Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC‑K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Volume-regulated anion channels (VRACs) are hexamers of LRRC8 proteins that are crucial for cell volume regulation. N termini (NTs) of the obligatory LRRC8A subunit modulate VRACs activation and ion selectivity, but the underlying mechanisms remain poorly understood. Here, we report a 2.8-Å cryo-electron microscopy structure of human LRRC8A that displays well-resolved NTs. Amino-terminal halves of NTs fold back into the pore and constrict the permeation path, thereby determining ion selectivity together with an extracellular selectivity filter with which it works in series. They also interact with pore-surrounding helices and support their compact arrangement. The C-terminal halves of NTs interact with intracellular loops that are crucial for channel activation. Molecular dynamics simulations indicate that low ionic strength increases NT mobility and expands the radial distance between pore-surrounding helices. Our work suggests an unusual pore architecture with two selectivity filters in series and a mechanism for VRAC activation by cell swelling.

Objective: In this study, we utilized bibliometric methods to assess the worldwide scientific output and identify hotspots related to the research on the volume-regulated anion channel (VRAC) from 2014 to 2022. Methods: From Web of Science, we obtained studies related to VRAC published from 2014 to 2022. To analyzed the data, we utilized VOSviewer, a tool for visualizing network, to create networks based on the collaboration between countries, institutions, and authors. Additionally, we performed an analysis of journal co-citation, document citation, and co-occurrence of keywords. Furthermore, we employed CiteSpace (6.1. R6 Advanced) to analyzed keywords and co-cited references with the strongest burst. Results: The final analysis included a total of 278 related articles and reviews, covering the period from 2014 to 2022. The United States emerged as the leading country contributing to this field, while the University of Copenhagen stood out as the most prominent institution. The author with most publications and most citations was Thomas J. Jentsch. Among the cited references, the article by Voss et al. published in Science (2014) gained significant attention for its identification of LRRC8 heteromers as a crucial component of the volume-regulated anion channel VRAC. Pflügers Archiv European Journal of Physiology and Journal of Physiology-London were the leading journals in terms of the quantity of associated articles and citations. Through the analysis of keyword co-occurrence, it was discovered that VRAC is involved in various physiological processes including cell growth, migration, apoptosis, swelling, and myogenesis, as well as anion and organic osmolyte transport including chloride, taurine, glutamate and ATP. VRAC is also associated with related ion channels such as TMEM16A, TMEM16F, pannexin, and CFTR, and associated with various diseases including epilepsy, leukodystrophy, atherosclerosis, hypertension, cerebral edema, stroke, and different types of cancer including gastric cancer, glioblastoma and hepatocellular carcinoma. Furthermore, VRAC is involved in anti-tumor drug resistance by regulating the uptake of platinum-based drugs and temozolomide. Additionally, VRAC has been studied in the context of pharmacology involving DCPIB and flavonoids. Conclusion: The aim of this bibliometric analysis is to provide an overall perspective for research on VRAC. VRAC has become a topic of increasing interest, and our analysis shows that it continues to be a prominent area. This study offers insights into the investigation of VRAC channel and may guide researchers in identifying new directions for future research.

Volume-regulated anion channels (VRACs) and the acid-sensitive outwardly rectifying anion channel (ASOR) mediate flux of chloride and small organic anions. Although known for a long time, they were only recently identified at the molecular level. VRACs are heteromers consisting of LRRC8 proteins A to E. Combining the essential LRRC8A with different LRRC8 paralogues changes key properties of VRAC such as conductance or substrate selectivity, which is how VRACs are involved in multiple physiological functions including regulatory volume decrease, cell proliferation and migration, cell death, purinergic signalling, fat and glucose metabolism, insulin signalling, and spermiogenesis. VRACs are also involved in pathological conditions, such as the neurotoxic release of glutamate and aspartate. Certain VRACs are also permeable to larger, organic anions, including antibiotics and anti-cancer drugs, making them an interesting therapeutic target. ASOR, also named proton-activated chloride channel (PAC), is formed by TMEM206 homotrimers on the plasma membrane and on endosomal compartments where it mediates chloride flux in response to extracytosolic acidification and plays a role in the shrinking and maturation of macropinosomes. ASOR has been shown to underlie neuronal swelling which causes cell death after stroke as well as promoting the metastasis of certain cancers, making them intriguing therapeutic targets as well.

