Biology subjects

Mongin, A. A.

Publications and source records attributed to Mongin, A. A..

8 recordsLinked to original sources

bioRxiv2026

LRRC8A/D-containing anion channels mediate swelling-activated taurine release in the mouse brain

Volume-regulated anion channels (VRACs) are swelling-activated Cl- channels formed by heteromers of LRRC8 proteins (LRRC8A-E). VRACs mediate regulatory volume decrease by releasing Cl- and organic osmolytes, including taurine. In the brain, taurine functions as both an osmolyte and an inhibitory neuromodulator, acting as an agonist at glycine and GABAA receptors. Here, we used RNAi and genetically modified mice to (i) determine the contribution of LRRC8 subunits to swelling-activated taurine release, and (ii) test whether brain VRACs contribute to systemic osmoregulation. In astrocyte cultures lacking the essential VRAC subunit LRRC8A, swelling-activated [3H]taurine release was reduced by ~90%. Similarly, in vivo brain microdialysis revealed a [≥]90% reduction in swelling-activated taurine release in brain-wide LRRC8A knockout mice (bLRRC8A KO). RNAi experiments further confirmed LRRC8D as a key determinant of VRAC-mediated taurine release, with Lrrc8d knockdown reducing swelling activated [3H]taurine efflux by >50%. RNA-seq revealed enrichment of Lrrc8d in the hypothalamus, where astrocytic taurine release is proposed to inhibit magnocellular neurons and suppress arginine vasopressin secretion during hypoosmotic conditions. We therefore tested whether disrupting brain VRACs impairs recovery from systemic hypoosmotic stress. bLRRC8A KO mice and mice with gene trap-mediated LRRC8D depletion were challenged with an intraperitoneal injection of sterile water (7.5% body weight, under anesthesia), and serum osmolality was monitored over time. Unexpectedly, neither model showed impaired recovery from hypoosmotic challenge. Overall, this study identifies LRRC8A/D-containing VRACs as the primary conduit for swelling-activated taurine release in the brain, yet indicates that this pathway is not essential for maintaining systemic fluid-electrolyte homeostasis.

neuroscience↗

bioRxiv2026

Binding to Albumin and Off-Target Toxicity Confound the Use of LRRC8/VRAC Channel Blockers in Cell Physiology Assays

Volume-regulated anion channels (VRACs), formed by leucine-rich repeat-containing 8 (LRRC8) proteins, are ubiquitously expressed chloride channels essential for cell volume regulation and implicated in diverse physiological and pathological processes. Small-molecule VRAC inhibitors have been reported to modulate paracrine signaling, proliferation, differentiation, migration, and apoptosis, and have been patented for potential therapeutic applications in stroke, cardiovascular and metabolic diseases, and cancer. However, growing evidence indicates that many commonly used VRAC blockers exert substantial off-target effects and frequently fail to reproduce phenotypes observed after deletion of the essential VRAC subunit LRRC8A. Here, we systematically compared effects of several widely used pharmacological VRAC inhibitors with outcomes of molecular downregulation of LRRC8A in limiting proliferation of malignant glioblastoma cells derived from surgical specimens. NIH/3T3 fibroblasts served as a non-malignant control. In serum-containing media, structurally diverse VRAC blockers (DCPIB, DIDS, carbenoxolone, phloretin, and bromadiolone) reduced proliferation in a non-uniform manner, with potencies that did not correlate with reported VRAC affinities and varied markedly among cell lines. Radiotracer-based measurements of VRAC activity indicated that these discrepancies were largely attributable to binding of inhibitors to serum albumin. When experiments were repeated under serum-free conditions, all inhibitors except DIDS and phloretin induced extensive death of both malignant and non-malignant cells, confirmed by microscopy and LDH release assays. This cytotoxicity was accompanied by a marked reduction in intracellular ATP levels, consistent with previously reported mitochondrial uncoupling effects. In contrast, LRRC8A knockdown reduced proliferation without substantial cell death. Together, these findings demonstrate that most commercially available VRAC blockers limit proliferation and viability predominantly through VRAC-independent mechanisms. Under standard culture conditions, serum albumin masks much of their intrinsic cytotoxicity. These results underscore the need for rigorous molecular controls in pharmacological studies and provide basis for developing more selective and less toxic VRAC-targeting agents.

