TRPV1 Channels Research Articles

Investigating the Impact of Estrogen Levels on Voiding Characteristics, Bladder Structure, and Related Proteins in a Mouse Model of Menopause-Induced Lower Urinary Tract Symptoms.

Lower urinary tract symptoms (LUTS) are common in postmenopausal women. These symptoms are often linked to decreased estrogen levels following menopause. This study investigated the relationship between estrogen levels, alterations in bladder tissue structure, bladder function, and the incidence of urinary frequency. An age-appropriate bilateral ovariectomized mouse model (OVX) was developed to simulate conditions of estrogen deficiency. Mice were divided into three groups: a sham-operated control group, OVX, and an estradiol-treated group. The assessments included estrogen level measurement, urination frequency, cystometry, histological analysis, immunofluorescence staining, and real-time quantitative PCR. Additionally, we quantified the expression of the mechanosensitive channel proteins Piezo1 and TRPV4 in mouse bladder tissues. Lower estrogen levels were linked to increased voiding episodes and structural changes in mouse bladder tissues, notably a significant increase in Collagen III fiber deposition. There was a detectable negative relationship between estrogen levels and the expression of Piezo1 and TRPV4, mechanosensitive proteins in mouse bladder tissues, which may influence voiding frequency and nocturia. Estrogen treatment could improve bladder function, decrease urination frequency, and reduce collagen deposition in the bladder tissues. This study explored the connection between estrogen levels and urinary frequency, potentially setting the stage for novel methods to address frequent urination symptoms in postmenopausal women.

20S-Ginsenoside Rh2, the major bioactive saponin in Panax notoginseng flowers, ameliorates cough by inhibition of NaV1.7 and TRPV1 channel currents and downregulation of TRPV1 expression

Ethnopharmacological relevancePanax notoginseng flowers, which are the buds of the traditional Chinese medicinal herb Sanqi, are widely used in China for their cough-ameliorating properties, with demonstrated therapeutic effects in the treatment of both acute and chronic coughs. However, both the antitussive mechanism and active compound basis of P. notoginseng flowers remain poorly understood. Aim of the studyWe investigated the antitussive effects of P. notoginseng flowers, identified the bioactive constituents responsible for alleviating cough symptoms, and elucidated the underlying pharmacological mechanisms. Materials and methodsWe analyzed the major chemical constituents of aqueous extracts of P. notoginseng flowers using liquid chromatography–mass spectrometry and quantitatively analyzed the key component, 20S-ginsenoside Rh2, using high-performance liquid chromatography. Using a cough reflex model in healthy mice and an ovalbumin-induced, highly sensitive guinea pig cough model, we verified the suppressive effects of P. notoginseng flowers and their saponin constituents on coughing. Furthermore, we explored the mechanisms of action of the key ion channels, NaV1.7 and TRPV1, using whole-cell patch-clamp techniques and molecular docking. Finally, the therapeutic mechanisms of P. notoginseng flowers on pathological cough were revealed using hematoxylin and eosin staining, immunohistochemistry, and western blotting. ResultsThe active components of P. notoginseng flowers were primarily protopanaxadiol-type saponins, among which 20S-ginsenoside Rh2 had the highest content (51.46 mg/g). In the mouse model, P. notoginseng flowers exhibited antitussive effects comparable to those of pentoxyverine citrate. Although its main saponin component, 20S-ginsenoside Rh2, showed slightly weaker effects, it still demonstrated concentration-dependent inhibition of channel activity. The whole-cell patch-clamp technique and virtual molecular docking showed that Rh2 might exert its effects by directly binding to the NaV1.7 and TRPV1 channels. In the guinea pig model, P. notoginseng flowers and their saponin components not only reduced cough frequency and prolonged the latency period before cough onset, but also significantly inhibited tracheal and pulmonary inflammation and the overexpression of TRPV1. Conclusions20S-Ginsenoside Rh2, the major bioactive saponin in P. notoginseng flowers, exhibits potent antitussive effects. The potential mechanism of action of 20S-Ginsenoside Rh2 in the treatment of cough may involve inhibiting NaV1.7 and TRPV1 channel currents through direct binding to core protein active sites and downregulating TRPV1 expression.