Cl- movement and Cl--sensitive signal pathways contributes to the survival and switch of inflammatory phenotype of microglia and are believed to play a key role in the inflammatory brain injury after ischemic stroke. Here, we demonstrated an important role of Cl- transmembrane transporter Swell1, in the survival and M2-like polarization of microglia in ischemic stroke. Knockdown or overexpression of Swell1 in cultured microglia inhibited or increased hypotonic-activated Cl- currents, respectively, and these changes were completely blocked by the volume-regulated anion channels (VRACs) inhibitor DCPIB. Swell1 conditional knock-in mice promoted microglia survival in ischemic brain region and resulted in significant reductions in neural cell death, infarction volume and neurological deficits following transient middle cerebral artery occlusion (tMCAO). Using gene manipulating technique and pharmacological inhibitors, we further revealed that Swell1 opening led to SGK1 (a Cl--sensitive kinase)-mediated activation of FOXO3a/CREB as well as WNK1 (another Cl--sensitive kinase)-mediated SPAK/OSR1-CCCs activation, which promoted microglia survival and M2-like polarization, thereby attenuating neuroinflammation and ischemic brain injury. Taken together, our results demonstrated that Swell1 is an essential component of microglia VRACs and its activation protects against ischemic brain injury through promoting microglia survival and M2-like polarization.

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In the nervous system, the concentration of Cl- in neurons that express GABA receptors plays a key role in establishing whether these neurons are excitatory, mostly during early development, or inhibitory. Thus, much attention has been dedicated to understanding how neurons regulate their intracellular Cl- concentration. However, regulation of the extracellular Cl- concentration by other cells of the nervous system, including glia and microglia, is as important because it ultimately affects the Cl- equilibrium potential across the neuronal plasma membrane. Moreover, Cl- ions are transported in and out of the cell, via either passive or active transporter systems, as counter ions for K+ whose concentration in the extracellular environment of the nervous system is tightly regulated because it directly affects neuronal excitability. In this book chapter, we report on the Cl- channel types expressed in the various types of glial cells focusing on the role they play in the function of the nervous system in health and disease. Furthermore, we describe the types of stimuli that these channels are activated by, the other solutes that they may transport, and the involvement of these channels in processes such as pH regulation and Regulatory Volume Decrease (RVD). The picture that emerges is one of the glial cells expressing a variety of Cl- channels, encoded by members of different gene families, involved both in short- and long-term regulation of the nervous system function. Finally, we report data on invertebrate model organisms, such as C. elegans and Drosophila, that are revealing important and previously unsuspected functions of some of these channels in the context of living and behaving animals.

Chondrocyte hypertrophy is a significant factor in cartilage development, yet the molecular mechanism for cell volume expand during the process is remains unclear. In the present study, the relationship between Swell1, a cell volume regulated anion channel, and chondrocyte hypertrophy was explored. The results reveal that the spatiotemporal expression of Swell1 was similar with the development process of hypertrophic chondrocytes in condyles. Through Col10a1 mediated knock out of Swell1 in hypertrophy chondrocytes, we found that there are less obvious boundary between different condylar cartilage layers in which increased hypertrophic chondrocytes were scattered in all three cartilage layers. The cortical bone mass and bone mineral density in the subchondral bone significantly increased. Additionally, knock out of Swell1 could increase the expression of OCN in the femur condyle. Based on the aforementioned findings, a conclusion could be drawn that Swell1 is a significant factor in chondrocyte hypertrophy during the condylar osteochondral development process, and there was some difference between the mandibular and femur condyles, which will provide some new clues for understanding the development of cartilage and related diseases.

