Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines.
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ABSTRACT:
SUBMITTER: Furuya K
PROVIDER: S-EPMC8401269 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The release of neuronal messengers outside synapses has broad biological implications, particularly with regard to communication between axons and glia. We identify a mechanism for nonsynaptic, nonvesicular release of adenosine triphosphate (ATP) from axons through volume-activated anion channels (VAACs) activated by microscopic axon swelling during action potential firing. We used a combination of single-photon imaging of ATP release, together with imaging for intrinsic optical signals, intracellular calcium ions (Ca(2+)), time-lapse video, and confocal microscopy, to investigate action potential-induced nonsynaptic release of this neurotransmitter. ATP release from cultured embryonic dorsal root ganglion axons persisted when bafilomycin or botulinum toxin was used to block vesicular release, whereas pharmacological inhibition of VAACs or prevention of action potential-induced axon swelling inhibited ATP release and disrupted activity-dependent signaling between axons and astrocytes. This nonvesicular, nonsynaptic communication could mediate various activity-dependent interactions between axons and nervous system cells in normal conditions, development, and disease.
| S-EPMC5023281 | biostudies-literature
Project description:Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA-based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel-dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.
| S-EPMC7170513 | biostudies-literature
Project description:ATP serves not only as an energy source for all cell types but as an 'extracellular messenger' for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg(2+) and/or H(+) salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP(4-) in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.
| S-EPMC2096548 | biostudies-other
Project description:To become fully functional effector T cells, naïve T cells undergo a drastic metabolic status shift from quiescence to substantial biosynthesis. The synthesis of biomass initiated by T cell receptor (TCR) signals drives the cell volume increase during T cell activation (T lymphoblast), which resembles the slowest extreme of hypotonic cell swelling. However, the fundamental role of cell volume regulation in TCR signaling during T lymphoblast and its underlying mechanisms remain a long-standing enigma. Here we show that optimal T cell activation and function require appropriate cell volume regulation by regulated volume decrease (RVD) mediated via volume-regulated anion channels (VRACs) during T cell blast. Pharmacological blockade of VRACs and genetic deletion of LRRC8A in T cells, the essential component of VRACs, impair T cell activation and function, particularly under weak TCR stimulation, such as low-affinity TCR-pMHC engagement and unsaturated anti-CD3e crosslinking. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles driven by various TCR signal strengths, revealing the prominent effects of cell volume regulation for T cell functions. More importantly, LRRC8A-deficient mice have defective antiviral immunity and obstacles in effector-memory transition of CD8+ T cells. Furthermore, LRRC8A escorts the thymic selection of T cells and shapes the T cell repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via RVD during T cell blast induced by activation-driven intracellular osmolytes accumulation to keep the proximal TCR signaling molecules at adequate density. T cell blast driven by TCR signal feedbacks to control T cell activation. Together, our results reveal a previously unappreciated layer of T cell activation regulation that LRRC8A acts as a cell volume controlling "valve" to facilitate T cell activation and function
2023-09-23 | GSE243574 | GEO
Project description:ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 muM. The OGD-induced ATP release was inhibited by Gd(3+) and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl(-) channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X(7) receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd(3+) and arachidonic acid. The channel was found to be permeable to ATP(4-) with a permeability ratio of P(ATP)/P(Cl) = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.
| S-EPMC2377326 | biostudies-other
Project description:The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ?800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (?) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic ?.
| S-EPMC4733249 | biostudies-literature
Project description:Background and purposeThe ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis.Experimental approachExperiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells.Key resultsIn cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons.Conclusion and implicationsOur results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.
| S-EPMC3594680 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B-E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.
| S-EPMC6451053 | biostudies-literature
Project description:Hypoxia and inhibitors of mitochondrial respiration impair the regulatory volume decrease (RVD) of cerebellar granule neurons after hypotonic swelling. RVD is linked to the opening of volume-regulated anion channels (VRACs). VRACs are outwardly rectifying, inactivate slowly during maintained depolarization, and are permeable to the cellular organic osmolyte taurine. Channel activation requires nonhydrolytic ATP binding and is not modulated by intracellular ADP. VRAC opening is reversibly depressed by hypoxia and by mitochondrial inhibitors such as oligomycin, rotenone, and antimycin A. These results demonstrate that neuronal VRAC activation and swelling are both tightly linked to cellular energy. Moreover, the findings reported in this work may have a particular significance for inherited mitochondrial human diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which cause brain swelling and edema.
| S-EPMC6792668 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat-containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.
| S-EPMC6120214 | biostudies-literature