Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

HSV-1 employs UL56 to antagonize expression and function of cGAMP channels.

DNA sensing is important for antiviral immunity. The DNA sensor cGAS synthesizes 2'3'-cyclic GMP-AMP (cGAMP), a second messenger that activates STING, which induces innate immunity. cGAMP not only activates STING in the cell where it is produced but cGAMP also transfers to other cells. Transporters, channels, and pores (including SLC19A1, SLC46A2, P2X7, ABCC1, and volume-regulated anion channels (VRACs)) release cGAMP into the extracellular space and/or import cGAMP. We report that infection with multiple human viruses depletes some of these cGAMP conduits. This includes herpes simplex virus 1 (HSV-1) that targets SLC46A2, P2X7, and the VRAC subunits LRRC8A and LRRC8C for degradation. The HSV-1 protein UL56 is necessary and sufficient for these effects that are mediated at least partially by proteasomal turnover. UL56 thereby inhibits cGAMP uptake via VRAC, SLC46A2, and P2X7. Taken together, HSV-1 antagonizes intercellular cGAMP transfer. We propose that this limits innate immunity by reducing cell-to-cell communication via the immunotransmitter cGAMP.

LRRC8A as a central mediator promotes colon cancer metastasis by regulating PIP5K1B/PIP2 pathway.

Colorectal cancer (CRC) has been the third most common malignancy and the second cause of cancer-related mortality. As the core of volume-sensitive chloride currents, leucine-rich repeat-containing 8A (LRRC8A) contributes to tumor progression but is not consistent, especially for whom the roles in colon carcinoma metastasis were not fully elucidated. Herein, LRRC8A proteins were found highly expressed in hematogenous metastasis from human colorectal cancer samples. The oxaliplatin-resistant HCT116 cells highly expressed LRRC8A, which was related to impaired proliferation and enhanced migration. The over-expressed LRRC8A slowed proliferation and increased migration ex vivo and in vivo. The elevated LRRC8A upregulated the focal adhesion, MAPK, AMPK, and chemokine signaling pathways via phosphorylation and dephosphorylation. Inhibition of LRRC8A impeded the TNF-α signaling cascade and TNF-α-induced migration. LRRC8A binding to PIP5K1B regulated the PIP2 formation, providing a platform for LRRC8A to mediate cell signaling transduction. Importantly, LRRC8A self-regulated its transcription via NF-κB1 and NF-κB2 pathways and the upregulation of NIK/NF-κB2/LRRC8A transcriptional axis was unfavorable for colon cancer patients. Collectively, our findings reveal that LRRC8A is a central mediator in mediating multiple signaling pathways to promote metastasis and targeting LRRC8A proteins could become a potential clinical biomarker-driven treatment strategy for colon cancer patients.

LRRC8A promotes Glaesserella parasuis cytolethal distending toxin-induced p53-dependent apoptosis in NPTr cells.

Glaesserella parasuis is an early colonizer of the swine upper respiratory tract and can break through the respiratory barrier for further invasion. However, the mechanisms underlying G. parasuis increases epithelial barrier permeability remain unclear. This study demonstrates that G. parasuis cytolethal distending toxin (CDT) induces p53-dependent apoptosis in new-born piglet tracheal (NPTr) cells. Moreover, we report for the first time that leucine-rich repeat-containing protein 8A (LRRC8A), an essential subunit of the volume-regulated anion channel (VRAC), involves in apoptosis of NPTr cells mediated by G. parasuis CDT. Pharmacological inhibition of VRAC with either PPQ-102 or NS3728 largely attenuated CDT-induced apoptosis in NPTr cells. Additionally, experiments with cells knocked down for LRRC8A using small interfering ribonucleic acid (siRNA) or knocked out LRRC8A using CRISPR/Cas9 technology showed a significant reduction in CDT-induced apoptosis. Conversely, re-expression of Sus scrofa LRRC8A in LRRC8A-/- NPTr cells efficiently complemented the CDT-induced apoptosis. In summary, these findings suggest that LRRC8A is pivotal for G. parasuis CDT-induced apoptosis, providing novel insights into the mechanism of apoptosis caused by CDT.

Inhibitor screening for volume-sensitive LRRC8A chloride channel.

Leucine-rich repeat-containing protein 8 A (LRRC8A) protein is a critical member of volume-regulated anion channels. It plays a critical roles in the regulation of cellular volume and involves in the development of diseases like osteoarthritis. Screening of lead compounds to modulate its function may provide potential therapeutics of related diseases. Here, we employ virtual screening techniques and molecular dynamics (MD) simulation to screen potential inhibitors against LRRC8A. LRRC8A was regarded as the drug target to investigate potential compounds from the ZINC15 database via molecular docking. The final compound was selected among the top 10 Autodock Vina score (-8.8 Kcal/mol) with the ZINC ID ZINC000018195627 after druggability prediction. The docked complex from the virtual screening was subjected to MD simulation to analyze the stability of the LRRC8A protein-ligand complex, with parameters including root mean square deviation, root mean square fluctuation and radius of gyration. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method was further employed to predict the binding free energies from MD simulation trajectory. Our study provides insightful analysis for the potential compound to modulate LRRC8A and lay the foundation of therapeutics development against osteoarthritis.Communicated by Ramaswamy H. Sarma.

