Impaired autophagy from TRPV4 activation drives α-synuclein pathology in a Parkinson's disease model: A toxicological insight.

Transient Receptor Potential Vanilloid-4 Has a Major Role in Visceral Hypersensitivity Symptoms

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca(2+) concentration and ATP release. Stretch stimulation evoked intracellular Ca(2+) increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca(2+) increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca(2+) increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca(2+). These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the transient receptor potential channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating the activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate or by application of bradykinin, which activates PKC via a G-protein-coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, bisindolylmaleimide I, and rottlerin or by mutation of the serine/threonine residues Ser(162), Thr(175), and Ser(189). The adenylate cyclase activator forskolin also enhanced activation of TRPV4, and the enhancement was antagonized by the selective cyclic AMP-dependent protein kinase (PKA) inhibitor H89 or by mutation of serine residue Ser(824). Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using small interfering RNA. We conclude that the serine/threonine kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Transient receptor potential cation channels of the vanilloid subfamily (TRPV) participate in the generation of Ca(2+) signals at different locations of the respiratory system, thereby controlling its correct functioning. TRPV1 expression and activity appear to be altered under pathophysiological conditions such as chronic cough and airway hypersensitivity, whereas TRPV4 single nucleotide polymorphisms (SNP) are associated with chronic obstructive pulmonary disease. However, to date, there is no information about the genetic impact of either TRPV1 or TRPV4 on asthma pathophysiology. We now report on the association of two functional SNPs, TRPV1-I585V and TRPV4-P19S, with childhood asthma. Both SNPs were genotyped in a population of 470 controls without respiratory symptoms and 301 asthmatics. Although none of the SNPs modified the risk of suffering from asthma, carriers of the TRPV1-I585V genetic variant showed a lower risk of current wheezing (odds ratio = 0.51; p = 0.01), a characteristic of active asthma, or cough (odds ratio = 0.57; p = 0.02). Functional analysis of TRPV1-I585V, using the Ca(2+)-sensitive dye fura-2 to measure intracellular [Ca(2+)] concentrations, revealed a decreased channel activity in response to two typical TRPV1 stimuli, heat and capsaicin. On the other hand, TRPV4-P19S, despite its loss-of-channel function, showed no significant association with asthma or the presence of wheezing. Our data suggest that genetically determined level of TRPV1 activity is relevant for asthma pathophysiology.

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder often accompanied by dementia in its advanced stages. The pathogenesis of PD dementia involves abnormal autophagy and the accumulation of phosphorylated at serine 129 α-synuclein (pS129 α-syn). Proper functioning of the autophagy-lysosomal pathway (ALP) is essential for the effective degradation of pS129 α-syn. Our previous studies implicated the calcium channel transient receptor potential vanilloid 4 (TRPV4) in dopaminergic neuron degeneration by demonstrating that its overexpression induces endoplasmic reticulum stress and inflammation, driving neuronal loss-a hallmark of PD. This study aimed to investigate the role of TRPV4 in ALP-mediated pS129 α-syn clearance in the 1-methyl-4-phenylpyridinium ion (MPP+)-induced PC12 cells. We observed that MPP+ upregulated TRPV4 expression, reduced cell viability, and increased pS129 α-syn levels. Critically, all these effects were reversed by TRPV4 siRNA. Furthermore, TRPV4 siRNA restored cellular autophagic flux, which was impaired by MPP+. Treatment with either TRPV4 siRNA or TRPV4 special inhibitor HC067047 attenuated the MPP+-induced elevation of microtubule associated protein 1 light chain 3B (LC3B) and p62, while restoring the expression of lysosome-associated membrane protein 1 (LAMP1) and mature cathepsin D - key indicators of ALP functionality. These results suggest that TRPV4 silencing enhances α-syn degradation via the ALP, highlighting its potential as a therapeutic target for PD.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-26513-z.

Cannabidiol is an ultra potent sensitizer for 2-APB responses in rTRPV2 and mTRPV3 channels but not in rTRPV1 through a mechanism that engages channel regions further from the cannabidiol binding site and the pore.