Rationale: Chronic ethanol consumption as a public health problem worldwide boosts the development of chronic liver diseases in hepatitis B virus (HBV)-infected patients. Arachidonic acid metabolite prostaglandin E2 (PGE2) activates regulatory T cells (Tregs) function. Here, we aim to investigate the underlying mechanism by which chronic ethanol consumption enriches the HBV-induced abnormal lipid metabolism and Tregs. Methods: The si-RNAs were used to weaken the expression of SWELL1 in HepG2, HepG2.2.15 and K180 cancer cell lines, followed by RNA sequencing from HepG2 cells. Arachidonic acid metabolite PGE2 and LTD4 were measured by ELISA assay in vivo and in vitro. Western blot analysis and RT-qPCR were used to examine HBx and SWELL1 and transcriptional factor Sp1 in clinical HCC samples and cell lines. The effect of chronic ethanol consumption on Tregs was tested by flow cytometry in HBV-Tg mice. The splenic Tregs were collected and analyzed by RNA sequencing. Results: The cooperative effect of ethanol and HBV in abnormal lipid metabolism was observed in vivo and in vitro. The depression of SWELL1 (or HBx) resulted in the reduction of lipid content and arachidonic acid metabolite, correlating with suppression of relative gene atlas. Ethanol and SWELL1 elevated the levels of PGE2 or LTD4 in the liver of mice and cell lines. Interestingly, the ethanol modulated abnormal lipid metabolism through activating HBx/Sp1/SWELL1/arachidonic acid signaling. Chronic ethanol consumption remarkably increased the population of PBL Tregs and splenic Tregs in HBV-Tg mice, consistently with the enhanced expression of PD-L1 in vivo and in vitro. Mechanically, RNA-seq data showed that multiple genes were altered in the transcriptomic atlas of Tregs sorting from ethanol-fed mice or HBV-Tg mice. Conclusion: The chronic ethanol intake enriches the HBV-enhanced abnormal lipid metabolism through HBx/SWELL1/arachidonic acid signaling and activates Tregs in mice.

BACKGROUND: SWELL1 was recently demonstrated to be an indispensable part of the volume-regulated anion channel (VRAC). VRAC is reported to participate in cell proliferation, survival, and migration. However, the correlation between SWELL1 and hepatocellular carcinoma (HCC) remains poorly-understood. In this study, we tried to explore the role of SWELL1 in HCC.
METHODS: Immunohistochemistry and quantitative real-time-PCR (qRT-PCR) was used to measure SWELL1 expression in HCC samples obtained from patients with HCC. The effects of SWELL1 on HCC cell proliferation, apoptosis, and metastasis were analysed by corresponding cytological experiments including Cell Counting Kit-8 (CCK8), colony-forming, 5-ethynyl-2\'-deoxyuridine (EdU), cell cycle analysis, TUNEL, Annexin V and PI staining, wound healing, transwell, and so on. BALB/c nude mice were used for the in vivo assays. qRT-PCR and western blotting was performed for molecular mechanisms.
RESULTS: SWELL1 was highly expressed in HCC tissues, and related to the poor prognosis. In vitro, the over-expression of SWELL1 significantly induced cell proliferation and migration, and inhibited apoptosis, whereas suppressing SWELL1 had the opposite effects. Moreover, knockdown of SWELL1 suppressed the growth and metastasis of HCC in vivo. Further experiments revealed that SWELL1 induced cell growth by activating the cyclinD1/CDK2 pathway via the connection with PKCa at the signalling level, and regulated cell migration through the JNK pathway in HCC.
CONCLUSIONS: SWELL1 acts as a promoter in the growth and metastasis of HCC cells and may be a potential intervention target for HCC. FUND: This work is supported by the National Natural Science Foundation of China (No. 81572422, 81700515).