Transient Receptor Potential Vanilloid Member Research Articles

TRPV4 Antagonist Treatment of Hydrocephalus Causes Cell and Molecular Changes in Multiple Brain Cell Types

Hydrocephalus is defined by cerebrospinal fluid (CSF) accumulation in the brain’s ventricles causing ventriculomegaly, an imbalance in CSF secretion, flow, and/or absorption. Treatments for hydrocephalus include invasive surgical procedures, which commonly fail throughout a patient’s life. Transient receptor potential vanilloid member 4 (TRPV4) is a mechanosensitive cation channel implicated in osmotic regulation. Our laboratory found that a TRPV4 antagonist, RN1734, ameliorates hydrocephalus in a rat model of congenital hydrocephalus. It was hypothesized that treatment slowed production of CSF by targeting choroid plexus (CP), a contiguous layer of tightly regulated epithelial cells, that is responsible for CSF production. However, hydrocephalus pathology can have various effects on the brain. This study focuses on changes occurring in CP epithelial cells (CPe) and glia, which have roles in brain-barrier maintenance and brain fluid/electrolyte regulation. Interestingly, CPe and glia contain many of the same channels and transporters, including TRPV4, that are important in regulating both CSF and brain interstitial fluid. These cells contain aquaporins (AQP), AQP1 in CPe, and AQP4 in glia. AQPs have been implicated in diseases associated with fluid regulation and may interact with TRPV4. We hypothesize that TRPV4 and AQPs are important in hydrocephalus pathology, and that changes may be attenuated with RN1734 treatment. A point mutation in TMEM67 results in hydrocephalus (hydro). Changes in cell morphology, gene transcription, protein expression, localization of TRPV4, AQP1, and AQP4 were observed. Postnatal day 15 (P15) untreated (unt) and RN1734-treated (RN;2mg/ml DMSO; 4mg/kg intraperitoneal injection daily P7-P14) wildtype and mutant ( Tmem67−/−) rats were utilized in this study. CPe and periventricular (PV) glia were visualized using fluorescent immunohistochemistry (IHC) of GFAP and a plasma membrane marker. Cell volume of CPe’s and process morphology of glia were altered in hydro rats. RN returned these changes to relatively normal in CPe, but not glia. Next, we used IHC to examine localization of TRPV4 and the AQPs. Increased immunoreactivity of TRPV4 and AQP1 was observed in CPe of unt and RN Tmem67−/− rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and PV white matter of hydro rats. TRPV4 immunoreactivity, but not AQP4, was normalized with RN. We then utilized RTqPCR to inspect transcriptional changes. The data showed increased AQP1 and decreased TRPV4 mRNA in PV cortex and CP of unt and RN Tmem67−/− rats. Previous data from our laboratory found no change in TRPV4 protein in unt Tmem67−/− CPe, but we were curious about AQP expression. The amount of AQP1 protein was increased in the CP of unt and RN Tmem67−/− rats. In PV cortex, AQP4, but not TRPV4, protein was increased in hydro rats. With RN, AQP4 protein was not normalized. In summary, TRPV4 changes in the Tmem67−/− rats returned to normal with RN, glial morphology and AQP changes did not. This may indicate residual inflammation in the Tmem67−/− rats, which we aim to address in future studies. To further elucidate TRPV4’s role in hydrocephalus pathology, future studies will explore post-translational and membrane localization changes. Overall, investigating cell and molecular changes that occur in hydrocephalus will help find better pharmacological targets for this disease. Funding: United States Department of Defense Congressionally Directed Medical Research Program Award; Hydrocephalus Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Localization of sensory nerve terminals containing calcitonin gene-related peptide (CGRP) on striated muscle fibers in the rat esophagus: Evidence for triple innervation via motor endplates

