scholarly journals Cryo-EM structure of the volume-regulated anion channel LRRC8

2018 ◽  
Author(s):  
Go Kasuya ◽  
Takanori Nakane ◽  
Takeshi Yokoyama ◽  
Yanyan Jia ◽  
Masato Inoue ◽  
...  
Keyword(s):  
Cell Volume ◽  
Molecular Mechanisms ◽  
Anion Channel ◽  
Membrane Topology ◽  
Cell Swelling ◽  
Cell Functions ◽  
Gap Junction Channel ◽  
Electron Microscopy Analysis ◽  
Extracellular Side ◽  
And Migration

AbstractMaintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

More than just a pressure relief valve: physiological roles of volume-regulated LRRC8 anion channels

2019 ◽  
Vol 400 (11) ◽  
pp. 1481-1496 ◽  
Author(s):  
Lingye Chen ◽  
Benjamin König ◽  
Tianbao Liu ◽  
Sumaira Pervaiz ◽  
Yasmin S. Razzaque ◽  
...  
Keyword(s):  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Anion Channel ◽  
Therapy Resistance ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Anion Channels ◽  
Relief Valve ◽  
Wide Range ◽  
And Migration

Abstract The volume-regulated anion channel (VRAC) is a key player in the volume regulation of vertebrate cells. This ubiquitously expressed channel opens upon osmotic cell swelling and potentially other cues and releases chloride and organic osmolytes, which contributes to regulatory volume decrease (RVD). A plethora of studies have proposed a wide range of physiological roles for VRAC beyond volume regulation including cell proliferation, differentiation and migration, apoptosis, intercellular communication by direct release of signaling molecules and by supporting the exocytosis of insulin. VRAC was additionally implicated in pathological states such as cancer therapy resistance and excitotoxicity under ischemic conditions. Following extensive investigations, 5 years ago leucine-rich repeat-containing family 8 (LRRC8) heteromers containing LRRC8A were identified as the pore-forming components of VRAC. Since then, molecular biological approaches have allowed further insight into the biophysical properties and structure of VRAC. Heterologous expression, siRNA-mediated downregulation and genome editing in cells, as well as the use of animal models have enabled the assessment of the proposed physiological roles, together with the identification of new functions including spermatogenesis and the uptake of antibiotics and platinum-based cancer drugs. This review discusses the recent molecular biological insights into the physiology of VRAC in relation to its previously proposed roles.

scholarly journals A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

1999 ◽  
Vol 113 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Dayue Duan ◽  
Suzanne Cowley ◽  
Burton Horowitz ◽  
Joseph R. Hume
Keyword(s):  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Phosphorylation Site ◽  
Cardiac Cells ◽  
Site Directed Mutagenesis ◽  
Cell Swelling ◽  
Transport Systems ◽  
Serine Residue ◽  
Channel Regulation

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

scholarly journals Cell Death Induction and Protection by Activation of Ubiquitously Expressed Anion/Cation Channels. Part 2: Functional and Molecular Properties of ASOR/PAC Channels and Their Roles in Cell Volume Dysregulation and Acidotoxic Cell Death

2021 ◽  
Vol 9 ◽  
Author(s):  
Yasunobu Okada ◽  
Kaori Sato-Numata ◽  
Ravshan Z. Sabirov ◽  
Tomohiro Numata
Keyword(s):  
Cell Death ◽  
Cell Volume ◽  
Apoptotic Cell ◽  
Cell Volume Regulation ◽  
Anion Channel ◽  
Cell Swelling ◽  
Dimensional Structure ◽  
Cation Channels ◽  
Anion Channels ◽  
Animal Cells

For survival and functions of animal cells, cell volume regulation (CVR) is essential. Major hallmarks of necrotic and apoptotic cell death are persistent cell swelling and shrinkage, and thus they are termed the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. A number of ubiquitously expressed anion and cation channels play essential roles not only in CVR but also in cell death induction. This series of review articles address the question how cell death is induced or protected with using ubiquitously expressed ion channels such as swelling-activated anion channels, acid-activated anion channels, and several types of TRP cation channels including TRPM2 and TRPM7. In the Part 1, we described the roles of swelling-activated VSOR/VRAC anion channels. Here, the Part 2 focuses on the roles of the acid-sensitive outwardly rectifying (ASOR) anion channel, also called the proton-activated chloride (PAC) anion channel, which is activated by extracellular protons in a manner sharply dependent on ambient temperature. First, we summarize phenotypical properties, the molecular identity, and the three-dimensional structure of ASOR/PAC. Second, we highlight the unique roles of ASOR/PAC in CVR dysfunction and in the induction of or protection from acidotoxic cell death under acidosis and ischemic conditions.