Sodium salicylate (SA), a cyclooxygenase inhibitor, has been shown to increase insulin sensitivity and to suppress inflammation in obese patients and animal models. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed in afferent nerve fibers. Cyclooxygenase-derived prostaglandins are involved in the activation and sensitization of TRPV1. This study tested whether the metabolic and renal effects of SA were mediated by the TRPV1 channel. Wild-type (WT) and TRPV1−/− mice were fed a Western diet (WD) for 4 months and received SA infusion (120mg/kg/day) or vehicle for the last 4 weeks of WD feeding. SA treatment significantly increased blood pressure in WD-fed TRPV1−/− mice (p < 0.05) but not in WD-fed WT mice. Similarly, SA impaired renal blood flow in TRPV1−/− mice (p < 0.05) but not in WT mice. SA improved insulin and glucose tolerance in both WT and TRPV1−/− mice on WD (all p < 0.05). In addition, SA reduced renal p65 and urinary prostaglandin E2, prostaglandin F1α, and interleukin-6 in both WT and TRPV1−/− mice (all p < 0.05). SA decreased urine noradrenaline levels, increased afferent renal nerve activity, and improved baroreflex sensitivity in WT mice (all p < 0.05) but not in TRPV1−/− mice. Importantly, SA increased serum creatinine and urine kidney injury molecule-1 levels and decreased the glomerular filtration rate in obese WT mice (all p < 0.05), and these detrimental effects were significantly exacerbated in obese TRPV1−/− mice (all p < 0.05). Lastly, SA treatment increased urine albumin levels in TRPV1−/− mice (p < 0.05) but not in WT mice. Taken together, SA-elicited metabolic benefits and anti-inflammatory effects are independent of TRPV1, while SA-induced sympathetic suppression is dependent on TRPV1 channels. SA-induced renal dysfunction is dependent on intact TRPV1 channels. These findings suggest that SA needs to be cautiously used in patients with obesity or diabetes, as SA-induced renal dysfunction may be exacerbated due to impaired TRPV1 in obese and diabetic patients.

The TRPV4 channel is a calcium-permeable channel (PCa/PNa ∼ 10). Its expression has been reported in ventricular myocytes, where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4+/+ and trpv4-/- mice. TRPV4 membrane expression and its colocalization with L-type calcium channels (Cav1.2) was confirmed using Western blot biotinylation, immunoprecipitation, and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4-/- compared with trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD90) in trpv4+/+ but not in trpv4-/- myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased calcium transient (CaT) amplitude in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 carries an inward Ca2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD90 in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 prolongs AP duration in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accord with what was found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.NEW & NOTEWORTHY Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4-/- but not in trpv4-/- ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4-/- myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

Understanding the mechanism of oxytocin-induced uterine contractility is critical for addressing conditions at both extremes of the uterine contractility spectrum, preterm labour and uterine atony. We hypothesized that oxytocin induces extracellular calcium influx and uterine contraction through activation of the transient receptor potential vanilloid 4 (TRPV4) channel. To test this hypothesis, uterine tissue was obtained with informed consent from pregnant patients undergoing term, non-labouring caesarean delivery. In human myometrial tissue and smooth muscle cells in primary culture (mSMCs), TRPV4 and oxytocin receptor (OXTR) proteins colocalize at distances less than 40nm. In mSMCs, both pharmacological blockade of TRPV4 and TRPV4 depletion via small interfering RNA prevent oxytocin-induced calcium influx and contraction. In contrast, voltage-gated calcium channel blockade does not diminish oxytocin-induced calcium transients. Pharmacological blockade of OXTR has no effect on TRPV4 agonist-induced calcium influx or contractility. In uterine tissue from patients with oxytocin-resistant uterine atony, there is a marked reduction in glycosylated OXTR expression and in proximity ligation between OXTR and TRPV4 compared with tissue from control patients with optimal postpartum contractility. Taken together, these findings demonstrate that in the gravid uterine smooth muscle, TRPV4 activation is required for oxytocin-induced uterine contraction. They also suggest reduced OXTR-TRPV4 protein-protein interaction as a novel pathophysiological mechanism underlying uterine atony in non-labouring parturients. These findings highlight the physiological importance of oxytocin signalling via the TRPV4 channel and may motivate the development of targeted, TRPV4-focused treatments to modulate uterine contractility. KEY POINTS: Oxytocin-induced contraction in smooth muscle cells from term pregnant human myometrium requires activation of the TRPV4 calcium channel. TRPV4 and oxytocin receptor (OXTR) colocalize at <40nM and interact functionally in myometrial smooth muscle cells. TRPV4 antagonism or siRNA-mediated TRPV4 knockdown abolishes oxytocin-induced calcium influx and contractility. In patients with oxytocin-resistant uterine atony, glycosylated OXTR quantity and TRPV4-OXTR colocalization are markedly reduced. These findings identify TRPV4 as a critical mediator of uterine contractility. TRPV4 antagonists may have a role as novel therapeutic agents for preventing or treating preterm labour.

The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition invitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis.

