Human TRPV1 Research Articles

Vital but vulnerable: Human TRPV6 is a trade-off of powerful Ca2+ uptake and susceptibility to epithelial barrier dysfunction

Robust Ca2+ absorption is controlled by the highly Ca2+-permeable channels TRPV5 and TRPV6 in the kidney and intestine of terrestrial vertebrates, to fulfill the body's demand for Ca2+. Recently, a pathogenic role of TRPV6 was revealed in alcohol-associated endotoxemia; the intestinal barrier function is compromised by oxidative stress via TRPV6-mediated Ca2+ dysregulation. TRPV6-mediated Ca2+ absorption vital to maintain homeostasis is vulnerable to alcohol-induced malactivation.

Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels

The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.

The Effect of Anti-Chemokine Oral Drug XC8 on Cough Triggered by The Agonists of TRPA1 But Not TRPV1 Channels in Guinea Pigs.

IntroductionChronic cough heavily affects patients’ quality of life, and there are no effective licensed therapies available. Cough is a complication of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection, asthma, and other diseases. Patients with various diseases have a different profile of tussive responses to diverse cough triggers, thereby suggesting sundry mechanisms of neuronal dysfunctions. Previously, we demonstrated that the small molecule drug XC8 shows a clinical anti-asthmatic effect. The objective of the present study was to investigate the effect of XC8 on cough.MethodsWe studied the antitussive effect of XC8 on cough induced by agonists activating human transient receptor potential (TRP) cation channels TRPA1 or TRPV1 in guinea pigs. We checked the agonistic/antagonistic activity of XC8 on the human cation channels TRPA1, TRPV1, TRPM8, P2X purinoceptor 2 (P2X2), and human acid sensing ion channel 3 (hASIC3) in Fluorescent Imaging Plate Reader (FLIPR) assay.ResultsXC8 demonstrated clear antitussive activity and dose-dependently inhibited cough in guinea pigs induced by citric acid alone (up to 67.1%) or in combination with IFN-γ (up to 76.4%). XC8 suppressed cough reflexes induced by the repeated inhalation of citric acid (up to 80%) or by cinnamaldehyde (up to 60%). No activity of XC8 against cough evoked by capsaicin was revealed. No direct agonistic/antagonistic activity of XC8 on human TRPA1, TRPV1, TRPM8, P2X2, or hASIC3 was detected.ConclusionsXC8 acts against cough evoked by the activation of TRPA1 (citric acid/cinnamaldehyde) but not TRPV1 (capsaicin) channels. XC8 inhibits the cough reflex and suppresses the cough potentiation by IFN-γ. XC8 might be of significant therapeutic value for patients suffering from chronic cough associated with inflammation.

Effects of butyrate\u2212 on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate− were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate− (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate− stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).

Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole

TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel’s periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel’s selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.

Transient Receptor Potential channels, TRPV1 and TRPA1 in melanocytes synergize UV-dependent and UV-independent melanogenesis.

Melanogenesis is essential for pigmentation and deregulated melanogenesis causes pigmentary diseases. Psoralen and ultraviolet A (PUVA) therapy strongly stimulates pigmentation, but the underlying molecular mechanisms are elusive. Melanin content of cultured human melanocytes was spectrophotometrically measured. Patch-clamp recordings were made in human melanocytes or HEK 293 cells transiently expressing wild type or mutant human TRPV1 and TRPA1 channels. Endogenous expression of TRPV1 and TRPA1 in melanocytes was analysed by western blotting and was knocked down with siRNA. In vivo pigmentary responses were measured by a colorimeter in mouse ear skin. The expression of TRPV1 and TRPA1 in human pigmented lesions was examined by immunohistochemical staining. PUVA strongly stimulated melanogenesis and PUVA-induced TRPV1 and TRPA1 channel activation in melanocytes and the resulting Ca2+ influx were required for the stimulated melanogenesis both in vitro and in vivo. Agonists-induced TRPV1 and TRPA1 activation alone did not stimulate melanogenesis, but it synergized UVA or intrinsic cAMP and NO signalling pathways to stimulate UV-dependent or UV-independent melanogenesis. Moreover, the expressions of TRPV1 and TRPA1 were increased in human melanocytic lesions and inhibition of both channels decreased melanin content in melanoma cells. TRPV1 and TRPA1 are key molecular sensors and enhancers of extrinsic and intrinsic melanogenic signals in both physiological and pathological conditions, and activation of both channels in melanocytes contributes to PUVA therapy-induced pigmentation. Our work provides a common mechanism of melanogenic regulation and highlights TRPV1 and TRPA1 as potential therapeutic targets for pigmentary disorders.

