scholarly journals Intracellular and extracellular loops of LRRC8 are essential for volume-regulated anion channel function

2018 ◽  
Vol 150 (7) ◽  
pp. 1003-1015 ◽  
Author(s):  
Toshiki Yamada ◽  
Kevin Strange
Keyword(s):  
Anion Channel ◽  
Transmembrane Domains ◽  
Channel Structure ◽  
Cell Swelling ◽  
Voltage Sensitivity ◽  
Intracellular Loop ◽  
Heteromeric Channel ◽  
Extracellular Loops ◽  
And Function ◽  
Essential Subunit

The volume-regulated anion channel (VRAC) is expressed ubiquitously in vertebrate cells and mediates swelling-induced release of Cl− and organic solutes. Recent studies by several laboratories have demonstrated conclusively that VRAC is encoded by members of the leucine-rich repeat containing 8 (Lrrc8) gene family, which comprises five members, termed Lrrc8a–e. Numerous observations indicate that VRAC is a heteromeric channel comprising the essential subunit LRRC8A and one or more of the other LRRC8 paralogs. Here we demonstrate that the intracellular loop (IL) connecting transmembrane domains 2 and 3 of LRRC8A and the first extracellular loop (EL1) connecting transmembrane domains 1 and 2 of LRRC8C, LRRC8D, or LRRC8E are both essential for VRAC activity. We generate homomeric VRACs by replacing EL1 of LRRC8A with that of LRRC8C and demonstrate normal regulation by cell swelling and shrinkage. We also observe normal volume-dependent regulation in VRAC homomers in which the IL of LRRC8C, LRRC8D, or LRRC8E is replaced with the LRRC8A IL. A 25–amino acid sequence unique to the LRRC8A IL is sufficient to generate homomeric VRAC activity when inserted into the corresponding region of LRRC8C and LRRC8E. LRRC8 chimeras containing these partial LRRC8A IL sequences exhibit altered anion permeability, rectification, and voltage sensitivity, suggesting that the LRRC8A IL plays a role in VRAC pore structure and function. Our studies provide important new insights into the structure/function roles of the LRRC8 EL1 and IL. Homomeric LRRC8 channels will simplify future studies aimed at understanding channel structure and the longstanding and vexing problem of how VRAC is regulated by cell volume changes.

scholarly journals A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel

2019 ◽  
Vol 151 (2) ◽  
pp. 100-117 ◽  
Author(s):  
Kevin Strange ◽  
Toshiki Yamada ◽  
Jerod S. Denton
Keyword(s):  
Structure Function ◽  
Cell Volume ◽  
Cell Volume Regulation ◽  
Anion Channel ◽  
Clear Understanding ◽  
Physiological Processes ◽  
Heteromeric Channel ◽  
Key Aspects ◽  
And Function ◽  
Vexing Problem

The swelling-activated anion channel VRAC has fascinated and frustrated physiologists since it was first described in 1988. Multiple laboratories have defined VRAC’s biophysical properties and have shown that it plays a central role in cell volume regulation and possibly other fundamental physiological processes. However, confusion and intense controversy surrounding the channel’s molecular identity greatly hindered progress in the field for >15 yr. A major breakthrough came in 2014 with the demonstration that VRAC is a heteromeric channel encoded by five members of the Lrrc8 gene family, Lrrc8A–E. A mere 4 yr later, four laboratories described cryo-EM structures of LRRC8A homomeric channels. As the melee of structure/function and physiology studies begins, it is critical that this work be framed by a clear understanding of VRAC biophysics, regulation, and cellular physiology as well as by the field’s past confusion and controversies. That understanding is essential for the design and interpretation of structure/function studies, studies of VRAC physiology, and studies aimed at addressing the vexing problem of how the channel detects cell volume changes. In this review we discuss key aspects of VRAC biophysics, regulation, and function and integrate these into our emerging understanding of LRRC8 protein structure/function.

scholarly journals Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel

2021 ◽  
Vol 12 ◽  
Author(s):  
Mikel Rezola ◽  
Aida Castellanos ◽  
Xavier Gasull ◽  
Núria Comes
Keyword(s):  
Glutamate Release ◽  
Anion Channel ◽  
Cell Swelling ◽  
Physical Interaction ◽  
Biophysical Properties ◽  
Hek 293 ◽  
Caveolin 1 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Essential Subunit

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

scholarly journals Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops

1991 ◽  
Vol 100 (3) ◽  
pp. 567-578 ◽  
Author(s):  
S. Rahman ◽  
W.H. Evans
Keyword(s):  
Gap Junctions ◽  
Cell Communication ◽  
Amino Acid Sequences ◽  
Transmembrane Domains ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Intracellular Loop ◽  
Extracellular Loops ◽  
Disulphide Bonds ◽  
Peptide Antibodies

A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplasmically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cell-cell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of connexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

2002 ◽  
Vol 283 (2) ◽  
pp. C569-C578 ◽  
Author(s):  
Alexander A. Mongin ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Atp Release ◽  
Anion Channel ◽  
P2y Receptors ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Medium Osmolarity ◽  
Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

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.

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding

1994 ◽  
Vol 267 (5) ◽  
pp. C1203-C1209 ◽  
Author(s):  
P. S. Jackson ◽  
R. Morrison ◽  
K. Strange
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Pipette Solution ◽  
Organic Osmolyte ◽  
Atp Levels ◽  
Patch Pipette ◽  
Taurine Efflux

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

scholarly journals PP2Cδ Controls the Differentiation and Function of Dendritic Cells Through Regulating the NSD2/mTORC2/ACLY Pathway

2022 ◽  
Vol 12 ◽  
Author(s):  
Nianyin Lv ◽  
Sufeng Jin ◽  
Zihao Liang ◽  
Xiaohui Wu ◽  
Yanhua Kang ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Experimental Autoimmune Encephalomyelitis ◽  
Essential Role ◽  
Proteasomal Degradation ◽  
Autoimmune Encephalomyelitis ◽  
And Function ◽  
Essential Subunit ◽  
Experimental Autoimmune ◽  
Sustained Activation

Dendritic cells (DCs) are recognized as a key orchestrator of immune response and homeostasis, deregulation of which may lead to autoimmunity such as experimental autoimmune encephalomyelitis (EAE). Herein we show that the phosphatase PP2Cδ played a pivotal role in regulating DC activation and function, as PP2Cδ ablation caused aberrant maturation, activation, and Th1/Th17-priming of DCs, and hence induced onset of exacerbated EAE. Mechanistically, PP2Cδ restrained the expression of the essential subunit of mTORC2, Rictor, primarily through de-phosphorylating and proteasomal degradation of the methyltransferase NSD2 via CRL4DCAF2 E3 ligase. Loss of PP2Cδ in DCs accordingly sustained activation of the Rictor/mTORC2 pathway and boosted glycolytic and mitochondrial metabolism. Consequently, ATP-citrate lyse (ACLY) was increasingly activated and catalyzed acetyl-CoA for expression of the genes compatible with hyperactivated DCs under PP2Cδ deletion. Collectively, our findings demonstrate that PP2Cδ has an essential role in controlling DCs activation and function, which is critical for prevention of autoimmunity.

scholarly journals Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

2018 ◽  
Author(s):  
David M. Kern ◽  
SeCheol Oh ◽  
Richard K. Hite ◽  
Stephen G. Brohawn
Keyword(s):  
Lipid Bilayers ◽  
Critical Role ◽  
Anion Channel ◽  
Lipid Binding ◽  
Channel Structure ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs ◽  
Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

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