BackgroundMany esophageal striated muscles of mammals are dually innervated by the vagal and enteric nerves. Recently, substance P (SP)-sensory nerve terminals with calcitonin gene-related peptide (CGRP) were found on a few striated muscle fibers in the rat esophagus, implying that these muscle fibers are triply innervated. In this study, we examined the localization and origin of CGRP-nerve endings in striated muscles to consider their possible roles in the esophagus regarding triple innervation. MethodsWholemounts of the rat esophagus were immunolabeled to detect CGRP-nerve endings in striated muscles. Also, retrograde tracing was performed by injecting Fast Blue (FB) into the esophagus, and cryostat sections of the medulla oblongata, nodose ganglion (NG), and the tenth thoracic (T10) dorsal root ganglion (DRG) were immunostained to identify the origin of the CGRP-nerve endings. ResultsCGRP-fine, varicose nerve endings were localized in motor endplates on a few esophageal striated muscle fibers (4 %), most of which received nitric oxide (NO) synthase nerve terminals, and most of the CGRP nerve endings were SP- and transient receptor potential vanilloid member 1 (TRPV1)-positive. Retrograde tracing showed many FB-labeled CGRP-neurons positive for SP and TRPV1 in the NG and T10 DGR. ConclusionsThis study suggests that the CGRP-varicose nerve endings containing SP and TRPV1 in motor endplates are sensory, and a few esophageal striated muscle fibers are triply innervated. The nerve endings may detect acetylcholine-derived acetic acid from the vagal motor nerve endings and NO from esophageal intrinsic nerve terminals in the motor endplates to regulate esophageal motility.

Remote Manipulation of TRPV1 Signaling by Near-Infrared Light-Triggered Nitric Oxide Nanogenerators for Specific Cancer Therapy.

Specific activation of transient receptor potential vanilloid member 1 (TRPV1) channels provides a new avenue for cancer treatment by inducing excessive Ca2+ influx. However, controllable manipulation of TRPV1 signaling for clinical application has remained elusive due to the challenge in finding a mild and effective method of exerting external stimulus without adverse side effects in living systems. Herein, a TRPV1-targeting near-infrared (NIR) triggered nitric oxide (NO)-releasing nanoplatform (HCuS@PDA-TRPV1/BNN6) based on polydopamine (PDA) coated hollow copper sulfide nanoparticles (HCuS NPs) is developed for specific cancer therapy. Upon NIR irradiation, the NO donor BNN6 encapsulated in NIR-responsive nanovehicles can locally generate NO to activate TRPV1 channels and induce Ca2+ influx. This NIR controlled mode enables the nanoplatform to exert its therapeutic effects below the apoptotic threshold temperature (43°C), minimizing the photothermal damage to normal tissue. Integrating this special NO-mediated therapy with HCuS NPs mediated chemodynamic therapy, the designed nanoplatform exhibits a boosted anticancer activity with negligible systematic toxicity. Together, this study provides a promising strategy for site-specific cancer therapy by spatiotemporally controlled activation of surface ion channels, thus offering a solution to an unmet clinical need in cancer treatment.

TRPV4‐mediated Ca2+ deregulation causes mitochondrial dysfunction via the AKT/α‐synuclein pathway in dopaminergic neurons

AbstractMutations in the gene encoding the transient receptor potential vanilloid member 4 (TRPV4), a Ca2+ permeable nonselective cation channel, cause TRPV4‐related disorders. TRPV4 is widely expressed in the brain; however, the pathogenesis underlying TRPV4‐mediated Ca2+ deregulation in neurodevelopment remains unresolved and an effective therapeutic strategy remains to be established. These issues were addressed by isolating mutant dental pulp stem cells from a tooth donated by a child diagnosed with metatropic dysplasia with neurodevelopmental comorbidities caused by a gain‐of‐function TRPV4 mutation, c.1855C > T (p.L619F). The mutation was repaired using CRISPR/Cas9 to generate corrected isogenic stem cells. These stem cells were differentiated into dopaminergic neurons and the pharmacological effects of folic acid were examined. In mutant neurons, constitutively elevated cytosolic Ca2+ augmented AKT‐mediated α‐synuclein (α‐syn) induction, resulting in mitochondrial Ca2+ accumulation and dysfunction. The TRPV4 antagonist, AKT inhibitor, or α‐syn knockdown, normalizes the mitochondrial Ca2+ levels in mutant neurons, suggesting the importance of mutant TRPV4/Ca2+/AKT‐induced α‐syn in mitochondrial Ca2+ accumulation. Folic acid was effective in normalizing mitochondrial Ca2+ levels via the transcriptional repression of α‐syn and improving mitochondrial reactive oxygen species levels, adenosine triphosphate synthesis, and neurite outgrowth of mutant neurons. This study provides new insights into the neuropathological mechanisms underlying TRPV4‐related disorders and related therapeutic strategies.

Phenolic Compounds and Capsaicinoids in Three Capsicum annuum Varieties: From Analytical Characterization to In Silico Hypotheses on Biological Activity.