Human TRPV1 and TRPA1 are receptors for bacterial quorum sensing molecules.

In this study, we investigated the activation of TRPV1 and TRPA1 by N-acyl homoserine lactones, quorum sensing molecules produced by Gram-negative bacteria, and the inhibitory effect of TRPV1 and TRPA1 by autoinducing peptides (AIPs), quorum sensing molecules produced by Gram-positive bacteria, using human embryonic kidney 293T cell lines stably expressing human TRPV1 and TRPA1, respectively. As a result, we found that some N-acyl homoserine lactones, such as N-octanoyl-L-homoserine lactone (C8-HSL), N-nonanoyl-L-homoserine lactone (C9-HSL) and N-decanoyl-L-homoserine lactone (C10-HSL), activated both TRPV1 and TRPA1. In addition, we clarified that some N-acyl homoserine lactones, such as N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), only activated TRPV1 and N-acyl homoserine lactones having saturated short acyl chain, such as N-acetyl-L-homoserine lactone (C2-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), only activated TRPA1. Furthermore, we found that an AIP, simple linear peptide CHWPR, inhibited both TRPV1 and TRPA1 and peptide having thiolactone ring DICNAYF, the thiolactone ring were formed between C3 to F7, strongly inhibited only the TRPV1. Although the specificity of TRPV1 and TRPA1 for quorum sensing molecules was different, these data suggest that both TRPV1 and TRPA1 would function as receptors for quorum sensing molecule produced by bacteria. Graphical Abstract.

Surfactant cocamide monoethanolamide causes eye irritation by activating nociceptor TRPV1 channels.

Cocamide monoethanolamide (CMEA) is commonly used as a surfactant-foam booster in cosmetic formulations. Upon contact with the eye or other sensitive skin areas, CMEA elicits stinging and lasting irritation. We hypothesized a specific molecular interaction with TRPV1 channels by which CMEA caused eye irritation. Eye irritancy was evaluated using eye-wiping tests in rabbits and mice. Intracellular Ca2+ concentrations and action potentials were measured using Ca2+ imaging and current clamp respectively. Voltage clamp, site-direct mutagenesis and molecular modelling were used to identify binding pockets for CMEA on TRPV1 channels. CMEA-induced eye irritation is ameliorated by selective ablation of TRPV1 channels.Rodents exhibit much stronger responses to CMEA than rabbits. In trigeminal ganglion neurons, CMEA induced Ca2+ influx and neuronal excitability, effects mitigated by a TRPV1 channel inhibition and absent in TRPV1 knockout neurons. In HEK-293 cells expressing TRPV1 channels, CMEA increased whole-cell currents by increasing channel open probability (EC50 = 10.2μM), without affecting TRPV2, TRPV3, TRPV4, and TRPA1 channel activities. Lauric acid monoethanolamide (LAMEA), the most abundant constituent of CMEA, was the most efficacious and potent TRPV1 channel activator, binding to the capsaicin-binding pocket of the channel. The T550I mutants of rabbit and human TRPV1 channels exhibit much lower sensitivity to LAMEA. CMEA directly activates TRPV1 channels to produce eye irritation. Rabbits, the standard animal used for eye irritancy tests are poor models for evaluating human eye irritants structurally related to CMEA. Our study identifies potential alternatives to CMEA as non-irritating surfactants.

Accelerated Production and Maturation of Human Induced Pluripotent Stem Cell Derived Nociceptors