scholarly journals GCK-3, a Newly Identified Ste20 Kinase, Binds To and Regulates the Activity of a Cell Cycle–dependent ClC Anion Channel

2005 ◽  
Vol 125 (2) ◽  
pp. 113-125 ◽  
Author(s):  
Jerod Denton ◽  
Keith Nehrke ◽  
Xiaoyan Yin ◽  
Rebecca Morrison ◽  
Kevin Strange
Keyword(s):  
Cell Cycle ◽  
Signaling Pathways ◽  
Cell Volume ◽  
Specific Binding ◽  
Anion Channel ◽  
Fluid Secretion ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Binding Motif ◽  
Dependent Manner

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

scholarly journals Drosophila Bestrophin-1 Chloride Current Is Dually Regulated by Calcium and Cell Volume

2007 ◽  
Vol 130 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Li-Ting Chien ◽  
H. Criss Hartzell
Keyword(s):  
Cell Volume ◽  
Anion Channel ◽  
Cell Swelling ◽  
Macular Dystrophy ◽  
Dependent Manner ◽  
S2 Cells ◽  
New Family ◽  
Drosophila S2 Cells ◽  
Cl Channels ◽  
Dose Dependent

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl− channels that are activated by Ca2+. While the regulation of bestrophin currents has focused on intracellular Ca2+, little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl− currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca2+ and cell volume. The bestrophin Cl− currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl− current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca2+ regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca2+ and the Ca2+-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca2+ potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca2+, Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.

scholarly journals Acid- and Volume-Sensitive Chloride Currents in Microglial Cells

2019 ◽  
Vol 20 (14) ◽  
pp. 3475 ◽  
Author(s):  
Michael Kittl ◽  
Katharina Helm ◽  
Marlena Beyreis ◽  
Christian Mayr ◽  
Martin Gaisberger ◽  
...  
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Cell Types ◽  
Microglial Cells ◽  
Cell Swelling ◽  
Mean Cell Volume ◽  
Current Activation ◽  
And Migration

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl− current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl− currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I− > Cl− > gluconate− permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl− conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

scholarly journals Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

2012 ◽  
Vol 302 (12) ◽  
pp. C1702-C1712 ◽  
Author(s):  
Hiroaki Miyazaki ◽  
Kevin Strange
Keyword(s):  
Amino Acid ◽  
Cell Volume ◽  
Conformational Changes ◽  
Anion Channel ◽  
Cell Swelling ◽  
Channel Activity ◽  
Wild Type ◽  
Multisite Phosphorylation ◽  
Cycle Dependent ◽  
Linker Domain

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.

The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology

2015 ◽  
Vol 396 (9-10) ◽  
pp. 975-990 ◽  
Author(s):  
Tobias Stauber
Keyword(s):  
Cell Biology ◽  
Regulatory Volume Decrease ◽  
Anion Channel ◽  
Cell Swelling ◽  
Transepithelial Transport ◽  
Organic Osmolytes ◽  
Physiological Processes ◽  
Starting Point ◽  
And Migration ◽  
Volume Decrease

Abstract Cellular volume regulation is fundamental for numerous physiological processes. The volume-regulated anion channel, VRAC, plays a crucial role in regulatory volume decrease. This channel, which is ubiquitously expressed in vertebrates, has been vastly characterized by electrophysiological means. It opens upon cell swelling and conducts chloride and arguably organic osmolytes. VRAC has been proposed to be critically involved in various cellular and organismal functions, including cell proliferation and migration, apoptosis, transepithelial transport, swelling-induced exocytosis and intercellular communication. It may also play a role in pathological states like cancer and ischemia. Despite many efforts, the molecular identity of VRAC had remained elusive for decades, until the recent discovery of heteromers of LRRC8A with other LRRC8 family members as an essential VRAC component. This identification marks a starting point for studies on the structure-function relation, for molecular biological investigations of its cell biology and for re-evaluating the physiological roles of VRAC. This review recapitulates the identification of LRRC8 heteromers as VRAC components, depicts the similarities between LRRC8 proteins and pannexins, and discussed whether VRAC conducts larger osmolytes. Furthermore, proposed physiological functions of VRAC and the present knowledge about the physiological significance of LRRC8 proteins are summarized and collated.

