Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

Background: Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV.

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl /H exchange, we now generate Clcn3 mice in which ClC-3 is converted into a Cl channel.

LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion.

Glucose homeostasis depends critically on insulin that is secreted by pancreatic β-cells. Serum glucose, which is directly sensed by β-cells, stimulates depolarization- and Ca-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels.

VRAC: molecular identification as LRRC8 heteromers with differential functions.

A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E.

Whirlin increases TRPV1 channel expression and cellular stability.

The expression and function of TRPV1 are influenced by its interaction with cellular proteins. Here, we identify Whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors.

Potentiation of the transient receptor potential vanilloid 1 channel contributes to pruritogenesis in a rat model of liver disease.

Persistent pruritus is a common disabling dermatologic symptom associated with different etiologic factors. These include primary skin conditions, as well as neuropathic, psychogenic, or systemic disorders like chronic liver disease. Defective clearance of potential pruritogenic substances that activate itch-specific neurons innervating the skin is thought to contribute to cholestatic pruritus.

Advances in modulating thermosensory TRP channels.

Introduction: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions.

Agonist- and Ca2+-dependent desensitization of TRPV1 channel targets the receptor to lysosomes for degradation.

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca(2+) cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood.

An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain.

Small peptides patterned after the N terminus of the synaptosomal protein of 25 kDa, a member of the protein complex implicated in Ca(2+)-dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107 (palmitoyl-EEMQRR-NH(2)), a 6-mer palmitoylated peptide that blocks the inflammatory recruitment of ion channels to the plasma membrane of nociceptors and the release of calcitonin gene-related peptide from primary sensory neurons, displays potent and long-lasting in vivo antihyperalgesia and antiallodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund's adjuvant, osteosarcoma, chemotherapy, and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent antihyperalgesic and antiallodynic activity that lasted ≥24 h.

Role of the transient receptor potential vanilloid 1 in inflammation and sepsis.

The transient receptor potential vanilloid 1 (TRPV1) is a thermoreceptor that responds to noxious temperatures, as well as to chemical agonists, such as vanilloids and protons. In addition, its channel activity is notably potentiated by proinflammatory mediators released upon tissue damage. The TRPV1 contribution to sensory neuron sensitization by proalgesic agents has signaled this receptor as a prime target for analgesic and anti-inflammatory drug intervention.

Triazine-based vanilloid 1 receptor open channel blockers: design, synthesis, evaluation, and SAR analysis.

The thermosensory transient receptor potential vanilloid 1 channel (TRPV1) is a polymodal receptor activated by physical and chemical stimuli. TRPV1 activity is drastically potentiated by proinflammatory agents released upon tissue damage. Given the pivotal role of TRPV1 in human pain, there is pressing need for improved TRPV1 antagonists, the development of which will require identification of new pharmacophore scaffolds.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min.

Complex regulation of TRPV1 and related thermo-TRPs: implications for therapeutic intervention.

The capsaicin receptor TRPV1 (Transient Receptor Potential, Vanilloid family member 1), the founding member of the heat-sensitive TRP ("thermo-TRP") channel family, plays a pivotal role in pain transduction. There is mounting evidence that TRPV1 regulation is complex and is manifest at many levels, from gene expression through post-translational modification and formation of receptor heteromers to subcellular compartmentalization and association with regulatory proteins. These mechanisms are believed to be involved both in disease-related changes in TRPV1 expression, and the long-lasting refractory state, referred to as "desensitization", that follows TRPV1 agonist treatment.

GABAA receptor associated protein (GABARAP) modulates TRPV1 expression and channel function and desensitization.

Transient receptor potential vanilloid (TRPV1) transduces noxious chemical and physical stimuli in high-threshold nociceptors. The pivotal role of TRPV1 in the physiopathology of pain transduction has thrust the identification and characterization of interacting partners that modulate its cellular function. Here, we report that TRPV1 associates with gamma-amino butyric acid A-type (GABA(A)) receptor associated protein (GABARAP) in HEK293 cells and in neurons from dorsal root ganglia coexpressing both proteins.

Chloride channelopathies.

Channelopathies, defined as diseases that are caused by mutations in genes encoding ion channels, are associated with a wide variety of symptoms. Impaired chloride transport can cause diseases as diverse as cystic fibrosis, myotonia, epilepsy, hyperekplexia, lysosomal storage disease, deafness, renal salt loss, kidney stones and osteopetrosis. These disorders are caused by mutations in genes belonging to non-related gene families, i.

Differential contribution of SNARE-dependent exocytosis to inflammatory potentiation of TRPV1 in nociceptors.

Potentiation of the pain-integrator ion channel transient receptor potential vanilloid type 1 (TRPV1) underlies thermal hyperalgesia mediated by a variety of proinflammatory factors. Two complementary mechanisms of TRPV1 inflammatory sensitization have been proposed, namely a decrease of its activation threshold and an increment of its surface expression in nociceptors. Here we investigated the involvement of regulated exocytosis to the inflammatory sensitization of TRPV1 in rat neonatal dorsal root ganglion neurons by proalgesic agents.

Activated spinal cord ependymal stem cells rescue neurological function.

Spinal cord injury (SCI) is a major cause of paralysis. Currently, there are no effective therapies to reverse this disabling condition. The presence of ependymal stem/progenitor cells (epSPCs) in the adult spinal cord suggests that endogenous stem cell-associated mechanisms might be exploited to repair spinal cord lesions.

Physiology and pharmacology of the vanilloid receptor.

The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia.

Differentiation of postnatal neural stem cells into glia and functional neurons on laminin-coated polymeric substrates.

A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp.

Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions.

Background: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.

Methodology And Principal Findings: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions.

Pharmacological intervention at ionotropic glutamate receptor complexes.

L-glutamate is considered the main excitatory neurotransmitter in the mammalian brain. Paradoxically, L-glutamate is also the most important excitotoxin pivotally involved in the aetiology of several neurodegenerative diseases such as stroke, Alzheimer, Parkinson, amyotropic lateral sclerosis, Huntington and neuropathic pain. L-glutamate signalling is transduced both presynaptically and postsynaptically by metabotropic and ionotropic receptors.

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.

Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies.