Vanilloid Subfamily Research Articles

Analysis of TRPV gene expression in the respiratory epithelium of asthma patients with osmotic airway hyperresponsiveness

Introduction . The phenomenon of airway hyperresponsiveness in response to changes in air humidity is widespread among patients with asthma. Transient receptor potential channels of the vanilloid subfamily (TRPV) are of interest in terms of study of specific mechanisms of bronchial osmotic hyperresponsiveness. Aim . The aim of the study was to evaluate the expression TRPV1, TRPV2 and TRPV4 genes at mRNA level in the epithelium of the upper and lower respiratory tract in asthma patients with osmotic airway hyperresponsiveness. Materials and methods . We examined 35 patients with mild and moderate asthma. All patients underwent bronchoprovocation tests with hypo- and hyperosmotic solutions. Expression of TRPV receptors was studied in brush biopsies of the nasal and bronchial epithelium by quantitative PCR with reverse transcription. Results . Airway hyperrresponsiveness to hypoosmotic and hyperosmotic stimuli was observed in 25% and 50% of cases, respectively. It was found that patients with lower baseline FEV 1 had overexpression of TRPV1 (3.2 times, p=0.05) and TRPV2 (6.2 times, p=0.013) in the bronchial epithelium. Hypoosmotic airway hyperresponsiveness was associated with increased expression of the TRPV1 (7.4 times, p=0.004) and TRPV2 (18.5 times, p=0.014) genes in the bronchial epithelium. Airway hyperresponsiveness to hypertonic stimulus was also characterized by higher expression of TRPV1 (4.7 times, p=0.013) and TRPV2 (7.6 times, p=0.024). No difference in TRPV4 expression levels was found. Conclusion . The obtained data indicate the up-regulation of TRPV1 and TRPV2 in the bronchial epithelium of asthma patients with osmotic airway hyperresponsiveness, what, in turn, may indicate the role of these receptors in the development of airway osmotic-induced responses and in the pathogenesis of asthma.

Phospholipase Cβ3 Expressed in Mouse DRGs is Involved in Inflammatory and Postoperative Pain.

BackgroundPrevious studies suggested that phospholipase Cβ3 (PLCβ3), which is a common downstream component in the signaling cascade, plays an important role in peripheral mechanisms of perception including nociception. However, detailed profiles of PLCβ3-expressing dorsal root ganglion (DRG) neurons and involvement of PLCβ3 in inflammatory and postoperative pain have not been fully investigated.PurposeWe evaluated neurochemical char0acteristics of PLCβ3-expressing DRG neurons in mice and then we examined the effects of selective knockdown of PLCβ3 expression in DRGs on inflammatory and postoperative pain.MethodsMale C57BL/6-strain mice were used. For the inflammatory model, each mouse received subcutaneous injection of complete Freund’s adjuvant (CFA) in the left hindpaw. For the postoperative pain model, a plantar incision was made in the left hindpaw. PLCβ3 antisense oligodeoxynucleotide or PLCβ3 mismatch oligodeoxynucleotide was intrathecally administered once a day for three consecutive days in each model. The time courses of thermal hyperalgesia and mechanical hyperalgesia were investigated. Changes in PLCβ3 protein levels in DRGs were evaluated by Western blotting.ResultsImmunohistochemical analysis showed that high proportion of the PLCβ3-positive profiles were biotinylated isolectin B4-positive or transient receptor potential vanilloid subfamily 1-positive. PLCβ3 protein level in DRGs during CFA-induced inflammation was comparable to that at baseline. Intrathecal administration of PLCβ3 antisense oligodeoxynucleotide, which significantly suppressed PLCβ3 expression in DRGs, did not affect pain thresholds in normal conditions but inhibited CFA-induced thermal and mechanical hyperalgesia both at the early and late phases compared to that in mismatch oligodeoxynucleotide-treated mice. Intrathecal administration of PLCβ3 antisense oligodeoxynucleotide also inhibited surgical incision-induced thermal and mechanical hyperalgesia.ConclusionOur results uncover a unique role of PLCβ3 in the development and maintenance of inflammatory pain induced by CFA application and in those of surgical incision-induced pain, although PLCβ3 does not play a major role in thermal nociception or mechanical nociception in normal conditions.