The scarce availability and prohibitive costs of primary human DRG tissues limits the scalability of in vitro models for pain drug discovery and disease modeling. Human induced pluripotent stem cell (hiPSC)-derived sensory neurons provide a near-limitless amount of tissue for these applications. Using an accelerated, step-wise, directed differentiation protocol that proceeds through a novel ‘primal ectoderm’ population, we are able to manufacture nociceptors rapidly and at scale by relying on a simplified chemically-defined basal medium, recombinant growth factors and small molecules. Following production and only one week of maturation, these nociceptors have a consistent molecular phenotype and express key therapeutic pain targets such as human voltage-gated sodium channels Nav1.7, 1.8, and 1.9, which are primarily responsible for eliciting nociceptor action potentials, as well as the human capsaicin receptor TRPV1. Using whole-cell patch clamp electrophysiology, we demonstrate that these neurons fire action potentials and possess burst-firing ability by day 9 in vitro as assessed by performing stepwise depolarizing current injections in current clamp mode. We also show these neurons exhibit inward and outward currents in response to voltage steps in voltage clamp mode. We have also demonstrated that these neurons are responsive to ligands such as capsaicin using calcium imaging region-of-interest analysis. Greater than 30% of neuronal cell bodies respond to 100 nM capsaicin following an additional 2 to 3 weeks of maturation. Importantly, this response was ablated by capsazepine, thus confirming response specificity. These data demonstrate that hiPSC nociceptors have the potential to screen and identify novel analgesic or anti-nociceptive compounds for use in humans.

Identification and characterization of piperine synthase from black pepper, Piper nigrum L.

Black pepper (Piper nigrum L.) is the world’s most popular spice and is also used as an ingredient in traditional medicine. Its pungent perception is due to the interaction of its major compound, piperine (1-piperoyl-piperidine) with the human TRPV-1 or vanilloid receptor. We now identify the hitherto concealed enzymatic formation of piperine from piperoyl coenzyme A and piperidine based on a differential RNA-Seq approach from developing black pepper fruits. This enzyme is described as piperine synthase (piperoyl-CoA:piperidine piperoyl transferase) and is a member of the BAHD-type of acyltransferases encoded by a gene that is preferentially expressed in immature fruits. A second BAHD-type enzyme, also highly expressed in immature black pepper fruits, has a rather promiscuous substrate specificity, combining diverse CoA-esters with aliphatic and aromatic amines with similar efficiencies, and was termed piperamide synthase. Recombinant piperine and piperamide synthases are members of a small gene family in black pepper. They can be used to facilitate the microbial production of a broad range of medicinally relevant aliphatic and aromatic piperamides based on a wide array of CoA-donors and amine-derived acceptors, offering widespread applications.

Gating of the capsaicin receptor TRPV1 by UVA-light and oxidants are mediated by distinct mechanisms

Redox-sensitivity is a common property of several transient receptor potential (TRP) ion channels. Oxidants and UVA-light activate TRPV2 by oxidizing methionine pore residues which are conserved in the capsaicin-receptor TRPV1. However, the redox-sensitivity of TRPV1 is regarded to depend on intracellular cysteine residues. In this study we examined if TRPV1 is gated by UVA-light, and if the conserved methionine residues are relevant for redox-sensitivity of TRPV1. Patch clamp recordings were performed to explore wildtype (WT) and mutants of human TRPV1 (hTRPV1).UVA-light induced hTRPV1-mediated membrane currents and potentiated both proton- and heat-evoked currents. The reducing agent dithiothreitol (DTT) prevented and partially reversed UVA-light induced sensitization of hTRPV1. UVA-light induced sensitization was reduced in the mutant hTRPV1-C158A/C387S/C767S (hTRPV1-3C). The remaining sensitivity to UVA-light of hTRRPV1-3C was not further reduced upon exchange of the methionine residues M568 and M645. While UVA-induced sensitization was reduced in the protein kinase C-insensitive mutant hTRPV1-S502A/S801A, the PKC-inhibitors chelerythrine chloride, staurosporine and Gö6976 did not reduce UVA-induced effects on hTRPV1-WT. While hTRPV1-3C was insensitive to the cysteine-selective oxidant diamide, it displayed a residual sensitivity to H2O2 and chloramine-T. However, the exchange of M568 and M645 in hTRPV1-3C did not further reduce these effects.Our data demonstrate that oxidants and UVA-light gate hTRPV1 by cysteine-dependent as well as cysteine-independent mechanisms. In contrast to TRPV2, the methionine residues 568 and 645 seem to be of limited relevance for redox-sensitivity of hTRPV1. Finally, UVA-light induced gating of hTRPV1 does not seem to require activation of protein kinase C.

Convergence of peptidergic and non-peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn.

Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long-standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1, and Nav1.7 proteins stained the entirety of lamina 1-2, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

Parasympathetic neurons in the human submandibular ganglion

The submandibular ganglion (SMG) contains parasympathetic neurons which innervate the submandibular gland. In this study, immunohistochemistry for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), choline acetyltransferase (ChAT), dopamine β-hydroxylase (DBH), tyrosine hydroxylase (TH), and the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2) was performed on the human SMG. In the SMG, 17.5 % and 8.9 % of parasympathetic neurons were immunoreactive for VIP and TRPV2, respectively. SMG neurons mostly contained ChAT- and DBH-immunoreactivity. In addition, subpopulations of SMG neurons were surrounded by VIP (69.6 %)-, TRPV2 (54.4 %)- and DBH (9.5 %)-immunoreactive (-ir) nerve fibers. SMG neurons with pericellular VIP- and TRPV2-ir nerve fibers were significantly larger than VIP- and TRPV2-ir SMG neurons, respectively. Other neurochemical substances were rare in the SMG. In the human submandibular gland, TRPV1- and TRPV2-ir nerve fiber profiles were seen around blood vessels. Double fluorescence method also demonstrated that TRPV2-ir nerve fiber profiles were located around myoepithelial and acinar cells in the submandibular gland. VIP and TRPV2 are probably expressed by both pre- and post-ganglionic neurons innervating the submandibular and sublingual glands. VIP, DBH and TRPV2 may have functions about regulation of salivary components in the salivary glands and neuronal activity in the SMG.

A capsaicinoid-based soft drug, AG1529, for attenuating TRPV1-mediated histaminergic and inflammatory sensory neuron excitability

TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.

Why endogenous TRPV6 currents are not detectable-what can we learn from bats?

TRPV6 is a calcium selective TRP channel and is expressed in many species. TRPV6 transcripts are abundantly expressed in few tissues but strangely channel properties are only accessible to electrophysiological recordings after overexpression whereas in native tissue functional channel currents seem not to be detectable. Another exceptional property of human and mouse TRPV6 proteins is that the initiation of translation starts from a non-canonical ACG triplet which is translated as methionine. This triplet is located 120 bp upstream of the first in-frame AUG codon of the human/mouse TRPV6 mRNA. In contrast, the TRPV6 gene of bats is initiated from an AUG triplet at the corresponding position of the human ACG. On the basis of these structural nucleotide differences between human and bats we studied the role of the absolute N-Terminus for the regulation of translation by developing chimera and mutants of human/bat TRPV6 channels. The human sequence which is located downstream of the initiation codon slows down ribosomal scanning in 3′ direction. We suggest that the mechanism involves most likely the deceleration of ribosome scanning by stem-loop formation and the use of the common initiator tRNA, tRNAiMet, which is placed onto the inappropriate ACG codon resulting in low protein synthesis. The reduced translation efficiency is important to protect TRPV6 expressing cells from toxic calcium overload. The regulation of the TRPV6 translation in bats may be an adaptation to low calcium amounts present in the natural nutrition. In addition, we show that also the GFP protein can be controlled using the translational mechanism of human TRPV6.

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

Gating of human TRPV3 in a lipid bilayer.

The TRPV3 channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle. The ligand-free, closed conformation reveals well-ordered lipids interacting with the channel and two physical constrictions along the ion conduction pore involving both the extracellular selectivity filter and intracellular helix bundle crossing. Both the selectivity filter and bundle crossing expand upon activation, accompanied by substantial structural rearrangements at the cytoplasmic inter-subunit interface. Transition to the inactivated state involves a secondary structure change of the pore-lining helix, which contains a π-helical segment in the closed and open conformations but becomes entirely α-helical upon inactivation. Together with electrophysiological characterization, structures of TRPV3 in a lipid membrane environment provide unique insights into channel activation and inactivation mechanisms.

The Zinc-Finger Domain Containing Protein ZC4H2 Interacts with TRPV4, Enhancing Channel Activity and Turnover at the Plasma Membrane.