Interactions between the Astrocytic Volume-Regulated Anion Channel and Aquaporin 4 in Hyposmotic Regulation of Vasopressin Neuronal Activity in the Supraoptic Nucleus.

We assessed interactions between the astrocytic volume-regulated anion channel (VRAC) and aquaporin 4 (AQP4) in the supraoptic nucleus (SON). Acute SON slices and cultures of hypothalamic astrocytes prepared from rats received hyposmotic challenge (HOC) with/without VRAC or AQP4 blockers. In acute slices, HOC caused an early decrease with a late rebound in the neuronal firing rate of vasopressin neurons, which required activity of astrocytic AQP4 and VRAC. HOC also caused a persistent decrease in the excitatory postsynaptic current frequency, supported by VRAC and AQP4 activity in early HOC; late HOC required only VRAC activity. These events were associated with the dynamics of glial fibrillary acidic protein (GFAP) filaments, the late retraction of which was mediated by VRAC activity; this activity also mediated an HOC-evoked early increase in AQP4 expression and late subside in GFAP-AQP4 colocalization. AQP4 activity supported an early HOC-evoked increase in VRAC levels and its colocalization with GFAP. In cultured astrocytes, late HOC augmented VRAC currents, the activation of which depended on AQP4 pre-HOC/HOC activity. HOC caused an early increase in VRAC expression followed by a late rebound, requiring AQP4 and VRAC, or only AQP4 activity, respectively. Astrocytic swelling in early HOC depended on AQP4 activity, and so did the early extension of GFAP filaments. VRAC and AQP4 activity supported late regulatory volume decrease, the retraction of GFAP filaments, and subside in GFAP-VRAC colocalization. Taken together, astrocytic morphological plasticity relies on the coordinated activities of VRAC and AQP4, which are mutually regulated in the astrocytic mediation of HOC-evoked modulation of vasopressin neuronal activity.

LRRC8A promotes the initial development of oxaliplatin resistance in colon cancer cells.

Leucine-rich repeat-containing 8 A (LRRC8A) is an essential component of the volume-regulated anion channel (VRAC), which plays a vital role in cell proliferation, migration, apoptosis, and drug resistance. In this study, we investigated the effects of LRRC8A on oxaliplatin resistance in colon cancer cells. The cell viability was measured after oxaliplatin treatment with cell counting kit-8 (CCK8) assay. RNA sequencing was used to analyze the differentially expressed genes (DEGs) between HCT116 and oxaliplatin-resistant HCT116 cell line (R-Oxa) cells. CCK8 assay and apoptosis assay indicated that R-Oxa cells significantly promoted drug resistance to oxaliplatin compared with native HCT116 cells. R-Oxa cells, deprived of oxaliplatin treatment for over six months (R-Oxadep), maintained a similar resistant property as R-Oxa cells. The LRRC8A mRNA and protein expression were markedly increased in both R-Oxa and R-Oxadep cells. Regulation of LRRC8A expression affected the resistance to oxaliplatin in native HCT116 cells, but not R-Oxa cells. Furthermore, The transcriptional regulation of genes in the platinum drug resistance pathway may contribute to the maintenance of oxaliplatin resistance in colon cancer cells. In conclusion, we propose that LRRC8A promotes the acquisition rather than the maintenance of oxaliplatin resistance in colon cancer cells.

LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

LRRC8A is responsible for exosome biogenesis and volume regulation in colon cancer cells.

Exosomes are vital mediators for intercellular communications in the tumor microenvironment to accelerate colon cancer progression. Leucine-rich repeat-containing 8A (LRRC8A), the core component of the volume-regulated anion channel, is closely associated with acquiring heterogeneity for tumor cells. However, the role of LRRC8A in the exosomes remains largely unknown. Here, we reported that LRRC8A was one of the compositions in the exosomes released from colon cancer HCT116 cells. Down-regulation of LRRC8A proteins inhibited ex vivo cell growth and induced apoptosis. Consistently, chloride channel blockers DCPIB and NPPB inhibited cell growth and induced cell apoptosis in a time or concentration-dependent manner. Interestingly, the total amounts and proportions of different diameter exosomes released in 6 h were not altered by the treatment of DCPIB and NPPB in HCT116 cells. In contrast with the inhibition of LRRC8A, overexpression of LRRC8A proteins in HCT116 cells released significantly more distinct populations of exosomes. Importantly, the switches of ratios for exosomes in a hypotonic challenge were eliminated by DCPIB treatment. Collectively, our results uncovered that LRRC8A proteins were responsible for the exosome generation and sorted into exosomes for monitoring the volume regulation.

Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

The contribution of spinal dorsal horn astrocytes in neuropathic pain at the early stage of EAE.

