Activation Of Transient Receptor Potential Vanilloid 1 Channels Research Articles

Increased TRPV4 Channel Expression Enhances and Impairs Blood Vessel Function in Hypertension.

Endothelial cell TRPV4 (transient receptor potential vanilloid 4) channels provide a control point that is pivotal in regulating blood vessel diameter by mediating the Ca2+-dependent release of endothelial-derived vasoactive factors. In hypertension, TRPV4-mediated control of vascular function is disrupted, but the underlying mechanisms and precise physiological consequences remain controversial. Here, using a comprehensive array of methodologies, endothelial TRPV4 channel function was examined in intact mesenteric resistance arteries from normotensive Wistar-Kyoto and spontaneously hypertensive rats. Our results show there is a notable shift in vascular reactivity in hypertension characterized by enhanced endothelium-dependent vasodilation at low levels of TRPV4 channel activation. However, at higher levels of TRPV4 activity, this vasodilatory response is reversed, contributing to the aberrant vascular tone observed in hypertension. The change in response, from dilation to constriction, was accompanied by a shift in intracellular Ca2+ signaling modalities arising from TRPV4 activity. Oscillatory TRPV4-evoked IP3 (inositol triphosphate)-mediated Ca2+ release, which underlies dilation, decreased, while the contraction inducing sustained Ca2+ rise, arising from TRPV4-mediated Ca2+ influx, increased. Our findings also reveal that while the sensitivity of endothelial cell TRPV4 to activation was unchanged, expression of the channel is upregulated and IP3 receptors are downregulated in hypertension. These data highlight the intricate interplay between endothelial TRPV4 channel expression, intracellular Ca2+ signaling dynamics, and vascular reactivity. Moreover, the data support a new unifying hypothesis for the vascular impairment that accompanies hypertension. Specifically, endothelial cell TRPV4 channels play a dual role in modulating blood vessel function in hypertension.

Complement Component C5a and Fungal Pathogen Induce Diverse Responses through Crosstalk between Transient Receptor Potential Channel (TRPs) Subtypes in Human Conjunctival Epithelial Cells.

The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.

Engineering magnetic nanosystem for TRPV1 and TRPV4 channel activation.

Recently, physical tools for remotely stimulating mechanical force-sensitive and temperature-sensitive proteins to regulate intracellular pathways have opened up novel and exciting avenues for basic research and clinical applications. Among the numerous modes of physical stimulation, magnetic stimulation is significantly attractive for biological applications due to the advantages of depth penetration and spatial-temporally controlled transduction. Herein, the physicochemical parameters (e.g., shape, size, composition) that influence the magnetic properties of magnetic nanosystems as well as the characteristics of transient receptor potential vanilloid-1 (TRPV1) and transient receptor potential vanilloid-4 (TRPV4) channels are systematically summarized, which offer opportunities for magnetic manipulation of cell fate in a precise and effective manner. In addition, representative regulatory applications involving magnetic nanosystem-based TRPV1 and TRPV4 channel activation are highlighted, both at the cellular level and in animal models. Furthermore, perspectives on the further development of this magnetic stimulation mode are commented on, with emphasis on scientific limitations and possible directions for exploitation. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

Amyloid β reduces mechanical sensation by attenuating the function of TRPV4 channels in rat small dorsal root ganglion neurons