Fluorescence labeling strategies for cell surface expression of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a trans-cyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Defective CFTR modulates mechanosensitive channels TRPV4 and PIEZO1 and drives endothelial barrier failure

Cystic fibrosis (CF) is a genetic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite reports of CFTR expression on endothelial cells, pulmonary vascular perturbations, and perfusion deficits in CF patients, the mechanism of pulmonary vascular disease in CF remains unclear. Here, our pilot study of 40 CF patients reveals a loss of small pulmonary blood vessels in patients with severe lung disease. Using a vessel-on-a-chip model, we establish a shear-stress-dependent mechanism of endothelial barrier failure in CF involving TRPV4, a mechanosensitive channel. Furthermore, we demonstrate that CFTR deficiency downregulates the function of PIEZO1, another mechanosensitive channel involved in angiogenesis and wound repair, and exacerbates loss of small pulmonary blood vessel. We also show that CFTR directly interacts with PIEZO1 and enhances its function. Our study identifies key cellular targets to mitigate loss of small pulmonary blood vessels in CF.

Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation.

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.

Acidity-Responsive Fe-PDA@CaCO3 Nanoparticles for Photothermal-Enhanced Calcium-Overload- and Reactive-Oxygen-Species-Mediated Tumor Therapy.

Calcium-overload-mediated tumor therapy has received considerable interest in oncology. However, its efficacy has been proven to be inadequate due to insufficient calcium ion concentration at the tumor site coupled with challenges in facilitating efficient calcium uptake by tumors, leading to unsatisfactory therapeutic outcomes. In the present study, calcium carbonate nanoshell mineralized ferric polydopamine nanoparticles (Fe-PDA@CaCO3 NPs) were prepared for achieving Ca2+-overload-mediated tumor therapy. Upon entering the tumor site, the pH-responsive CaCO3 layer, acting as a "Ca2+ storage pool", rapidly degraded and released high quantities of free Ca2+ within the weakly acidic environment. The Fe-PDA core, with its excellent photothermal conversion properties, could significantly increase the temperature upon exposure to near-infrared (NIR) light irradiation, thereby activating the TRPV1 channel and leading to a large influx of released Ca2+ into the cytoplasm. Furthermore, the exposed Fe-PDA core could react with the tumor-overexpressed hydrogen peroxide (H2O2) to efficiently produce hydroxyl radicals (•OH), significantly increasing intracellular reactive oxygen species (ROS) levels and thus inhibiting the activity of the Ca2+ efflux pump, resulting in a high intracellular Ca2+ concentration. Ultimately, the increase in calcium/ROS levels could disrupt mitochondrial homeostasis and activate the apoptosis pathway. The current work presents a promising approach for tumor therapy using photothermal-enhanced calcium-overload-mediated ion interference therapy and chemodynamic therapy.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

TRPV1 signaling of perirenal adipose tissue promotes DOCA-Salt-induced hypertension and kidney injury.