Role of TRPV1 in colonic mucin production and gut microbiota profile

This study focuses on exploring the role of sensory cation channel Transient Receptor Potential channel subfamily Vanilloid 1 (TRPV1) in gut health, specifically mucus production and microflora profile in gut.We employed resiniferatoxin (ultrapotent TRPV1 agonist) induced chemo-denervation model in rats and studied the effects of TRPV1 ablation on colonic mucus secretion patterns. Histological and transcriptional analysis showed substantial decrease in mucus production as well as in expression of genes involved in goblet cell differentiation, mucin production and glycosylation. 16S metagenome analysis revealed changes in abundance of various gut bacteria, including decrease in beneficial bacteria like Lactobacillus spp and Clostridia spp. Also, TRPV1 ablation significantly decreased the levels of short chain fatty acids, i.e. acetate and butyrate.The present study provides first evidence that systemic TRPV1 ablation leads to impairment in mucus production and causes dysbiosis in gut. Further, it suggests to address mucin production and gut microbiota related adverse effects during the development of TRPV1 antagonism/ablation-based therapeutic and preventive strategies.

Kainic Acid Activates TRPV1 via a Phospholipase C/PIP2-Dependent Mechanism in Vitro.

Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca2+ influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEKTRPV1 cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca2+ induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca2+-induced neurotoxicity.

Role of TRPV4 in matrix stiffness-induced expression of EMT-specific LncRNA.

Long non-coding RNAs (LncRNAs) are long (> 200 bases), non-coding, single-stranded RNAs that have emerged as major regulators of gene expression, cell differentiation, development, and oncogenesis. In view of the fact that matrix stiffness plays a role in cellular functions associated with these processes, it is important to ask what role matrix stiffness plays in regulating expression of LncRNAs. In this report, we show that (i) matrix stiffness causes differential expression of epithelial-mesenchymal transition (EMT)-related LncRNAs and mRNAs in primary mouse normal epidermal keratinocytes, (ii) differential expression of EMT-related LncRNAs and mRNAs occurs in response to combined stimulation of transforming growth factor β1 and matrix stiffness, and (iii) transient receptor potential (TRP) channel of the vanilloid subfamily, TRPV4, a matrix stiffness-sensitive ion channel, plays a role in differential expression of EMT-related LncRNAs and mRNAs in response to combined stimulation by TGFβ1 and matrix stiffness. These data identify TRPV4 as a candidate plasma membrane mechanosensor that transmits matrix-sensing signals essential to LncRNA expression. Our results also show that we have established and validated an assay system capable of discovering novel LncRNAs and mRNAs sensitive to matrix stiffening.

A Closer Look at Anandamide Interaction With TRPV1.

The transient receptor potential subfamily vanilloid type 1 ion channel (TRPV1), located in the peripheral nervous system has been implicated in the perception of pain and possesses the ability to be modulated by various cannabinoid ligands. Because of its location, TRPV1 is an ideal target for the development of novel pain therapeutics. Literature precedent suggests a wide range of cannabinoid ligands can activate TRPV1, but the location and mode of entry is not well understood. Understanding the modes in which cannabinoids can enter and bind to TRPV1 can aid in rational drug design. The first endogenous ligand identified for TRPV1 was the endocannabinoid, anandamide (AEA). The Molecular Dynamics (MD) studies discussed here investigate the entry mode of AEA into TRPV1. During the course of the 10+ microsecond MD simulations, two distinct binding modes were observed: AEA binding in the tunnel formed by the S1–S4 region, and AEA binding in the vanilloid binding pocket, with preference for the former. Unbiased MD simulations have revealed multiple spontaneous binding events into the S1–S4 region, with only one event of AEA binding the vanilloid binding pocket. These results suggest that AEA enters TRPV1 via a novel location between helices S1–S4 via the lipid bilayer.

Treatments for Childhood Atopic Dermatitis: an Update on Emerging Therapies.

