Comprehensive review on the novel immunotherapy target: Leucine-rich repeat-containing 8A/volume-regulated anion channel.

Leucine-rich repeat-containing 8A (LRRC8A) is a key component of the volume-regulated anion channel (VRAC) that influences essential homeostatic processes in various immune cells. These processes include the regulation of cell volume and membrane potential and the facilitation of the transport of organic agents used as anticancer drugs and immune-stimulating factors. Therefore, understanding the structure-function relationship of LRRC8A, exploring its physiological role in immunity, assessing its efficacy in treating diseases, and advancing the development of compounds that regulate its activity are important research frontiers. This review emphasized the emerging field of LRRC8A, outlined its structure and function, and summarized its role in immune cell development and immune cell-mediated antiviral and antitumor effects. Additionally, it explored the potential of LRRC8A as an immunotherapeutic target, offering insights into resolving persistent challenges and future research directions.

Piezo1, the new actor in cell volume regulation.

All animal cells control their volume through a complex set of mechanisms, both to counteract osmotic perturbations of the environment and to enable numerous vital biological processes, such as proliferation, apoptosis, and migration. The ability of cells to adjust their volume depends on the activity of ion channels and transporters which, by moving K+, Na+, and Cl- ions across the plasma membrane, generate the osmotic gradient that drives water in and out of the cell. In 2010, Patapoutian's group identified a small family of evolutionarily conserved, Ca2+-permeable mechanosensitive channels, Piezo1 and Piezo2, as essential components of the mechanically activated current that mediates mechanotransduction in vertebrates. Piezo1 is expressed in several tissues and its opening is promoted by a wide range of mechanical stimuli, including membrane stretch/deformation and osmotic stress. Piezo1-mediated Ca2+ influx is used by the cell to convert mechanical forces into cytosolic Ca2+ signals that control diverse cellular functions such as migration and cell death, both dependent on changes in cell volume and shape. The crucial role of Piezo1 in the regulation of cell volume was first demonstrated in erythrocytes, which need to reduce their volume to pass through narrow capillaries. In HEK293 cells, increased expression of Piezo1 was found to enhance the regulatory volume decrease (RVD), the process whereby the cell re-establishes its original volume after osmotic shock-induced swelling, and it does so through Ca2+-dependent modulation of the volume-regulated anion channels. More recently we reported that Piezo1 controls the RVD in glioblastoma cells via the modulation of Ca2+-activated K+ channels. To date, however, the mechanisms through which this mechanosensitive channel controls cell volume and maintains its homeostasis have been poorly investigated and are still far from being understood. The present review aims to provide a broad overview of the literature discussing the recent advances on this topic.

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.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Hypoxia, Ion Channels and Glioblastoma Malignancy.

The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.

Trends in volume-regulated anion channel (VRAC) research: visualization and bibliometric analysis from 2014 to 2022.

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.

Fluid Osmolarity Modulates the Rate of Spontaneous Contraction of Lymphatic Vessels and Lymph Flow by Means of a Cooperation between TRPV and VRAC Channels.

Lymphatic vessels are capable of sustaining lymph formation and propulsion via an intrinsic mechanism based on the spontaneous contraction of the lymphatic muscle in the wall of lymphatic collectors. Exposure to a hyper- or hypo-osmolar environment can deeply affect the intrinsic contraction rate and therefore alter lymph flow. In this work, we aimed at defining the putative receptors underlying such a response. Functional experiments were conducted in ex vivo rat diaphragmatic specimens containing spontaneously contracting lymphatic vessels that were exposed to either hyper- or hypo-osmolar solutions. Lymphatics were challenged with blockers to TRPV4, TRPV1, and VRAC channels, known to respond to changes in osmolarity and/or cell swelling and expressed by lymphatic vessels. Results show that the normal response to a hyperosmolar environment is a steady decrease in the contraction rate and lymph flow and can be prevented by blocking TRPV1 channels with capsazepine. The response to a hyposmolar environment consists of an early phase of an increase in the contraction rate, followed by a decrease. The early phase is abolished by blocking VRACs with DCPIB, while blocking TRPV4 mainly resulted in a delay of the early response. Overall, our data suggest that the cooperation of the three channels can shape the response of lymphatic vessels in terms of contraction frequency and lymph flow, with a prominent role of TRPV1 and VRACs.

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.

Syringin inhibits endogenous volume regulated anion channel currents of HEK293 cells in hypotonic circumstances.

Syringin is a natural chemical compound first isolated from the bark of lilac and is known to have neuroprotective effects in middle cerebral artery occlusion (MCAO). Volume regulated anion channel (VRAC) is a cell swelling-activated anion channel, which is implicated in brain ischemia. However, the mechanism underlying the syringin protecting the neuron from damage in MCAO is still unclear. We hypothesized that syringin has an inhibitory effect on the opening of VRAC channels. To access the effect of syringin on VRAC currents and predict how syringin interacts with VRAC proteins, we performed whole-cell patch-clamp experiments using HEK293 cells. Initially, HEK293 cells were perfused with isotonic extracellular solution, followed by hypotonic extracellular solution to stimulate endogenous VRAC currents. Once the VRAC currents reached a steady state, the hypotonic solution containing syringin was perfused to study the effect of syringin on VRAC currents. The potential interaction between syringin and the VRAC protein was investigated using molecular docking as a predictive model. In this study, we found that syringin moderately inhibited VRAC currents in a dose-dependent manner. The potential binding of syringin to LRRC8 protein was predicted through in silico molecular docking, which suggests an affinity of -6.6 kcal/mol and potential binding sites of arginine 103 and leucine 101. Our results herein characterize syringin as an inhibitor of the VRAC channels, which provides valuable insights for the future development of VRAC channel inhibitors.