Amyloid β (Aβ) is well known to be a key trigger for the progression of Alzheimer's disease and dementia. Aβ forms oligomers that develop into amyloid plaques, causing neuronal death and dysfunction. Aβ-induced neuronal dysfunction has been reported in peripheral neurons as well as in the brain. We previously reported that transient receptor potential vanilloid 4 (TRPV4) channels play an important role in mechanical sensation in sensory neurons innervating skeletal muscle. However, to date, it remains unclear whether Aβ modulates mechanosensation or TRPV4 channel activity in peripheral sensory neurons. As such, we hypothesized that Aβ acutely impairs the response of small dorsal root ganglion (DRG) neurons to mechanical stimuli by attenuating the function of TRPV4 channels. Purpose: The aim of this investigation was to determine the effects of Aβ1-40 and 1-42 on mechanosensation in small DRG neurons. Method: In in vitro whole-cell patch clamp recordings from cultured rat DRG neurons (Sprague-Dawley rat, n = 4, body weight: 316 ± 57 g), mechanically-activated (MA) current amplitudes or GSK1016790A (a selective agonist of TRPV4, 1 μM)-induced inward currents were measured before and 5 min after application of either HEPES buffered solution (control), Aβ1-40 (2.5 ng/μL), Aβ1-42 (2.5 ng/μL) or Aβ1-40/42 solution (2.5 ng/μL, respectively). Results: MA current amplitude was significantly decreased 5 min after application of both Aβ1-40 (Before vs. After: -71.7 ± 14.5 vs. -13.5 ± 4.8 pA. P < 0.01) and Aβ1-42 (Before vs. After: -63.7 ± 16.2 vs. -14.5 ± 5.9 pA. P < 0.05) while HEPES application did not alter MA currents (Before vs. After: -69.5 ± 17.9 ± -66.2 ± 13.9 pA. P = 0.36). Likewise, GSK1016790A-induced inward current was significantly suppressed by Aβ1-40/42 solution (Fold change in total charge transfer: HEPES vs. Aβ1-40/42: 0.92 ± 0.47 vs. 0.33 ± 0.06. P < 0.05). Conclusion: The data demonstrate that both Aβ1-40 and 1-42 suppress 1) the cellular response to mechanical stimulation and 2) TRPV4 currents in rat small DRG neurons. These findings suggest that both Aβ1-40 and 1-42 attenuate mechanical sensation in DRG neurons by inhibiting TRPV4 channels. Supported by NIH HL-151632 and JSPS 23KJ2207. 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.

Reactive Oxygen Species Activate a Ferritin-Linked TRPV4 Channel under a Static Magnetic Field.

Magnetogenetics has shown great potential for cell function and neuromodulation using heat or force effects under different magnetic fields; however, there is still a contradiction between experimental effects and underlying mechanisms by theoretical computation. In this study, we aimed to investigate the role of reactive oxygen species (ROS) in mechanical force-dependent regulation from a physicochemical perspective. The transient receptor potential vanilloid 4 (TRPV4) cation channels fused to ferritin (T4F) were overexpressed in HEK293T cells and exposed to static magnetic fields (sMF, 1.4-5.0 mT; gradient: 1.62 mT/cm). An elevation of ROS levels was found under sMF in T4F-overexpressing cells, which could lead to lipid oxidation. Compared with the overexpression of TRPV4, ferritin in T4F promoted the generation of ROS under the stimulation of sMF, probably related to the release of iron ions from ferritin. Then, the resulting ROS regulated the opening of the TRPV4 channel, which was attenuated by the direct addition of ROS inhibitors or an iron ion chelator, highlighting a close relationship among iron release, ROS production, and TRPV4 channel activation. Taken together, these findings indicate that the produced ROS under sMF act on the TRPV4 channel, regulating the influx of calcium ions. The study would provide a scientific basis for the application of magnetic regulation in cellular or neural regulation and disease treatment and contribute to the development of the more sensitive regulatory technology.

Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog.

Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Carcinogenesis and Metastasis: Focus on TRPV1-Positive Neurons and Immune Cells.