Atopic dermatitis (AD) is generally considered a T helper type 2-dominated disease. Pediatric AD is usually less severe than adult AD, but it may present as moderate to severe lesions that are inadequately managed by current modalities including emollients/moisturizers, topical corticosteroids (TCSs), topical calcineurin inhibitors (TCIs), and even systemic immunosuppressants (such as cyclosporine, azathioprine, methotrexate, and mycophenolate mofetil). In addition, systemic immunosuppressants are often not recommended for childhood AD by the current guidelines due to their toxicities. Therefore, there is still an unmet need for a safe and effective long-term therapy for pediatric AD patients whose disease is inadequately controlled or who are intolerant to current treatments. The emerging therapeutics for AD focuses on intervening in the inflammatory pathway by targeting specific cytokines/chemokines or their receptors. Monoclonal antibodies against immunoglobulin E (IgE), interleukin (IL)-4 receptor subunit α, IL-5, IL-13, IL-31 receptor subunit α, IL-33, and thymic stromal lymphopoietin (TSLP) have been evaluated clinically for AD. Encouraging results have been reported for many of the biologics, of which the most exciting is dupilumab. Other emerging systemic therapies include small molecules such as baricitinib, abrocitinib, upadacitinib, and tradipitant. Several novel topical agents are under clinical investigation for the treatment of AD, including topical phosphodiesterase 4 (PDE4) inhibitors, Janus kinase (JAK) inhibitors, aryl hydrocarbon receptor (AhR) modulating agents, and transient receptor potential vanilloid subfamily member 1 (TRPV1) antagonists. Accompanied by thorough characterization of different phenotype and endotype subsets, the application of precision medicine could provide new prospects for the optimal treatment of AD.

Identification of a new functional domain of Nogo-A that promotes inflammatory pain and inhibits neurite growth through binding to NgR1.

Nogo-A is a key inhibitory molecule to axon regeneration, and plays diverse roles in other pathological conditions, such as stroke, schizophrenia, and neurodegenerative diseases. Nogo-66 and Nogo-Δ20 fragments are two known functional domains of Nogo-A, which act through the Nogo-66 receptor (NgR1) and sphingosine-1-phosphate receptor 2 (S1PR2), respectively. Here, we reported a new functional domain of Nogo-A, Nogo-A aa 846-861, was identified in the Nogo-A-specific segment that promotes complete Freund's adjuvant (CFA)-induced inflammatory pain. Intrathecal injection of its antagonist peptide 846-861PE or the specific antibody attenuated the CFA-induced inflammatory heat hyperalgesia. The 846-861 PE reduced the content of transient receptor potential vanilloid subfamily member 1 (TRPV1) in dorsal root ganglia (DRG) and decreased the response of DRG neurons to capsaicin. These effects were accompanied by a reduction in LIMK/cofilin phosphorylation and actin polymerization. GST pull-down and fluorescence resonance energy transfer (FRET) assays both showed that Nogo-A aa 846-861 bound to NgR1. Moreover, we demonstrated that Nogo-A aa 846-861 inhibited neurite outgrowth from cortical neurons and DRG explants. We concluded that Nogo-A aa 846-861 is a novel ligand of NgR1, which activates the downstream signaling pathways that inhibit axon growth and promote inflammatory pain.

The structure of lipid nanodisc-reconstituted TRPV3 reveals the gating mechanism.

Transient receptor potential vanilloid subfamily member 3 (TRPV3) is a temperature-sensitive cation channel. Previous cryo-EM analyses of TRPV3 in detergent micelles or amphipol proposed that the lower gate opens by α-to-π helical transitions of the nearby S6 helix. However, it remains unclear how physiological lipids are involved in the TRPV3 activation. Here we determined the apo state structure of mouse (Mus musculus) TRPV3 in a lipid nanodisc at 3.3 Å resolution. The structure revealed that lipids bound to the pore domain stabilize the selectivity filter in the narrow state, suggesting that the selectivity filter of TRPV3 affects cation permeation. When the lower gate is closed in nanodisc-reconstituted TRPV3, the S6 helix adopts the π-helical conformation without agonist- or heat-sensitization, potentially stabilized by putative intra-subunit hydrogen bonds and lipid binding. Our findings provide insights into the lipid-associated gating mechanism of TRPV3.