Reactive astrocytes play a complex role in multiple sclerosis, and the astrocytes reactivity is an important factor in the pathogenesis of pain. It is of great significance to explore the genesis and development mechanism of pain in the early stage of multiple sclerosis (MS) for early intervention of the disease. This study aims to explore astrocyte reactivity at different stages of the experimental autoimmune encephalomyelitis (EAE) model, a mouse model of MS, and the role of astrocytes in the pain in the early stage of the EAE. In this study, we demonstrated that spinal dorsal horn astrocytes were activated in the pre-clinical stage of EAE mice, and the inhibition of spinal cord astrocyte reactivity effectively alleviates pain symptoms in EAE mice. On the other hand, spinal cord microglia were not directly participated in the early EAE pain. Moreover, the ion channel LRRC8A mediated the reactivity of spinal dorsal horn astrocytes by regulating the STAT3 pathway, therefore playing a role in the early pain of EAE.

LRRC8A Is a Promising Prognostic Biomarker and Therapeutic Target for Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor of the digestive system with increasing morbidity and mortality. The lack of sensitive and reliable biomarkers is one of the main reasons for the poor prognosis. Volume-regulated anion channels (VRAC), which are ubiquitously expressed in the vertebrate cell membrane, are composed of leucine-rich repeat-containing 8A (LRRC8A) and four other homologous family members (LRRC8B-E). VRAC heterogeneous complex is implicated in each of the six "hallmarks of cancer" and represents a novel therapeutic target for cancer. In this study, LRRC8A was speculated to be a promising prognostic biomarker and therapeutic target for PAAD based on a series of bioinformatics analyses. Additional cell experiments and immunohistochemical assays demonstrated that LRRC8A can affect the prognosis of PAAD and is correlated to cell proliferation, cell migration, drug resistance, and immune infiltration. Functional analysis indicated that LRRC8A influences the progression and prognosis of patients with PAAD by the regulation of CD8+ T cells immune infiltration. Taken together, these results can help in the design of new therapeutic drugs for patients with PAAD.

Structure-function relationships of the LRRC8 subunits and subdomains of the volume-regulated anion channel (VRAC).

Volume Regulated Anion Channels (VRAC) are critical contributors to cell volume homeostasis and are expressed ubiquitously in all vertebrate cells. VRAC sense increases in cell volume, and act to return cells to baseline volume in a process known as regulatory volume decrease (RVD) through the efflux of anions and organic osmolytes. This review will highlight seminal studies that elucidated the role of VRAC in RVD, their characteristics as a function of subunit specificity, and their clinical relevance in physiology and pathology. VRAC are also known as volume-sensitive outward rectifiers (VSOR) and volume-sensitive organic osmolyte/anion channels (VSOAC). In this review, the term VRAC will be used to refer to this family of channels.

The expanding toolbox to study the LRRC8-formed volume-regulated anion channel VRAC.

The volume-regulated anion channel (VRAC) is activated upon cell swelling and facilitates the passive movement of anions across the plasma membrane in cells. VRAC function underlies many critical homeostatic processes in vertebrate cells. Among them are the regulation of cell volume and membrane potential, glutamate release and apoptosis. VRAC is also permeable for organic osmolytes and metabolites including some anti-cancer drugs and antibiotics. Therefore, a fundamental understanding of VRAC's structure-function relationships, its physiological roles, its utility for therapy of diseases, and the development of compounds modulating its activity are important research frontiers. Here, we describe approaches that have been applied to study VRAC since it was first described more than 30 years ago, providing an overview of the recent methodological progress. The diverse applications reflecting a compromise between the physiological situation, biochemical definition, and biophysical resolution range from the study of VRAC activity using a classic electrophysiology approach, to the measurement of osmolytes transport by various means and the investigation of its activation using a novel biophysical approach based on fluorescence resonance energy transfer.

Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel.

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl- currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

Volume-activated chloride channels contribute to lipopolysaccharide plus nigericin-induced pyroptosis in bone marrow-derived macrophages.

The representative morphological features of pyroptosis are excessive cell swelling and subsequent membrane rupture. However, the mechanism underlying the cell's inherent inability to regulate volume during the progression of pyroptosis is poorly understood. In the current study, we found that both volume-activated chloride currents (Icl, vol) and the regulatory volume decrease (RVD) were markedly decreased in bone marrow-derived macrophages (BMDMs) undergoing pyroptosis induced by lipopolysaccharides (LPS) and nigericin. The inhibition of ICl, vol and RVD by the chloride channel blockers, tamoxifen or DCPIB, led to the emergence of pyroptosis-like phenotypes such as activated-caspase-1, pyroptotic-body-like bubbles, and a fried-egg-like appearance. Moreover, the expression of the volume-activated chloride channel (VRAC) constituent protein Leucine-Rich Repeat-Containing 8A (LRRC8A) was significantly down-regulated in pyroptotic BMDMs treated with LPS and nigericin. The silencing of LRRC8A expression by small interfering RNA (si)-LRRC8A transfection not only reduced ICl, vol and RVD, but also caused BMDMs to show pyroptosis-like manifestations such as activated-caspase-1, membrane bubbles, and have a fried-egg-like appearance. These results reveal a new mechanism for the loss of volume regulation in the process of pyroptotic cell swelling and strongly suggest that a functional deficiency of VRAC/LRRC8A plays a key role in this disorder.

Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines.

High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.