NH₂ stimulated PAR₂-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR₂ in a variety of systems and pathologies.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Neutrophil Elastase and Proteinase-3 Trigger G Protein-biased Signaling through Proteinase-activated Receptor-1 (PAR1)

Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gα_i-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.     

Furanocoumarins Are a Novel Class of Modulators for the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel

Furanocoumarin imperatorin is the major active component of Angelica dahurica root extracts, widely used in traditional medicine to treat headache, toothache, and orbital eye pain. In this study, we investigated the mechanisms that may underlie the pain-relieving effects of the compound. We found that imperatorin significantly inhibited formalin- and capsaicin-induced nocifensive responses but did not alter baseline thermal withdrawal thresholds in the rat. We established that imperatorin is a weak agonist of TRPV1, a channel implicated in detecting several noxious stimuli, exhibiting a 50% effective concentration (EC₅₀) of 12.6 ± 3.2 μm. A specific TRPV1 antagonist, JNJ-17203212 (0.5 μm), potently inhibited imperatorin-induced TRPV1 activation. Site-directed mutagenesis studies revealed that imperatorin most likely acted via a site adjacent to or overlapping with the TRPV1 capsaicin-binding site. TRPV1 recovery from desensitization was delayed in the presence of imperatorin. Conversely, imperatorin sensitized TRPV1 to acid activation but did not affect the current amplitude and/or the activation-inactivation properties of Na_v1.7, a channel important for transmission of nociceptive information. Thus, our data indicate that furanocoumarins represent a novel group of TRPV1 modulators that may become important lead compounds in the drug discovery process aimed at developing new treatments for pain management.     

Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

The β₁-adrenergic receptor (β₁AR) is the predominant βAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human β₁AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293_i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn¹⁵. Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg³¹ and Leu³² and Pro⁵² and Leu⁵³, which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the βAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg³¹–Leu³² cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface β₁ARs.     

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.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

Breaking down the barriers: fMRI applications in pain, analgesia and analgesics

Background

The vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described.

Results

In the present studies we investigated the hypothesis that the μ opioid receptor (MOP) agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca²⁺) responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca²⁺ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca²⁺ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1.

Conclusion

The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.     

Carboxyl-terminal Domain of Transient Receptor Potential Vanilloid 1 Contains Distinct Segments Differentially Involved in Capsaicin- and Heat-induced Desensitization

Multiple Ca²⁺-dependent processes are involved in capsaicin-induced desensitization of transient receptor potential vanilloid 1 (TRPV1), but desensitization of TRPV1 by heat occurs even in the absence of extracellular Ca²⁺, although the mechanisms are unknown. In this study, we tested the hypothesis that capsaicin and heat desensitize TRPV1 through distinct mechanisms involving distinct structural segments of TRPV1. In HEK293 cells that heterologously express TRPV1, we found that heat-induced desensitization was not affected by the inclusion of intracellular ATP or alanine mutation of Lys¹⁵⁵, both of which attenuate capsaicin-induced desensitization, suggesting that heat-induced desensitization occurs through mechanisms distinct from capsaicin-induced desensitization. To determine protein domains involved in heat-induced desensitization, we generated chimeric proteins between TRPV1 and TRPV3, a heat-gated channel lacking heat-induced desensitization. We found that TRPV1 with the carboxyl-terminal domain (CTD) of TRPV3 retained heat activation but was impaired in heat-induced desensitization. Further experiments using chimeric or deletion mutants within TRPV1 CTD indicated that the distal half of CTD regulates the activation and desensitization of TRPV1 in modality-specific manners. Within the distal CTD, we identified two segments that distinctly regulated capsaicin- and heat-induced desensitization. The results suggest that the activation and desensitization of TRPV1 by capsaicin and heat can be modulated differentially and disproportionally through different regions of TRPV1 CTD. Identifying the domains involved in thermal regulation of TRPV1 may facilitate the development of novel anti-hyperalgesic approaches aimed at attenuating activation and enhancing desensitization of TRPV1 by thermal stimuli.     

Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca2+-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

The epithelial Ca²⁺ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca²⁺-induced inactivation that protects the cell from toxic Ca²⁺ overload and may also limit intestinal Ca²⁺ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca²⁺, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P₂, and Ca²⁺-CaM inhibited the channel at physiologically relevant concentrations. Ca²⁺ alone also inhibited TRPV6 at high concentrations (IC₅₀ = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca²⁺-induced inactivation. Providing excess PI(4,5)P₂ reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P₂ did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P₂ and show that Ca²⁺, CaM, and the depletion of PI(4,5)P₂ all contribute to inactivation of TRPV6.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK₁R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca²⁺ signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK₁R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK₁R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK₁R Ca²⁺ signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK₁R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK₁R signaling from the plasma membrane that drives inflammation.     

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.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.