The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.

Research focus and thematic trends of transient receptor potential vanilloid member 1 research: a bibliometric analysis of the global publications (1990-2023).

Recently, various studies have been devoted to the study of transient receptor potential vanilloid member 1 (TRPV1)-related diseases, potential drugs, and related mechanisms. The objective of this investigation was to examine the significant areas and cutting-edge developments in TRPV1 study within recent decades. Articles or reviews were obtained from the Web of Science Core Collection. VOSviewer 1.6.18 and CiteSpace 6.1 R2 software were utilized to examine publication growth, distribution by country/region, institution, journal, authorship, references, and keywords. The software identified keywords with a high citation burstiness to determine emerging topics. From 1990 to 2023, the annual global publications increased by 62,000%, from 1 to 621. Journal of neuroscience published the most manuscripts and Nature produced the highest citations. The USA, Seoul National University and Di marzo V were the most productive and impactful institution, country, and author, respectively. "TRPV1," "Capsaicin receptor," "Activation," and "Pain" are the most important keywords. The burst keywords "TRPV1 channel," "Oxidative stress," "TRPV1 structure," and "Cancer" are supposed to be the research frontiers. The present study offers valuable insights into the understanding of TRPV1 and pain-related conditions. The research on TRPV1 has demonstrated a steady increase in studies related to pain-related diseases in the past few decades. The significance of TRPV1 in cancer pathogenesis and the resolution of its structure will emerge as a new academic trend in this field, providing direction for more widespread and comprehensive studies in the future.

FRET analysis of the temperature-induced structural changes in human TRPV3

Transient receptor potential vanilloid member 3 (TRPV3) is an ion channel that plays a critical role in temperature sensing in skin. There have been active studies on how TRPV3, which is also known as one of the temperature-sensitive transient receptor potential (thermoTRP) channels, responds to temperature. However, the previous studies were mostly based on TRPV3 originating from mice or rats. Here, we focus on human TRPV3 (hTRPV3) and show that which domain of hTRPV3 undergoes conformational changes as temperature increases by Förster resonance energy transfer (FRET) assay. During the heat-induced activation of hTRPV3, the linker domain close to C-terminus, that is, the C-terminal domain shows a largest structural change whereas there is little change in the ankyrin repeat domain (ARD). Interestingly, the activation of hTRPV3 by an agonist shows structural change patterns that are completely different from those observed during activation by heat; we observe structural changes in ARD and S2–S3 linker after ligand stimulation whereas relatively little change is observed when stimulated by heat. Our results provide insight into the thermal activation of hTRPV3 channel.

Nanoparticle-mediated TRPV1 channel blockade amplifies cancer thermo-immunotherapy via heat shock factor 1 modulation

The survival of malignant tumors is highly dependent on their intrinsic self-defense pathways such as heat shock protein (HSP) during cancer therapy. However, precisely dismantling self-defenses to amplify antitumor potency remains unexplored. Herein, we demonstrate that nanoparticle-mediated transient receptor potential vanilloid member 1 (TRPV1) channel blockade potentiates thermo-immunotherapy via suppressing heat shock factor 1 (HSF1)-mediated dual self-defense pathways. TRPV1 blockade inhibits hyperthermia-induced calcium influx and subsequent nuclear translocation of HSF1, which selectively suppresses stressfully overexpressed HSP70 for enhancing thermotherapeutic efficacy against a variety of primary, metastatic and recurrent tumor models. Particularly, the suppression of HSF1 translocation further restrains the transforming growth factor β (TGFβ) pathway to degrade the tumor stroma, which improves the infiltration of antitumor therapeutics (e.g. anti-PD-L1 antibody) and immune cells into highly fibrotic and immunosuppressive pancreatic cancers. As a result, TRPV1 blockade retrieves thermo-immunotherapy with tumor-eradicable and immune memory effects. The nanoparticle-mediated TRPV1 blockade represents as an effective approach to dismantle self-defenses for potent cancer therapy.