Both sensory neurons and immune cells, albeit at markedly different levels, express the vanilloid (capsaicin) receptor, Transient Receptor Potential, Vanilloid-1 (TRPV1). Activation of TRPV1 channels in sensory afferent nerve fibers induces local effector functions by releasing neuropeptides (most notably, substance P) which, in turn, trigger neurogenic inflammation. There is good evidence that chronic activation or inactivation of this inflammatory pathway can modify tumor growth and metastasis. TRPV1 expression was also demonstrated in a variety of mammalian immune cells, including lymphocytes, dendritic cells, macrophages and neutrophils. Therefore, the effects of TRPV1 agonists and antagonists may vary depending on the prominent cell type(s) activated and/or inhibited. Therefore, a comprehensive understanding of TRPV1 activity on immune cells and nerve endings in distinct locations is necessary to predict the outcome of therapies targeting TRPV1 channels. Here, we review the neuro-immune modulation of cancer growth and metastasis, with focus on the consequences of TRPV1 activation in nerve fibers and immune cells. Lastly, the potential use of TRPV1 modulators in cancer therapy is discussed.

Magnolol alleviates pulmonary fibrosis inchronic obstructive pulmonary disease by targeting transient receptor potential vanilloid 4-ankyrin repeat domain.

Transient receptor potential vanilloid 4 (TRPV4) plays a role in regulating pulmonary fibrosis (PF). While several TRPV4 antagonists including magnolol (MAG), have been discovered, the mechanism of action is not fully understood. This study aimed to investigate the effect of MAG on alleviating fibrosis in chronic obstructive pulmonary disease (COPD) based on TRPV4, and to further analyze its mechanism of action on TRPV4. COPD was induced using cigarette smoke and LPS. The therapeutic effect of MAG on COPD-induced fibrosis was evaluated. TRPV4 was identified as the main target protein of MAG using target protein capture with MAG probe and drug affinity response target stability assay. The binding sites of MAG at TRPV4 were analyzed using molecular docking and small molecule interaction with TRPV4-ankyrin repeat domain (ARD). The effects of MAG on TRPV4 membrane distribution and channel activity were analyzed by co-immunoprecipitation, fluorescence co-localization, and living cell assay of calcium levels. By targeting TRPV4-ARD, MAG disrupted the binding between phosphatidylinositol 3 kinase γ and TRPV4, leading to hampered membrane distribution on fibroblasts. Additionally, MAG competitively impaired ATP binding to TRPV4-ARD, inhibiting TRPV4 channel opening activity. MAG effectively blocked the fibrotic process caused by mechanical or inflammatory signals, thus alleviating PF in COPD. Targeting TRPV4-ARD presents a novel treatment strategy for PF in COPD.

Abstract P138: Novel Smooth Muscle Ca 2+ -signaling Nanodomains In Blood Pressure Regulation

Introduction: Transient receptor potential vanilloid 4 (TRPV4) channel is a crucial Ca 2+ influx pathway in arterial smooth muscle cells (SMCs). However, the effect of SMC TRPV4 (TRPV4 SMC ) channel activity on resting blood pressure has not been investigated. Hypothesis: We hypothesized that SMC TRPV4 (TRPV4 SMC ) channels regulate blood pressure under homeostatic and hypertensive conditions. Results: We provide first evidence that TRPV4 SMC deletion decreases the mean arterial pressure of mice (control: 104.8 ± 2.2 mm Hg; TRPV4 SMC -/- : 101.4 ± 1 mm Hg). α1AR stimulation activated TRPV4 SMC channels through protein kinase Cα (PKCα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Surprisingly, intraluminal pressure-induced TRPV4 SMC channel activity opposed vasoconstriction through activation of Ca 2+ -sensitive K + (BK) channels, indicating functionally opposite pools of TRPV4 SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR-TRPV4 SMC and intraluminal pressure-TRPV4 SMC -BK channel signaling have opposite effects on blood pressure, with α1AR-TRPV4 SMC signaling dominating under resting conditions. Further, in hypertensive patients and a mouse model of hypertension, constrictor α1AR-PKCα-TRPV4 signaling was upregulated, whereas dilator pressure-TRPV4-BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. Conclusions: Collectively, our data identify novel smooth muscle Ca 2+ -signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

Role of Src kinase in regulating protein kinase C mediated phosphorylation of TRPV1.

Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation. We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist. Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund's Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition. Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions. Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized.

Transient Receptor Potential Vanilloid-1 (TRPV1) Alleviates Hepatic Fibrosis via TGF-β Signaling.

Hepatic fibrosis is a major global health problem and considered a leading cause of liver-related morbidity and mortality worldwide. Although previous studies have suggested that transient receptor potential vanilloid-1 (TRPV1) is protective against cardiac and renal fibrosis, its functional role in hepatic fibrosis has remained elusive. Herein, we characterize the effects of TRPV1 on carbon tetrachloride- (CCl4-) induced mice, in vitro transforming growth factor-β- (TGF-β-) treated hepatic stellate cells (HSCs), and human fibrosis specimens. Finally, our results demonstrated the significant TRPV1 downregulation in human liver fibrosis tissues. Knocking out TRPV1 significantly increased the expression of various hepatic fibrosis markers, while the expression of these biomarkers declined markedly in capsaicin-activated mice. Moreover, our study revealed that knocking down TRPV1 would enhance the promotive effect of TGF-β on HSC proliferation, cell cycle, cell apoptosis, and ECM expression. Also, such promotive effect can be partially reversible by capsaicin, an exogenous activator of TRPV1. Collectively, the obtained data suggest that TRPV1 may alleviate CCl4-induced hepatic fibrosis and attenuate the effect of TGF-β on HSC activation, proliferation, and apoptosis, which overall implies that targeting TRPV1 channel activity may be an effective therapeutic strategy for treating hepatic fibrosis.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

Smooth Muscle TRPV4 Channels Signaling Nanodomains in Blood Pressure Regulation

Transient receptor potential vanilloid 4 (TRPV4) channel is a crucial Ca2+influx pathway in arterial smooth muscle cells (SMCs). However, the effect of SMC TRPV4 (TRPV4SMC) channel activity on resting blood pressure has not been investigated. We hypothesized that SMC TRPV4 (TRPV4SMC) channels regulate blood pressure under homeostatic and hypertensive conditions. Radiotelemetric recordings showed lower resting blood pressure in SMC‐specific TRPV4‐/‐ (TRPV4SMC‐/‐) mice than control mice, suggesting that TRPV4SMC channels elevate the resting blood pressure. α1 adrenergic receptor (α1AR) stimulation–induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4SMC‐/‐ mice, whereas intraluminal pressure–induced (myogenic) constriction was increased. Moreover, both α1AR stimulation and increased intraluminal pressure activated elementary Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) in MAs from control mice. These data supported the concept that TRPV4SMC channels contribute to α1AR–induced vasoconstriction but oppose myogenic vasoconstriction. Inhibition of Ca2+‐sensitive K+ (BK) channels abolished the effect of TRPV4SMC channels on myogenic vasoconstriction, indicating a crucial role for TRPV4SMC–BK channel signaling in limiting myogenic vasoconstriction. Accordingly, a direct activation of TRPV4SMC channels with GSK1016790A (30 nM) increased BK channel currents in SMC patch‐clamp experiments. α1AR stimulation, however, was unable to increase BK channel currents, implying two functionally opposite pools of TRPV4SMC channels. Direct STORM superresolution imaging revealed a1AR:TRPV4SMC and TRPV4SMC:BK channel co‐localization, and almost no co‐localization of a1ARs and BK channels. Together, these data supported the idea of spatially separated and functionally opposite nanodomains of TRPV4SMC channels. We further hypothesized that an imbalance between these two nanodomains contributes to elevated blood pressure in hypertension. In skeletal muscle arteries from hypertensive patients, a1AR activation of TRPV4SMC channel currents was accentuated whereas TRPV4SMC activation of BK channel currents was impaired. Similarly, MAs from angiotensin II‐infused hypertensive mice showed increased activity of constrictor a1AR–TRPV4SMC signaling and impairment of dilatory TRPV4SMC–BK channel signaling. Collectively, our data identify novel smooth muscle Ca2+ signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