The sacral dorsal commissural nucleus (SDCN) in the spinal cord receives both somatic and visceral primary afferents. Transient receptor potential vanilloid 1 (TRPV1) channels are preferentially expressed in certain fine primary afferents. However, knowledge of the SDCN neurons postnatal excitability development and their contacts with TRPV1 fibers remains elusive. Here, whole-cell recordings were conducted in spinal cord slices to evaluate the postnatal development of SDCN neurons and their possible contacts with functional TRPV1-expressing terminals. SDCN neurons in neonatal (postnatal day (P) 1-2), young (P8-10), and adult rats (P35-40) have different electrophysiological properties. SDCN neurons in neonatal rats have higher frequency of spontaneous firing, higher resting membrane potential, and lower presynaptic glutamate release probability. However, no difference in quantal release was found. At all developmental stages, TRPV1 activation with the selective agonist capsaicin increases glutamate release in the presence of tetrodotoxin, which blocks action potential-dependent and polysynaptic neurotransmission, indicating that functional TRPV1 fibers innervate SDCN neurons directly. Capsaicin-induced presynaptic glutamate release onto SDCN neurons depends on external Ca2+ influx through TRPV1 channels; voltage-dependent calcium channels had a slighter impact. In contrast, capsaicin blocked C fiber-evoked synaptic transmission, indicating that TRPV1 activation has opposite effects on spontaneous asynchronous and action potential-dependent synchronous glutamate release. These data indicate that excitability of SDCN neurons undergoes a developmental shift, and these neurons receive functional TRPV1 terminals from early postnatal stage. The opposite action of capsaicin on asynchronous and synchronous glutamate release should be taken into account when TRPV1 channels are considered as therapeutic targets.

Molecular and functional insights into Transient Receptor Potential Vanilloid 1 (TRPV1) in bull spermatozoa

BackgroundNoxious lifestyle factors including spicy diets and hot baths may lead to scar formation and recurrence. These phenomena are related to the activation of the transient receptor potential vanilloid-1 (TRPV1) cation channel. Our previous study revealed significant upregulation of TRPV1 expression in the dermis of hypertrophic scar (HS), while the exact underlying mechanism of TRPV1 activation in HS remains ill-defined. This study aims to clarify the contribution of TRPV1 activation to HS pathogenesis, particularly in relation to aberrant angiogenesis.MethodsFirst, this study employs single-cell RNA sequencing technology to analyze the association between vascular endothelial cells and the development of HS. Complementarily, bioinformatics analysis combined with histological validation is utilized to investigate the relationship between TRPV1 channels and aberrant angiogenesis within HS formation. Furthermore, the correlation between TRPV1 activation and HS phenotypes is rigorously validated at the in vivo level. In parallel, in vitro experiments are conducted to elucidate the impact of TRPV1 channel activation on the biological behaviors and functions of vascular endothelial cells. Subsequently, key downstream signaling pathways of TRPV1 are screened, and their molecular mechanisms in regulating vascular endothelial cell-mediated angiogenesis are systematically verified. Finally, a comprehensive analysis is performed to establish the clinical relevance of the TRPV1/nuclear factor kappa-B (NF-κB)/interleukin-6 (IL-6) axis with vascularization severity and adverse prognostic outcomes in hypertrophic scarring.ResultsSingle-cell RNA sequencing revealed significant cellular heterogeneity in vascular endothelial cells between normal skin and HS, indicating activated angiogenesis and substantial vascular endothelial cell alterations during HS development. Bulk RNA-seq and clinical analyses further confirmed this angiogenesis activation, demonstrating a close association with TRPV1 channel activation. In vivo studies established that capsaicin (CAP)-induced TRPV1 activation exacerbated HS progression through enhanced angiogenesis, whereas TRPV1 ablation or local inhibition markedly attenuated this effect. In vitro experiments demonstrated that TRPV1 activation regulated angiogenesis by promoting pro-angiogenic phenotypes. Transcriptomic analysis and functional validation identified the IL-6/signal transducer and activator of transcription 3 pathway as a downstream NF-κB-dependent pro-angiogenic axis mediated by TRPV1 in HS vascular endothelial cells. Critically, dermal overexpression of the TRPV1/NF-κB/IL-6 axis in HS patients correlated strongly with both disease severity and recurrence.ConclusionsHere, we show that the development of HS is strongly correlated with endothelial angiogenic activity. TRPV1 activation by CAP enhances proangiogenic processes including endothelial proliferation, migration, and tubule formation, while reducing apoptosis through the TRPV1/NF-κB/IL-6 axis. In a rabbit ear HS model, stimulation of TRPV1 contributes to the formation of HS via the TRPV1/NF-κB/IL-6 axis, whereas pharmacological ablation of TRPV1 significantly reversed these phenotypes. These findings shed light on the underlying molecular mechanisms and provide a potential therapeutic target for HS.