Association of TRPV5, CASR, and CALCR genetic variants with kidney stone disease susceptibility in Egyptians through main effects and gene–gene interactions

Kidney stone disease (KSD) represents an urgent medical problem because of increasing its prevalence. Several functional polymorphisms in genes involved in the renal handling of calcium were associated with KSD pathogenesis. Among those, the rs4236480 of transient receptor potential vanilloid member 5 (TRPV5) gene, the rs1801725 of calcium-sensing receptor (CASR) gene, and the rs1801197 of calcitonin receptor (CALCR) gene appear to be of great importance. Due to the scarce data on the Egyptians, this study aimed to evaluate the association of these candidate genetic variants with the risk of developing KSD in an Egyptian population. To do so, the biochemical parameters were measured along with the genotyping of the three polymorphisms using allelic discrimination assay in 134 KSD patients and 86 age and sex-matched healthy subjects. The results showed that the genotypic distributions and allelic frequencies of the studied variants were significantly different between cases and controls. The three polymorphisms increased the risk of KSD significantly under all the tested genetic models (OR ranges from 2.152 to 5.994), except for the recessive model of the CALCR rs1801197 polymorphism after Bonferroni correction. The gene–gene interaction analyzed by multifactor dimensionality reduction selected the three-locus combination as the best model associated with the susceptibility to KSD with OR 9.706. Further, synergistic interactions were identified between TRPV5 rs4236480 and CALCR rs1801197 variants and CASR rs1801725 and CALCR rs1801197 variants. In conclusion, the TRPV5 rs4236480, CASR rs1801725, and CALCR rs1801197 polymorphisms showed a significant association with the risk of KSD in the Egyptian population. Furthermore, their complex interactions might have an impact on the genetic susceptibility to develop KSD.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein–umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Yellow Chaste Weed and Its Components, Apigenin and Galangin, Affect Proliferation and Oxidative Stress in Blue Light-Irradiated HaCaT Cells.

While harmful effects of blue light on skin cells have been recently reported, there are few studies regarding natural products that alleviate its negative effects. Therefore, we investigated ameliorating effects of yellow chaste weed (YCW) (Helichrysum arenarium) extract and its components, apigenin and galangin, on blue light-irradiated HaCaT cells. In this study, we found that YCW extract improved the reduced proliferation of HaCaT cells induced by blue light-irradiation and reduced blue light-induced production of reactive oxygen species (ROS) levels. We also found that apigenin and galangin, the main components of YCW extract, showed the same activities as YCW extract. In experiments examining molecular mechanisms of YCW extract and its components such as apigenin and galangin, they all reduced expression of transient receptor potential vanilloid member 1 (TRPV1), its phosphorylation, and calcium ion (Ca2+) influx induced by blue light irradiation. In addition, apigenin and galangin regulated phosphorylation of mitogen-activated protein kinases (MAPKs). They also reduced phosphorylation of mammalian sterile 20-like kinase-1/2 (MST-1/2), inducing phosphorylation of Akt (protein kinase B), one downstream molecule of MST-1/2. Moreover, apigenin and galangin promoted translocation of Forkhead box O3 (FoxO3a) from the nucleus to the cytosol by phosphorylating FoxO3a. Besides, apigenin and galangin interrupted blue light influences on expression of nuclear and secretory clusterin. Namely, they attenuated both upregulation of nuclear clusterin and downregulation of secretory clusterin induced by blue light irradiation. We also found that they downregulated apoptotic protein Bcl-2 associated X protein (Bax) and conversely upregulated anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). Collectively, these findings indicate that YCW extract and its components, apigenin and galangin, antagonize the blue light-induced damage to the keratinocytes by regulating TRPV1/clusterin/FoxO3a and MAPK signaling.

Heat-dependent opening of TRPV1 in the presence of capsaicin

Noxious heat (> 42°C) and capsaicin are two well-known stimuli for nociception. Our ability to sense noxious heat and spiciness is owed to the transient receptor potential vanilloid member 1 (TRPV1), a calcium-permeable ion channel expressed in primary sensory nerve terminals. TRPV1 also serves as a sensor for other vanilloid compounds and is a member of the TRP channel superfamily. Here we present six high-resolution cryo-EM structures of apo and capsaicin-bound full-length TRPV1 reconstituted into lipid nanodiscs over a range of temperatures.