pharmacology and toxicology↗

bioRxiv2025

Subunit-specific roles of LRRC8 proteins in determining glutamate permeability of astrocytic volume-regulated anion channels

Volume-regulated anion channels (VRACs) are ubiquitous chloride channels that play important roles in cell volume regulation and numerous other physiological processes. VRACs are heteromeric complexes composed of leucine-rich repeat-containing proteins LRRC8A-E. LRRC8 subunit composition determines biophysical properties of VRACs, including permeability to small signaling molecules. Here, we used primary astrocyte cultures from wild-type and genetically modified C57BL/6 mice to investigate (i) subunit composition of native VRACs in the brain and (ii) subunit determinants of VRAC permeability to the excitatory neurotransmitter glutamate. qRT-PCR and RNA-seq analyses revealed high expression of Lrrc8a-d in mouse forebrain and astrocytes. Genetic deletion of the essential LRRC8A protein abolished swelling-activated glutamate release, measured as efflux of the non-metabolizable D-[3H]aspartate, confirming the crucial role of VRACs in this process. RNAi-mediated knockdown of individual subunits identified LRRC8A and LRRC8C as key components of glutamate-permeable astrocytic VRACs. qRT-PCR and Western blot analyses further showed that knockdown of LRRC8A or LRRC8C reciprocally altered the protein stability of the partner subunit without affecting their mRNA levels. A similar pattern of mutual regulation was observed between LRRC8A and LRRC8D. In contrast to LRRC8C, downregulation of LRRC8D had a more limited impact on glutamate release. Additional double-knockdown experiments demonstrated that LRRC8C- and LRRC8D-containing channels form distinct VRAC populations. This model was further supported by Western blot results showing no reciprocal regulation of LRRC8C and LRRC8D stability. Together, these findings refine our understanding of how the subunit organization of native brain VRACs governs gliotransmitter release, with implications for normal brain function and neurological disease. NEW & NOTEWORTHYVolume-regulated anion channels (VRACs) are ubiquitously expressed chloride channels composed of LRRC8A-E proteins. In human disorders, gain or loss of VRAC function leads to severe neurological phenotypes, potentially due to altered release of amino acid neurotrans-mitters. Here, we show that glutamate-permeable VRACs in brain astrocytes are primarily composed of LRRC8A and LRRC8C proteins. These findings provide insight into subunit organization of native VRACs in the CNS, with implications for normal brain function and neurological disease.

neuroscience↗

bioRxiv2025

LRRC8A-containing anion channels promote glioblastoma proliferation via a WNK1/mTORC2-dependent mechanism

Leucine-rich repeat-containing protein 8A (LRRC8A) is the essential subunit of ubiquitous volume-regulated anion channels (VRACs). LRCC8A is overexpressed in several cancers and promotes negative survival outcomes via a poorly defined mechanism. Here, we explored the role of LRRC8A and VRACs in the progression of glioblastoma (GBM), the most common and deadly primary brain tumor. We found that, as compared to healthy controls, LRRC8A mRNA was strongly upregulated in surgical GBM specimens, patient-derived GBM cell lines, and GBM datasets from The Cancer Genome Atlas (TCGA). Our in-silico analysis indicated that patients belonging to the lowest LRRC8A expression quartile demonstrated a trend for extended life expectancy. In patient-derived GBM cultures, siRNA-driven LRRC8A knockdown reduced cell proliferation and additionally decreased intracellular chloride levels and inhibited activity of mTOR complex 2. The antiproliferative effect of LRRC8A downregulation was recapitulated with a pharmacological inhibitor of VRAC. Our ensuing biochemical and molecular biology analyses established that the LRRC8A-containing VRACs facilitate GBM proliferation via a new mechanism involving non-enzymatic actions of the chloride-sensitive protein kinase WNK1. Accordingly, the chloride-bound WNK1 stimulates mTORC2 and the mTORC2-dependent protein kinases AKT and SGK, which promote proliferation. These findings establish the new mTORC2-centric axis for VRAC dependent regulation of cellular functions and uncover potential targets for GBM intervention. SUBJECT AREASCell biology Molecular biology Cancer

cancer biology↗

bioRxiv2024

Critical role of Gα12 and Gα13 proteins in TGF-β-induced myofibroblast differentiation.