Denervation of renal or perirenal adipose tissue (PRAT) can reduce arterial blood pressure in various hypertensive experimental models. Trpv1 (transient receptor potential vanillin 1) channel is highly expressed in the renal sensory nerves and the dorsal root ganglias (DRGs) projected by PRAT. However, it is currently unclear whether Trpv1 in DRGs projected from PRAT can regulate renal hypertension. We used resintoxin (RTX) to block the afferent sensory nerves of rat PRAT. We also constructed Trpv1 -/- mice and Trpv1 +/- mice or used the injection of AAV2-retro-shTrpv1 to detect the effects of Trpv1 knockout or knockdown of PRAT-projected DRGs on deoxycorticosterone acetate (DOCA)-Salt-induced hypertension and kidney injury. Blocking the afferent sensory nerves of PRAT with RTX can alleviate DOCA-Salt-induced hypertension and renal injury in rats. And this blockade reduces the expression of Trpv1 in the DRGs projected by PRAT. Injecting AAV2-retro-shTrpv1 into the PRAT of DOCA-Salt mice also achieved the same therapeutic effect. However, DOCA-Salt-induced hypertension and renal injury can be treated in Trpv1 +/- mice but not alleviated or even worsened in Trpv1 -/- mice, possibly because of compensatory increase of Trpv5 in DRG of Trpv1 -/- mice. Reducing, rather than eliminating, Trpv1 in DRG from PRAT-projection can reduce blood pressure and kidney damage in DOCA-Salt in rats or mice. Trpv1 in PRAT-DRGs may serve as a therapeutic target for salt-sensitive hypertension and its renal complications.

Three-dimensional matrix stiffness modulates mechanosensitive and phenotypic alterations in oral squamous cell carcinoma spheroids.

Extracellular biophysical cues such as matrix stiffness are key stimuli tuning cell fate and affecting tumor progression in vivo. However, it remains unclear how cancer spheroids in a 3D microenvironment perceive matrix mechanical stiffness stimuli and translate them into intracellular signals driving progression. Mechanosensitive Piezo1 and TRPV4 ion channels, upregulated in many malignancies, are major transducers of such physical stimuli into biochemical responses. Most mechanotransduction studies probing the reception of changing stiffness cues by cells are, however, still limited to 2D culture systems or cell-extracellular matrix models, which lack the major cell-cell interactions prevalent in 3D cancer tumors. Here, we engineered a 3D spheroid culture environment with varying mechanobiological properties to study the effect of static matrix stiffness stimuli on mechanosensitive and malignant phenotypes in oral squamous cell carcinoma spheroids. We find that spheroid growth is enhanced when cultured in stiff extracellular matrix. We show that the protein expression of mechanoreceptor Piezo1 and stemness marker CD44 is upregulated in stiff matrix. We also report the upregulation of a selection of genes with associations to mechanoreception, ion channel transport, extracellular matrix organization, and tumorigenic phenotypes in stiff matrix spheroids. Together, our results indicate that cancer cells in 3D spheroids utilize mechanosensitive ion channels Piezo1 and TRPV4 as means to sense changes in static extracellular matrix stiffness, and that stiffness drives pro-tumorigenic phenotypes in oral squamous cell carcinoma.

Crucial role for sensory nerves and Na/H exchanger inhibition in dapagliflozin and empagliflozin-induced arterial relaxation.

Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) from sensory nerves independent of glucose transport. The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using Wire-Myography. SGLT2, CGRP, TRPV1 and NHE1, expression was determined by Western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin.All SGLT inhibitors (1µM-100µM) and cariporide (30µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner.

Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Enhanced solubility and calcium absorption of calcium gluconate and calcium citrate in the presence of aspartic acid and glutamic acid

In the present study, the effects of aspartic acid and glutamic acid on the solubility and absorption of calcium gluconate and calcium citrate, two common forms of calcium supplements, were investigated. The results revealed that both amino acids increased the solubility of calcium gluconate and calcium citrate by binary or ternary complex formation, not by ligand exchange, as confirmed by calcium specification and density function theory calculations. The same mechanism applied to the calcium binding peptides. Furthermore, the addition of aspartic acid and glutamic acid resulted in a remarkable increase in calcium absorption compared to calcium gluconate and calcium citrate alone. This might be due to the altered properties (solubility, stability, and charges) of the complexes, as well as their enhanced interaction with the calcium channel TRPV6 through hydrogen bonding. Our findings provide new insights into the development of effective calcium supplementation strategies based on calcium-amino acids/calcium binding peptides complexes.