TRPV2 channel-based therapies in the cardiovascular field. Molecular underpinnings of clinically relevant therapies

The transient receptor potential (TRP) ion channel family is composed of twenty-seven channel proteins that are ubiquitously expressed in the human body. The TRPV (vanilloid) subfamily has been a recent target of investigation within the cardiovascular field. TRPV1, which is sensitive to heat as well as vanilloids, is the best characterized TRPV channel and is the namesake for the subfamily that includes six members. Research into the function of TRPV2 has suggested that it plays an important role in cardiovascular function.Over the last twenty years a greater understanding of the differences among the TRPV channels has allowed for more precise experimentation and has opened various translational opportunities. TRPV2 has been found to be a both a mechanosensor and a mediator of calcium handling and has been found to play important roles in healthy and diseased cardiomyocytes. These roles have been translated into clinical studies in patients with muscular dystrophy (both agonism and antagonism) as well as in patients with cardiomyopathy and heart failure with reduced ejection fraction. Its role as a structural protein has also been elucidated, though the clinical significance of this finding has yet to be established.Despite the clinical progress that has been made there is still a need for large, prospective randomized studies with TRPV2 channel agonists and antagonists in order to bring these basic and translational science findings to the bedside.

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.

Capsaicin and Its Analogues Impede Nocifensive Response of Caenorhabditis elegans to Noxious Heat.

Capsaicin is the most abundant pungent molecule identified in red chili peppers, and it is widely used for food flavoring, in pepper spray for self-defense devices and recently in ointments for the relief of neuropathic pain. Capsaicin and several other related vanilloid compounds are secondary plant metabolites. Capsaicin is a selective agonist of the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). After exposition to vanilloid solution, Caenorhabditis elegans wild type (N2) and mutants were placed on petri dishes divided in quadrants for heat stimulation. Thermal avoidance index was used to phenotype each tested C. elegans experimental groups. The data revealed for the first-time that capsaicin can impede nocifensive response of C. elegans to noxious heat (32-35 °C) following a sustained exposition. The effect was reversed 6 h post capsaicin exposition. Additionally, we identified the capsaicin target, the C. elegans transient receptor potential channel OCR-2 and not OSM-9. Further experiments also undoubtedly revealed anti-nociceptive effect for capsaicin analogues, including olvanil, gingerol, shogaol and curcumin.

Immunomodulatory and anti-oxidative effect of the direct TRPV1 receptor agonist capsaicin on Schwann cells

BackgroundOnly few studies describe the impact of nutritive factors on chronic inflammatory demyelinating polyneuropathy (CIDP), an inflammatory disease of the peripheral nervous system. The active component of chili pepper, capsaicin, is the direct agonist of the transient receptor potential channel vanilloid subfamily member 1. Its anti-inflammatory effect in the animal model experimental autoimmune neuritis (EAN) has been previously demonstrated.MethodsIn the present study, we describe the anti-inflammatory and anti-oxidative influence of capsaicin on Schwann cells (SCs) in an in vitro setting. Hereby, we analyze the effect of capsaicin on Schwann cells’ gene expression pattern, major histocompatibility complex class II (MHC-II) presentation, and H2O2-induced oxidative stress. Furthermore, the effect of capsaicin on myelination was examined in a SC-dorsal root ganglia (DRG) coculture by myelin basic protein staining. Finally, in order to investigate the isolated effect of capsaicin on SCs in EAN pathology, we transplant naïve and capsaicin pre-treated SCs intrathecally in EAN immunized rats and analyzed clinical presentation, electrophysiological parameters, and cytokine expression in the sciatic nerve.ResultsIn SC monoculture, incubation with capsaicin significantly reduces interferon gamma-induced MHC-II production as well as toll-like receptor 4 and intercellular adhesion molecule 1 mRNA expression. Calcitonin gene-related peptide mRNA production is significantly upregulated after capsaicin treatment. Capsaicin reduces H2O2-induced oxidative stress in SC in a preventive, but not therapeutic setting. In a SC-DRG coculture, capsaicin does not affect myelination rate. After intrathecal transplantation of naïve and capsaicin pre-treated SCs in EAN-immunized rats, naïve, but not capsaicin pre-treated intrathecal SCs, ameliorated EAN pathology in rats.ConclusionsIn conclusion, we were able to demonstrate a direct immunomodulatory and anti-oxidative effect of capsaicin in a SC culture by reduced antigen presentation and expression of an anti-inflammatory profile. Furthermore, capsaicin increases the resistance of SCs against oxidative stress. A primary effect of capsaicin on myelination was not proven. These results are in concordance with previous data showing an anti-inflammatory effect of capsaicin, which might be highly relevant for CIDP patients.

TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation.

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography, and gallstones. In the pancreas, excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis, ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel Piezo1 in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force was insufficient to activate these pathological changes, whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium, leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered transient receptor potential vanilloid subfamily 4 (TRPV4) channel opening, which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene-KO mice were protected from Piezo1 agonist- and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which a Piezo1-induced TRPV4 channel opening causes pancreatitis.

TRPV4 helps Piezo1 put the squeeze on pancreatic acinar cells.

Alterations in calcium signaling in pancreatic acinar cells can result in pancreatitis. Although pressure changes in the pancreas can elevate cytosolic calcium (Ca2+) levels, it is not known how transient pressure-activated elevations in calcium can cause prolonged calcium changes and consequent pancreatitis. In this issue of the JCI, Swain et al. describe roles for the mechanically activated plasma membrane calcium channels Piezo1 and transient receptor potential vanilloid subfamily 4 (TRPV4) in acinar cells. The authors used genetic deletion models and cell culture systems to investigate calcium signaling. Notably, activation of the Piezo1-dependent TRPV4 pathway was independent of the cholecystokinin (CCK) stimulation pathway. These results elegantly resolve an apparent discrepancy in calcium signaling and the pathogenesis of pancreatitis in pancreatic acinar cells.

Identification and functional characterization of a biflavone as a novel inhibitor of TRPV4‐dependent proatherogenic processes in macrophages

Atherosclerosis, a chronic inflammatory disease, is the major cause of mortality and morbidity in the United States. Studies suggest that arterial stiffness is a marker and a risk factor for atherosclerosis. Recent reports from our laboratory showed that TRPV4 (transient receptor potential channel of the vanilloid subfamily 4), a mechanosensitive ion channel, plays a role in oxidized LDL (oxLDL)‐induced macrophage foam cell formation, a critical process in atherogenesis. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 natural compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay. We identified ginkgetin, a biflavone, as a novel small molecule chemical inhibitor of TRPV4. Primary and secondary screening was performed with mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐dependent Ca2+ influx. In our hand’s, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐dependent Ca2+ influx. Furthermore, we found that antagonism of TRPV4 by ginkgetin blocks oxLDL‐induced macrophage foam cell formation. Moreover, we found that ginkgetin treatment 1) attenuated oxLDL uptake but not its cell surface binding in BMDMs, 2) inhibited lipopolysaccharide‐induced phosphorylation of JNK, and 3) inhibited oxLDL‐induced expression of proinflammatory cytokines. Taken together, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated proatherogenic processes in macrophages.Support or Funding InformationThis work was supported by NIH (1R01EB024556‐01) and NSF (CMMI‐1662776) grants to Shaik O. Rahaman.

Structure and function of the calcium-selective TRP channel TRPV6.

Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca2+ ; it shows constitutive activity regulated negatively by Ca2+ and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca2+ selectivity of TRPV6 originates from the narrow selectivity filter, where Ca2+ ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca2+ and Ba2+ permeate TRPV6 pore according to the knock-off mechanism, while tight binding of Gd3+ to the aspartate ring blocks the channel and prevents Na+ from permeating the pore. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2-aminoethoxydiphenyl borate (2-APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helical bundle and shifts open-closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca2+ inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca2+ -dependent inactivation. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM C-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.