Multiubiquitination of TRPV4 reduces channel activity independent of surface localization

Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Regulation of TRPV1 channel activities by intracellular ATP in the absence of capsaicin

Transient receptor potential vanilloid 1 (TRPV1) is a voltage-dependent non-selective cation channel activated by capsaicin, the main pungent ingredient of chili peppers, and noxious heat. Although TRPV1 channels produce outwardly rectifying currents even in the absence of capsaicin, little is known about the regulation mechanism of the TRPV1 currents. In the present study, we found that intracellular ATP regulates the basal activities of TRPV1 channels in a concentration-dependent manner. The ATP-dependent regulation of TRPV1 channels was mediated by phosphoinositides. Moreover, an increase in intracellular ATP concentration negatively shifted voltage-dependent activation of TRPV1 channels. These results suggest that the ATP-dependent production of phosphoinositides regulates the voltage-dependent gating of the basal TRPV1 channel activities in the absence of capsaicin.

Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the “tail-up, head-down” configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.

Dichotomous effects of smooth muscle TRPV4 channels on vascular contractility

Ca2+ signaling mechanisms in smooth muscle cells (SMCs) determine vascular contractility. Transient receptor potential vanilloid 4 (TRPV4) ion channels have been described as a crucial Ca2+ influx pathway in SMCs from small, resistance-sized arteries. However, the impact of SMC TRPV4 (TRPV4SMC) channel activity on blood pressure remains unknown, mostly due to the lack of studies in SMC-specific knockout mice. We hypothesized that TRPV4SMC channels control the resting blood pressure. Radiotelemetric blood pressure recordings showed reduced daytime and nighttime blood pressures in the inducible, SMC-specific TRPV4 knockout (TRPV4SMC-/-) mice compared to the control mice. The effect of TRPV4SMC-/- on vascular contractility was assessed using two endogenous vasoconstrictor mechanisms- 1) SMC α1 adrenergic receptor (α1AR) stimulation and 2) increased intraluminal pressure. Phenylephrine (α1AR agonist)-induced constriction of pressurized mesenteric arteries (MAs, 80 mm Hg, 1 nM-10 mM phenylephrine) and blood pressure elevation (10 mg/kg phenylephrine, i.p.) were blunted in TRPV4SMC-/- mice, identifying a central role of TRPV4SMC channels in α1AR-induced vasoconstriction and blood pressure elevation. Individual Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) were recorded using spinning disk confocal imaging in fluo-4-loaded MAs pressurized to 80 mm Hg. Phenylephrine (1 mM) activated TRPV4SMC sparklets, an effect abolished by protein kinase C (PKC) inhibitor (Go6976, 1 mM). These results indicated that PKC-TRPV4SMC channel signaling is a major contributor to α1AR-induced vasoconstriction. In direct contrast with PE-induced constriction, intraluminal pressure-induced constriction was higher in MAs from TRPV4SMC-/- mice, suggesting that SMC TRPV4 channels limit pressure-induced vasoconstriction. Previous studies have shown that TRPV4SMC channels can activate large-conductance, Ca2+-sensitive K+ (BK) channels, thereby decreasing pressure-induced vasoconstriction. Consistent with these findings, vasoconstriction in response to BK channel inhibitor (paxilline, 1 mM) was reduced in MAs from TRPV4SMC-/- mice, supporting a dilatory TRPV4SMC channel-BK channel signaling under resting conditions. In situ proximity ligation assay revealed co-localization of TRPV4SMC channels with both α1ARs and BK channels. Overall, TRPV4SMC channels increase the resting blood pressure but have divergent effects on vascular contractility. TRPV4SMC channels contribute to α1AR-induced vasoconstriction but limit pressure-induced vasoconstriction. Therefore, selective vasoactive stimulus-TRPV4SMC channel coupling may determine the impact of TRPV4SMC channels on vascular resistance.