Capsaicin suppresses interleukin-31-induced itching partially involved in inhibiting the expression of dorsal root ganglion interleukin-31 receptor A in male mice

To elucidate the mechanisms underlying the antipruritic effect of capsaicin, we investigated how topical application of capsaicin (0.01, 0.1 and 1.0% w/v) affects spontaneous scratching in NC/Nga mice, inerleukin-31 (IL-31) induced in BALB/c mice, and IL-31 receptor A (IL-31RA) and transient receptor potential vanilloid member 1 (TRPV1) mRNA expression in dorsal root ganglia (DRG). Capsaicin concentration-dependently suppressed long-lasting scratching (over 1.0 s, itch-associated scratching) and short-lasting scratching (0.3–1.0 s, locomotor activity) immediately after the application. Total long-lasting scratching and short-lasting scratching counts for 24 h and IL-31RA mRNA expression in the DRG significantly decreased with increasing concentration of capsaicin. Furthermore, 1.0% capsaicin suppressed long-lasting scratching and short-lasting scratching for more than 72 h. At this point, DRG IL-31RAmRNA was significantly decreased, but there was no change in cutaneous IL-31RA and TRPV1 mRNA. Thus capsaicin suppresses long-lasting scratching by inhibiting IL-31RA mRNA expression in the DRG. Next, we examined the effect of capsaicin on IL-31-induced long-lasting scratching in BALB/c mice. Repeated administration of IL-31 (50 μg/kg, subcutaneous) every 12 h for 3 days apparently increased long-lasting scratching counts and IL-31RA mRNA in the DRG. These increases were significantly suppressed by pretreatment with 1.0% capsaicin. TRPV1 mRNA in the DRG was also decreased within 1–24 h after capsaicin application. These results suggest that the strong and prolonged antipruritic action for IL-31-induced itching of capsaicin was caused by desensitization of C-fibers, and, in addition, the long-lasting inhibition of IL-31RA mRNA expression in the DRG.

Antioxidative and Analgesic Effects of Naringin through Selective Inhibition of Transient Receptor Potential Vanilloid Member 1.

Transient receptor potential vanilloid member 1 (TRPV1) is activated in response to capsaicin, protons, temperature, and free reactive oxygen species (ROS) released from inflammatory molecules after exposure to harmful stimuli. The expression level of TRPV1 is elevated in the dorsal root ganglion, and its activation through capsaicin and ROS mediates neuropathic pain in mice. Its expression is high in peripheral and central nervous systems. Although pain is a response evolved for survival, many studies have been conducted to develop analgesics, but no clear results have been reported. Here, we found that naringin selectively inhibited capsaicin-stimulated inward currents in Xenopus oocytes using a two-electrode voltage clamp. The results of this study showed that naringin has an IC50 value of 33.3 μM on TRPV1. The amino acid residues D471 and N628 of TRPV1 were involved in its binding to naringin. Our study bridged the gap between the pain suppression effect of TRPV1 and the preventive effect of naringin on neuropathic pain and oxidation. Naringin had the same characteristics as a model selective antagonist, which is claimed to be ideal for the development of analgesics targeting TRPV1. Thus, this study suggests the applicability of naringin as a novel analgesic candidate through antioxidative and analgesic effects of naringin.

Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor.

Background & AimsChronic abdominal pain is a common symptom of inflammatory bowel diseases (IBDs). Peripheral and central mechanisms contribute to the transition from acute to chronic pain during active disease and clinical remission. Lower mechanical threshold and hyperexcitability of visceral afferents induce gliosis in central pain circuits, leading to persistent visceral hypersensitivity (VHS). In the spinal cord, microglia, the immune sentinels of the central nervous system, undergo activation in multiple models of VHS. Here, we investigated the mechanisms of microglia activation to identify centrally acting analgesics for chronic IBD pain.MethodsUsing Designer Receptors Exclusively Activated by Designer Drugs (DREADD) expressed in transient receptor potential vanilloid member 1-expressing visceral neurons that sense colonic inflammation, we tested whether neuronal activity was indispensable to control microglia activation and VHS. We then investigated the neuron-microglia signaling system involved in visceral pain chronification.ResultsWe found that chemogenetic inhibition of transient receptor potential vanilloid member 1+ visceral afferents prevents microglial activation in the spinal cord and subsequent VHS in colitis mice. In contrast, chemogenetic activation, in the absence of colitis, enhanced microglial activation associated with VHS. We identified a purinergic signaling mechanism mediated by neuronal adenosine triphosphate (ATP) and microglial P2Y12 receptor, triggering VHS in colitis. Inhibition of P2RY12 prevented microglial reactivity and chronic VHS post-colitis.ConclusionsOverall, these data provide novel insights into the central mechanisms of chronic visceral pain and suggest that targeting microglial P2RY12 signaling could be harnessed to relieve pain in patients with IBD who are in remission.