The Study of TRPV1 Channels of the Central Nervous System and Their Effect on Anxiety in ICR Mice.

The TRPV1 channel is actively involved in various neuronal processes and is found in various structures of the nervous system, including peripheral and central neurons, sensory ganglia, spinal cord, and various parts of the brain. Due to its ability to respond to various stimuli, TRPV1 can have a significant impact on the body's responses to stress. Studies indicate the involvement of TRPV1 in the regulation of anxiety behavior. Suppression of TRPV1 activity leads to a decrease in the level of anxiety in animals, which indicates the importance of this channel in psychoemotional regulation. A promising compound for inhibiting this channel is the APHC3 peptide, which is a selective receptor antagonist. The results obtained this study show that this peptide has a pronounced anxiolytic effect, reducing the level of anxiety in the studied animals.

TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton.

Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses.

Inflammation-induced mast cell-derived nerve growth factor: a key player in chronic vulvar pain?

Provoked vulvodynia (PV) is characterized by localized chronic vulvar pain. It is associated with a history of recurrent inflammation, mast cell (MC) accumulation, and neuronal sprouting in the vulva. However, the mechanism of how vulvar-inflammation promotes neuronal sprouting and gene-expression adaptation in the spinal cord, leading to hypersensitivity and painful sensations, is unknown. Here, we found that vulvar tissue from women with PV (n=8) is characterized by MC accumulation and neuronal sprouting compared to women without PV (n=4). In addition, we observed these changes in an animal study of PV. Thus, we found that repeated vulvar zymosan-inflammation challenges lead to long-lasting mechanical and thermal vulvar hypersensitivity, which was mediated by MC accumulation, neuronal sprouting, overexpression of the pain channels (TRPV1 and TRPA1) in vulvar neurons, as well as a long-term increase of gene expression related to neuroplasticity, neuroinflammation, and nerve growth factor (NGF) in the spinal cord/DRG(L6-S3). However, regulation of the NGF pathway by stabilization of MC activity with ketotifen fumarate (KF) during vulvar inflammation attenuated the local increase of NGF and histamine, as well as the elevated transcription of pro-inflammatory cytokines, and NGF pathway in the spinal cord. Additionally, KF treatment during inflammation modulates MC accumulation, neuronal hyperinnervation, and overexpression of the TRPV1 and TRPA1 channels in the vulvar neurons, consequently preventing the development of vulvar pain. A thorough examination of the NGF pathway during inflammation revealed that blocking NGF activity by using an NGF-non-peptide-inhibitor (Ro08-2750) regulates the upregulation of genes related to neuroplasticity, and NGF pathway in the spinal cord, as well as modulates neuronal sprouting and overexpression of the pain channels, resulting in a reduced level of vulvar hypersensitivity. On the other hand, stimulation of the NGF pathway in the vulvar promotes neuronal sprouting, overexpression of pain channels, and increase of gene expression related to neuroplasticity, neuroinflammation, and NGF in the spinal cord, resulting in long-lasting vulvar hypersensitivity. In conclusion, our findings suggest that vulvar allodynia induced by inflammation is mediated by MC accumulation, neuronal sprouting, and neuromodulation in the vulvar. Additionally, chronic vulvar pain may involve a long-term adaptation in gene expression in the spinal cord, which probably plays a critical role in central sensitization and pain maintenance. Strikingly, regulating the NGF pathway during the critical period of inflammation prevents vulvar pain development via modulating the neuronal changes in the vestibule and spinal cord, suggesting a fundamental role for the NGF pathway in PV development.

Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl− (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

Molecular mechanisms of two novel and selective TRPV1 channel activators

Venomous toxins hold immense value as tools in elucidating the intricate structure and underlying mechanisms of ion channels. In this article, we identified of two novel toxins, Hainantoxin-XXI (HNTX-XXI) and Hainantoxin-XXII (HNTX-XXII), derived from the venom of the Chinese spider Ornithoctonus hainana. HNTX-XXI, boasting a molecular weight of 6869.095 Da, comprises 64 amino acid residues and contains 8 cysteines. Meanwhile, HNTX-XXII, with a molecular weight of 8623.732 Da, comprises 77 amino acid residues and contains 12 cysteines. Remarkably, we discovered that both HNTX-XXI and HNTX-XXII possess the ability to activate TRPV1. They activated TRPV1 with EC50 values of 3.6 ± 0.19 μM and 862 ± 56 nM, respectively. Furthermore, the current generated by the activation of TRPV1 by these toxins can be rapidly blocked by ruthenium red. Intriguingly, our analysis revealed that the interaction between HNTX-XXI and TRPV1 is mediated by three key amino acid residues: L465, V469, and D471. Similarly, the interaction between HNTX-XXII and TRPV1 is facilitated by four key amino acid residues: A657, F659, E600, and R601. These findings provide profound insights into the molecular basis of toxin-TRPV1 interactions and pave the way for future research exploring the therapeutic potential of these toxic peptides.

Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site (~5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Sertraline Alleviates Chronic Prostatitis by Regulating the TRPV1 Channel.

Although sertraline has been widely used for chronic prostatitis (CP), the mechanisms are unclear. Herein, we explored the mechanisms of sertraline in treating CP. Network pharmacology methods were used to explore the potential targets and molecular mechanisms. LPS was used to stimulate RWPE-1 cells to construct an in vitro model of CP. An experimental autoimmune prostatitis (EAP) mice model was built. CCK-8 assay, EdU assay, BrdU detection, and Tunel assay were performed to evaluate the proliferation and apoptosis process of cells or tissues, respectively. DCFH-DA and Fluo-4 fluorescence probes were used to detect intracellular ROS and calcium concentrations. Von Frey filaments and open-field tests were utilized to evaluate pain response and depressive-like behavior of mice. Histopathology was evaluated through hematoxylin and eosin staining. RT-qPCR, Western blot, immunofluorescence, and immunohistochemistry were utilized to evaluate the transcription, expression, and location of related proteins. Molecular dynamics (MD) simulation and surface plasmon resonance (SPR) assay were performed to measure the binding capacity of sertraline and related proteins. Through a network pharmacology analysis, 27 potential targets of sertraline for CP were obtained, and 5 key targets (CHRM1, ADRA1B, HTR2B, HTR2A, and TRPV1) were finally identified. Functional experiments suggested that TRPV1 was involved in the proliferation, apoptosis inhibition, and ROS production of LPS-induced RWPE-1 cells. In vitro experiments showed that sertraline significantly inhibited cell proliferation, ROS generation, and transcription of inflammation cytokines of LPS-induced RWPE-1 cells. Additionally, sertraline markedly promoted the apoptosis level of LPS-stimulated RWPE-1 cells and elevated the expression level of BAX while reducing the expression levels of Bcl2 and Caspase-3. MD simulation and SPR assay confirmed the direct binding of sertraline to TRPV1. Moreover, sertraline significantly down-regulated the expression level of TRPV1 and inhibited calcium influx of LPS-induced RWPE-1 cells. TRPV1 agonist (Capsaicin) significantly restored the effects on proliferation, apoptosis, ROS production, and calcium influx of sertraline on LPS-induced RWPE-1 cells. Mice experiments demonstrated that sertraline treatment could reduce pain response, improve depression-like symptoms, and relieve local prostate inflammation of EAP mice, as well as down-regulated the expression level of TRPV1, inhibit the proliferation, and promote apoptosis of prostate tissues in EAP mice. The results revealed the anti-inflammatory effect of sertraline for RWPE-1 cells and EAP mice, and the potential mechanism was regulating the TRPV1 channel. It indicated that sertraline might serve as a complementary anti-inflammatory agent for CP.