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 brainwide conditional LRRC8A knockout mice (LRRC8A bKO) using Nestin Cre -driven Lrrc8a flox/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

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is indispensable for cell volume regulation but its broader physiological functions remain under investigation. Astrocyte-targeted Lrrc8a deletion in the nervous system reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against ischemic damage. Here we show that deletion of LRRC8A in all brain cells, using Nestin Cre -driven Lrrc8a fl/fl excision, is lethal. Mice devoid of brain LRRC8A are born close to the expected Mendelian ratio and develop without overt histological abnormalities. Nevertheless, they all die between 5 to 8 weeks of age with a seizure-like phenotype. Consistent with seizures, we found disruptions in cell excitability, GABAergic signaling, and astrocytic production of the GABA precursor glutamine, all of which might contribute to mortality. This work provides the first evidence of a critical role for VRAC in control of brain excitability during maturation.

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Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Several profibrotic factors drive pulmonary fibrosis, with transforming growth factor-beta (TGF-beta) being the most established. TGF-beta promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. Anoctamin-1 (ANO1), also known as TMEM16A, is a calcium-activated chloride channel. We found that TGF-beta robustly upregulates ANO1 expression in human lung fibroblasts (HLF) at mRNA and protein levels. Consistent, ANO1 was readily detected in fibrotic areas of IPF lungs. TGF-beta treatment of HLF resulted in a significant increase in steady state accumulation of intracellular chloride concentration, which was prevented by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 or ANO1 siRNA significantly inhibited TGF-beta-induced myofibroblast differentiation as determined by the expression of smooth muscle alpha-actin, collagen-1 and fibronectin. Mechanistically, pharmacological or knockdown-mediated inhibition of ANO1 did not have an effect on the initial TGF-beta signaling (Smad2 phosphorylation), but it blocked downstream TGF-beta signaling including Rho pathway (assessed by phosphorylation of myosin light chain) and AKT activation. Together, these data demonstrate that ANO1 is a TGF-beta-inducible chloride channel that largely contributes to the increase in intracellular chloride concentration in TGF-beta-treated cells. Furthermore, ANO1 mediates TGF-beta-induced myofibroblast differentiation, at least partially through activation of Rho pathway and of AKT.