The Ca2+-permeable Transient Receptor Potential channel vanilloid subfamily member 4 (TRPV4) is involved in a broad range of physiological processes, including the regulation of systemic osmotic pressure, bone resorption, vascular tone, and bladder function. Mutations in the TRPV4 gene are the cause of a spectrum of inherited diseases (or TRPV4-pathies), which include skeletal dysplasias, arthropathies, and neuropathies. There is little understanding of the pathophysiological mechanisms underlying these variable disease phenotypes, but it has been hypothesized that disease-causing mutations affect interaction with regulatory proteins. Here, we performed a mammalian protein–protein interaction trap (MAPPIT) screen to identify proteins that interact with the cytosolic N terminus of human TRPV4, a region containing the majority of disease-causing mutations. We discovered the zinc-finger domain-containing protein ZC4H2 as a TRPV4-interacting protein. In heterologous expression experiments, we found that ZC4H2 increases both the basal activity of human TRPV4 as well as Ca2+ responses evoked by ligands or hypotonic cell swelling. Using total internal reflection fluorescence (TIRF) microscopy, we further showed that ZC4H2 accelerates TRPV4 turnover at the plasma membrane. Overall, these data demonstrate that ZC4H2 is a positive modulator of TRPV4, and suggest a link between TRPV4 and ZC4H2-associated rare disorders, which have several neuromuscular symptoms in common with TRPV4-pathies.

Effect of 158 herbal remedies on human TRPV1 and the two-pore domain potassium channels KCNK2, 3 and 9

Background and aimHerbal medicines are used to treat a broad number of maladies. However, the pharmacological profile of most remedies is poorly understood. We investigated the effect of herbal remedies from kampo, traditional Chinese medicine (TCM) and other phytotherapies on human two-pore domain potassium channels (KCNK channels; TREK-1, TASK-1 and TASK-3) as well as the human TRPV1 channel. KCNK channels are responsible for the background potassium current of excitable cells, thus essential for the maintenance of the resting membrane potential. Hence, modulators of KCNK channels are of medical significance, e.g. for the treatment of sleep disorders and pain. The transient receptor potential channel TRPV1 is a pain detector for noxious heat. Agonists of this receptor are still used for the treatment of pain in ectopic applications. Experimental procedureWe evaluated the effect of 158 herbal remedies on these channels in a heterologous expression system (Xenopus laevis oocytes) using the two-electrode voltage-clamp technique with the aim of increasing the comprehension of their pharmacological profile. Results and conclusionSome remedies with modulating effects were identified such as Angelica pubescens (radix), which inhibit TASK-1 and TASK-3 channels. Furthermore, the modulatory effects of the most effective remedies on the two TASK family members TASK-1 and TASK-3 correlate positively, reflecting their close relation. For the TRPV1 channel Terminalia chebula and Alchemilla xanthochlora were identified as potentiators. This study identifies a variety of herbal remedies as modulators of human K2P and TRPV1 channels and gives new insights into the pharmacological profile of these herbal remedies.

Extract of Foeniculum vulgare seed effect on UV‐induced photoaging‐associated ion channel and collagenase

Skin aging is mediated by genetics, hormonal changes, metabolic processes, and environmental stress. UV radiation is concerned as a major risk factor for skin aging, including wrinkles, and melanoma. Studies suggested that calcium ion channels, such as TRPV1 and ORAI1, are related with melanogenesis, skin wrinkling, and inflammation. Foeniculum vulgare has been used to treat inflammatory diseases. Recent studies showed that it protects against UV radiation‐induced skin photoaging. However, its effect on the ion channels related to UV‐induced photoaging is unclear. Therefore, this study aimed to evaluate the antagonistic effects of F. vulgare extract, fractions, and constituents on TRPV1.Electrophysiological analysis results showed that, at 100 μM, F. vulgare extract and fractions potently inhibited capsaicin‐induced TRPV1 [methanol extract (91%), n‐hexane (94%), and methylene chloride (100%) at −60 mV]. The methanol extract of F. vulgare inhibited MMP‐1 (collagenase) production (35% at 3.75 μg/mL), compared with control. These results suggested that the compounds in F. vulgare extract and fractions possessed anti‐aging effects that can decrease wrinkles and maintain elasticity by disrupting MMP‐1 production activated by the MAPK pathway. Unfortunately, compounds isolated from the hexane and methylene chloride fractions could not suppress ITRPV1. Surprisingly, even though capsaicin increased ITRPV1, the γ‐terpinene, limonene, scopoletin, and bergapten isolated from these two fractions slightly potentiated ITRPV1. Although 4 compounds of F. vulgare exerted an agonistic effect on TRPV1, the compounds with antagonistic effect have not been discovered. Our study suggested that F. vulgare had potential therapeutic effects on UV‐induced photoaging for therapeutic and cosmetic applications.Effect of F. vulgare extract and fractions on human TRPV1 (hTRPV1) current under capsaicin (CAP) treatmentFigure 1Effects of F. vulgare extract and fractions on UV‐induced melanogenesisFigure 2