Heat-dependent opening of TRPV1 in the presence of capsaicin.

Transient receptor potential vanilloid member 1 (TRPV1) is a Ca2+-permeable cation channel that serves as the primary heat and capsaicin sensor in humans. Using cryo-EM, we have determined the structures of apo and capsaicin-bound full-length rat TRPV1 reconstituted into lipid nanodiscs over a range of temperatures. This has allowed us to visualize the noxious heat-induced opening of TRPV1 in the presence of capsaicin. Notably, noxious heat-dependent TRPV1 opening comprises stepwise conformational transitions. Global conformational changes across multiple subdomains of TRPV1 are followed by the rearrangement of the outer pore, leading to gate opening. Solvent-accessible surface area analyses and functional studies suggest that a subset of residues form an interaction network that is directly involved in heat sensing. Our study provides a glimpse of the molecular principles underlying noxious physical and chemical stimuli sensing by TRPV1, which can be extended to other thermal sensing ion channels.

Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs.

Ca2+ influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2, to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double-knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and, following sperm entry or Plcz (also known as Plcz1) cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wild-type eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting that the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also showed that they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

Impaired neurite development and mitochondrial dysfunction associated with calcium accumulation in dopaminergic neurons differentiated from the dental pulp stem cells of a patient with metatropic dysplasia

Transient receptor potential vanilloid member 4 (TRPV4) is a Ca2+ permeable nonselective cation channel, and mutations in the TRPV4 gene cause congenital skeletal dysplasias and peripheral neuropathies. Although TRPV4 is widely expressed in the brain, few studies have assessed the pathogenesis of TRPV4 mutations in the brain. We aimed to elucidate the pathological associations between a specific TRPV4 mutation and neurodevelopmental defects using dopaminergic neurons (DNs) differentiated from dental pulp stem cells (DPSCs). DPSCs were isolated from a patient with metatropic dysplasia and multiple neuropsychiatric symptoms caused by a gain-of-function TRPV4 mutation, c.1855C>T (p.L619F). The mutation was corrected by CRISPR/Cas9 to obtain isogenic control DPSCs. Mutant DPSCs differentiated into DNs without undergoing apoptosis; however, neurite development was significantly impaired in mutant vs. control DNs. Mutant DNs also showed accumulation of mitochondrial Ca2+ and reactive oxygen species, low adenosine triphosphate levels despite a high mitochondrial membrane potential, and lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial content. These results suggested that the persistent Ca2+ entry through the constitutively activated TRPV4 might perturb the adaptive coordination of multiple mitochondrial functions, including oxidative phosphorylation, redox control, and biogenesis, required for dopaminergic circuit development in the brain. Thus, certain mutations in TRPV4 that are associated with skeletal dysplasia might have pathogenic effects on brain development, and mitochondria might be a potential therapeutic target to alleviate the neuropsychiatric symptoms of TRPV4-related diseases.

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

Activities of Ca2+-related ion channels during the formation of kidney stones in an infection-induced urolithiasis rat model.

Bacterial infection has long been recognized to contribute to struvite urinary stone deposition; however, its contribution to the development of chronic kidney stones has not been extensively investigated. In the present study, we hypothesized another possible method of bacteria contributing to the formation of calcium oxalate (CaOx) that accounts for the biggest part of the kidney stone. Bacteria may play important roles by influencing renal Ca2+-related ion channel activities, resulting in chronic inflammation of the kidney along with rapid aggregation of stones. We examined the correlation among infection-promoted CaOx kidney stones and alterations in Ca2+-related ion channels in an animal model with experimentally induced Proteus mirabilis and foreign body infection. After the bladder was infected for 7 days, the data demonstrated that stones were presented and induced severe renal tubular breakage as well as altered levels of monocyte chemoattractant protein-1, cyclooxygenase-2, osteopontin, and transient receptor potential vanilloid member 5 expression, reflecting responses of kidney ion channels. Monocyte chemoattractant protein-1, osteopontin, and transient receptor potential vanilloid member 5 expression was significantly downregulated over time, indicating the chronic inflammation phase of the kidney and accelerated aggregation of CaOx crystals, respectively, whereas cyclooxygenase-2 exhibited no differences. These results indicated that bacterial infection is considerably correlated with an alteration in renal Ca2+-related ion channels and might support specific and targeted Ca2+-related ion channel-based therapeutics for urolithiasis and related inflammatory renal damage.