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.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines.

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4-/- knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4-/- cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride.

OBJECTIVE: A simple and robust head space/gas chromatography with flame ionisation sensor (HS/GC/FIS) approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride (TRD) drug ingredient and its formulation is described. METHOD: This HS/GC/FIS approach was based on separation and analysis of CH3Cl content on DB-624 [75.0m - length, 0.53mm - internal diameter, 3.0µm - film thickness] column using nitrogen as carrier gas flowing through the column at 3mL/min stream rate. Detection of eluted CH3Cl was accomplished with flame ionization sensor at a set temperature of 260̊C. RESULTS: The optimised HS/GC/FIS methodological approach was thoroughly validated, demonstrating that it was linear with range of 5.0ppm to 1508.4ppm, sensitive with detection limit of 1.65ppm and quantification limit of 5.01ppm, reproducible with RSD values of 2.10-2.35%, accurate with recoveries of 81.9-99.0%, robust with percent variation of 7.5-12.22% with respect to changes in oven temperature, injector temperature, detector temperature and practical for regular TRD quality control. CONCLUSION: The findings revealed that with this optimised HS/GC/FIS methodological approach, the trace amounts of carcinogenic impurity (methyl chloride) in TRD drug ingredient and formulation could be successfully measured.

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 CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

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.

Novel Perspective of Cardiovascular Diseases: Volume-Regulatory Anion Channels in the Cell Membrane.

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide. Although there are established mechanisms and preventions for CVDs, they are not totally elucidative and effective. Emerging evidence suggests that the dysregulation of ion channels in the cell membranes underpins the dysfunction of the cardiovascular system. To date, a variety of cation channels have been widely recognized as important targets for the treatment of CVDs. As a critical component of the anion channels, the volume-regulated anion channel (VRAC) is involved in a series of cell functions by the volume regulation and maintenance of membrane homeostasis. It has been confirmed to play crucial roles in cell action potential generation, cell proliferation, differentiation and apoptosis, and the VRAC appears to be a major participant in metabolic processes during CVDs. This review summarizes the current evidence and progress concerning the VRAC, to determine the future directions and challenges for CVDs for both preventive and therapeutic purposes.

Molecular determinants underlying volume-regulated anion channel subunit-dependent oxidation sensitivity.

The volume-regulated anion channel (VRAC) is formed by LRRC8 subunits. Besides their role in the maintenance of cell homeostasis, VRACs are critically involved in oxidative stress mechanisms: reactive oxygen species directly modulate VRACs in a subunit-dependent manner. It was reported that LRRC8A-LRRC8E heteromeric channels are activated by oxidation, whereas LRRC8A-LRRC8C heteromers are inhibited. Here we adopted chimeric- as well as concatemeric-based strategies to identify residues responsible for the divergent effect of oxidants. We identified two cysteines in the first two leucine rich repeats of LRRC8E, C424 and C448, as the targets of oxidation. Oxidation likely results in the formation of a disulfide bond between the two cysteines, which in turn induces a conformational change leading to channel activation. Additionally, we found that LRRC8C inhibition is caused by oxidation of the first methionine. We thus identified crucial molecular elements involved in channel activation, which are conceivably relevant in determining physiological ROS effects. KEY POINTS: Volume-regulated anion channels (VRACs) are heterohexameric complexes composed of an essential LRRC8A subunit and a variable number of LRRC8B-E subunits. VRACs are directly regulated by oxidation, with LRRC8A-LRRC8E heteromers being potentiated and LRRC8A-LRRC8C heteromers being inhibited by oxidation. We identified two LRRC8E specific intracellular cysteines that form a disulfide bond upon oxidation leading to LRRC8A-LRRC8E potentiation. Inhibition of LRRC8A-LRRC8C heteromers is mediated by the oxidation of the start methionine, being additionally dependent on the identity of the LRR domain. Besides providing physiological insights concerning the outcome of reactive oxygen species modulation, the results point to key structural elements involved in VRAC activation.

Recent Advances in the Structural Biology of the Volume-Regulated Anion Channel LRRC8.

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC), which is activated by cell swelling and releases chloride ions (Cl-) or other osmolytes to counteract cell swelling. Although the LRRC8 protein family was identified as the molecular entity of VRAC only in 2014, due to recent advances in cryo-electron microscopy (cryo-EM), various LRRC8 structures, including homo-hexameric LRRC8A and LRRC8D structures, as well as inhibitor-bound and synthetic single-domain antibody-bound homo-hexameric LRRC8A structures, have been reported, thus extending our understanding of the molecular mechanisms of this protein family. In this review, we describe the important features of LRRC8 provided by these structures, particularly the overall architectures, and the suggested mechanisms underlying pore inhibition and allosteric modulation by targeting the intracellular leucine-rich repeat (LRR) domain.