Lithocholic acid increases intestinal phosphate and calcium absorption in a vitamin D receptor dependent but transcellular pathway independent manner

Phosphate/calcium homeostasis is crucial for health maintenance. Lithocholic acid, a bile acid produced by intestinal bacteria, is an agonist of vitamin D receptor. However, its effects on phosphate/calcium homeostasis remain unclear. Here, we demonstrated that lithocholic acid increases intestinal phosphate/calcium absorption in an enterocyte vitamin D receptor-dependent manner. Lithocholic acid was found to increase serum phosphate/calcium levels and thus to exacerbate vascular calcification in animals with chronic kidney disease. Lithocholic acid did not affect levels of intestinal sodium-dependent phosphate transport protein 2b, Pi transporter-1, -2, or transient receptor potential vanilloid subfamily member 6. Everted gut sac analyses demonstrated that lithocholic acid increased phosphate/calcium absorption in a transcellular pathway-independent manner. Lithocholic acid suppressed intestinal mucosal claudin 3 and occludin in wild-type mice, but not in vitamin D receptor knockout mice. Everted gut sacs of claudin 3 knockout mice showed an increased permeability for phosphate, but not calcium. In patients with chronic kidney disease, serum 1,25(OH)2 vitamin D levels are decreased, probably as an intrinsic adjustment to reduce phosphate/calcium burden. In contrast, serum and fecal lithocholic acid levels and fecal levels of bile acid 7α-dehydratase, a rate-limiting enzyme involved in lithocholic acid production, were not downregulated. The effects of lithocholic acid were eliminated by bile acid adsorptive resin in mice. Thus, lithocholic acid and claudin 3 may represent novel therapeutic targets for reducing phosphate burden.

A TRPV1 antagonist, PAC-14028 does not increase the risk of tumorigenesis in chemically induced mouse skin carcinogenesis

PAC-14028 (Asivatrep: C21H22F5N3O3S) cream is a novel, topical nonsteroidal, anti-inflammatory, and TRPV1 (transient receptor potential vanilloid subfamily, member 1) antagonist for the treatment of mild to moderate atopic dermatitis. Concerns about the risk of tumor development by TRPV1 blockade in the skin have been prompted, but these findings were proved to be indirect or are still controversial. This study was tested to determine whether TRPV1 selective antagonist, PAC-14028 cream is safe from the promotion of skin tumorigenesis in the two-stage carcinogenesis model. PAC-14028 cream, 0.25%, 0.5%, or 1.0% was applied once daily topically to mouse skin for up to 24 weeks in two-stage chemical carcinogenesis testing using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Morbidity/death, clinical signs, tumor formation, activity of EGFR/Akt/mTOR signaling, and systemic exposure to PAC-14028 were investigated. Daily dermal administration of PAC-14028, was not skin carcinogenic. There was also no evidence on the activation of EGFR/Akt/mTOR signaling pathway by the topical treatment of PAC-14028. On Day 169, 1.0% (20 mg/kg/day) of PAC-14028 in female mice resulted in a Cmax and AUC0-τ of 12916.0 ng/mL and 78962.9 ng‧hr/mL, respectively. PAC-14028 cream was well tolerated and did not increase the risk of skin tumorigenesis in two-stage carcinogenesis study.

Structural Insights on TRPV2 Gating by Exogenous Modulators

Transient receptor potential vanilloid 2 (TRPV2) belongs to the transient receptor potential vanilloid (TRPV) subfamily that consists of six members (TRPV1-6). TRPV2 has been proposed to play a critical role in a variety of physiological and pathophysiological processes. These findings placed TRPV2 on the list of important drug targets, but the molecular details of the mechanism underlying TRPV2 gating is not fully understood. TRPV2 is insensitive to vanilloids but can be activated by cannabidiol, 2-aminoethoxydiphenyl borate and probenecid. In addition, tranilast, an antiallergic drug, has been used in several reports as a TRPV2 antagonist. Here we present structures of TRPV2 in apo and ligand-bound forms resolved by cryo-electron microscopy and propose the molecular mechanism of TRPV2 channel activation and inhibition by synthetic molecules. This newly obtained information could guide us towards the design of novel TRPV2 specific therapeutic molecules.