Acquired cisplatin resistance in human ovarian A2780 cancer cells correlates with shift in taurine homeostasis and ability to volume regulate

2014 ◽  
Vol 307 (12) ◽  
pp. C1071-C1080 ◽  
Author(s):  
Belinda Halling Sørensen ◽  
Unnur Arna Thorsteinsdottir ◽  
Ian Henry Lambert
Keyword(s):  
Cell Volume ◽  
Cisplatin Resistance ◽  
Cell Damage ◽  
Anion Channel ◽  
Cell Swelling ◽  
Taurine Release ◽  
Drug Induced ◽  
Leukotriene Receptor ◽  
Taurine Uptake ◽  
Anion Channel Blocker

Cisplatin resistance is a major challenge in the treatment of cancer and develops through reduced drug accumulation and an increased ability to avoid drug-induced cell damage, cell shrinkage, and hence initiation of apoptosis. Uptake and release of the semiessential amino acid taurine contribute to cell volume homeostasis, and taurine has been reported to have antiapoptotic effects. Here we find that volume-sensitive taurine release in cisplatin-sensitive [wild-type (WT)] human ovarian cancer A2780 cells is reduced in the presence of the phospholipase A2 inhibitor bromenol lactone, the 5-lipoxygenase (5-LO) inhibitor ETH 615–139, and the cysteine leukotriene receptor 1 (CysLT1) antagonist zafirlukast and impaired by the anion channel blocker DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonate). Comparing WT and cisplatin-resistant (RES) A2780 cells we also find that evasion of cisplatin-induced cell death in RES A2780 cells correlates with an increased accumulation of taurine, due to an increased taurine uptake and a concomitant impairment of the volume-sensitive taurine release pathway, as well an inability to reduce cell volume after osmotic cell swelling. Downregulation of volume-sensitive taurine release in RES A2780 cells correlates with reduced expression of the leucine-rich repeat-containing protein 8A (LRRC8A). Furthermore, acute (18 h) exposure to cisplatin (5–10 μM) increases taurine release and LRRC8A expression in WT A2780 cells whereas cisplatin has no effect on LRRC8A expression in RES A2780 cells. It is suggested that shift in LRRC8A activity can be used as biomarker for apoptotic progress and acquirement of drug resistance.

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

2012 ◽  
Vol 303 (4) ◽  
pp. C416-C426 ◽  
Author(s):  
Venkanna Pasham ◽  
Anand Rotte ◽  
Wenting Yang ◽  
Christine Zelenak ◽  
Madhuri Bhandaru ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dendritic Cells ◽  
Cell Volume ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Ros Production ◽  
Ros Formation ◽  
The Difference ◽  
And Migration ◽  
The Impact

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice ( osr KI) and wild-type mice ( osr WT). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pHi) from 2′,7′- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na+/H+ exchanger activity from Na+-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr KI DCs. Cell volume and pHi were similar in osr KI and osr WT DCs, but Na+/H+ exchanger activity and ROS production were higher in osr KI than in osr WT DCs. Before LPS treatment, migration was similar in osr KI and osr WT DCs. LPS (1 μg/ml), however, increased migration of osr WT DCs but not of osr KI DCs. Na+/H+ exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na+/H+ exchanger activity, and pHi to a greater extent in osr KI than in osr WT DCs. LPS increased cell volume, Na+/H+ exchanger activity, and ROS formation in osr WT DCs but not in osr KI DCs and blunted the difference between osr KI and osr WT DCs. Na+/H+ exchanger activity in osr WT DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr KI DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na+/H+ exchanger activity in osr WT DCs but not in osr KI DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na+/H+ exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr WT mice. In conclusion, partial OSR1 deficiency influences Na+/H+ exchanger activity, ROS formation, and migration of dendritic cells.

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