Myofibroblast differentiation, characterized by accumulation of cytoskeletal and extracellular matrix proteins by fibroblasts, is a key process in wound healing and pathogenesis of tissue fibrosis. Transforming growth factor-{beta} (TGF-{beta}) is the most powerful known driver of myofibroblast differentiation. TGF-{beta} signals through transmembrane receptor serine/threonine kinases that phosphorylate Smad transcription factors (Smad2/3) leading to activation of transcription of target genes. Heterotrimeric G proteins mediate a distinct signaling from seven-transmembrane G protein coupled receptors, not commonly linked to Smad activation. We asked if G protein signaling plays any role in TGF-{beta}-induced myofibroblast differentiation, using primary cultured human lung fibroblasts. Activation of Gs by cholera toxin blocked TGF-{beta}-induced myofibroblast differentiation without affecting Smad2/3 phosphorylation. Inhibition of Gi by pertussis toxin, or siRNA-mediated combined knockdown of Gq and G11 had no significant effect on TGF-{beta}-induced myofibroblast differentiation. A combined knockdown of G12 and G13 resulted in a drastic inhibition of TGF-{beta}-stimulated expression of myofibroblast marker proteins (collagen-1, fibronectin, smooth-muscle -actin), with siG12 being significantly more potent than siG13. Mechanistically, a combined knockdown of G12 and G13 resulted in a substantially reduced phosphorylation of Smad2 and Smad3 in response to TGF-{beta}, which was accompanied by a significant decrease in the expression of TGF{beta} receptors (TGFBR1, TGFBR2) and of Smad3 under siG12/13 conditions. In conclusion, our study uncovers a novel role of G12/13 proteins in the control of TGF-{beta} signaling and myofibroblast differentiation.

cell biology↗

bioRxiv2023

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-{beta}) is one of the most established drivers of fibrotic processes. TGF-{beta} promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-{beta} robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-{beta}-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-{beta} signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-{beta} treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-{beta}-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-{beta}-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-{beta}; and (ii) ANO1 mediates TGF-{beta}-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity. NEW & NOTEWORTHYThis study describes a novel mechanism of differentiation of human lung fibroblasts (HLF) to myofibroblasts - the key process in the pathogenesis of pulmonary fibrosis. TGF-{beta} drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive With-No-Lysine (K) kinase (WNK1) to activate pro-fibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

cell biology↗

bioRxiv2022

Conditional deletion of LRRC8A in the brain protects against stroke damage independently of effect on swelling-activated glutamate release

The ubiquitous volume-regulated anion channels (VRACs), which are composed of LRRC8 proteins, facilitate cell volume homeostasis, and contribute to many other physiological processes. Prior studies demonstrated that treatment with non-specific VRAC blockers, or brain-specific deletion of the essential VRAC subunit LRRC8A, are highly protective in rodent stroke. In this work, we tested the widely accepted idea that harmful effects of VRACs in the brain are mediated by pathological release of the excitatory transmitter glutamate. We used two molecular genetic strategies to ablate LRRC8A expression in either brain astrocytes only (inducible deletion of Lrrc8aflox/flox with Aldh1l1CreERT2) or the majority of brain cells (neurons, astrocytes, and oligodendrocytes with NestinCre). To produce stroke, genetically modified mice were subjected to a 40-minute occlusion of the middle cerebral artery. The inducible deletion of astrocytic LRRC8A yielded no histological or behavioral protection. In contrast, the brain-wide LRRC8A knockout reduced ischemic infarction by ~50% in both heterozygotes (Het) and the full Lrrc8a knockout (KO) as compared to the control Lrrc8aflox/+ genotype. However, despite identical brain damage, Het and KO mice dramatically differed in their VRAC activities. Het mice had full swelling-activated glutamate release, while KO animals showed its virtual absence. These new findings refute the notion that VRAC-mediated glutamate release plays significant role in ischemic brain injury.

neuroscience↗

bioRxiv2020

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 brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated the brain-wide conditional LRRC8A knock-out 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.

neuroscience↗