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.

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.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

scholarly journals Cell swelling increases bile flow and taurocholate excretion into bile in isolated perfused rat liver

1992 ◽  
Vol 281 (3) ◽  
pp. 593-595 ◽  
Author(s):  
C Hallbrucker ◽  
F Lang ◽  
W Gerok ◽  
D Häussinger
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Cell Volume ◽  
Rat Liver ◽  
Bile Flow ◽  
Cell Swelling ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Volume Changes ◽  
Close Relationship

The effects of aniso-osmotically and amino-acid-induced cell-volume changes on bile flow and biliary taurocholate excretion were studied in isolated perfused rat liver. With taurocholate (100 microM) in the influent perfusate, hypo-osmotic exposure (225 mosmol/l) increased taurocholate excretion into bile and bile flow by 42 and 27% respectively, whereas inhibition by 32 and 47% respectively was observed after hyperosmotic (385 mosmol/l) exposure. The effects of aniso-moticity on taurocholate excretion into bile was observed throughout aniso-osmotic exposure, even after completion of volume-regulatory ion fluxes and were fully reversible upon re-exposure to normo-osmotic media. Hypo-osmotic cell swelling (225 mosmol/l) increased the Vmax. of taurocholate translocation from the sinusoidal compartment into bile about 2-fold. Also, cell swelling induced by glutamine and glycine stimulated both bile flow and biliary taurocholate excretion. There was a close relationship between the aniso-osmotically and amino-acid-induced change of cell volume and taurocholate excretion into bile. The data suggest that liver cell volume plays an important role in regulating bile-acid-dependent bile flow and biliary taurocholate excretion.

scholarly journals OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

2016 ◽  
Vol 38 (4) ◽  
pp. 1652-1662 ◽  
Author(s):  
Bernat Elvira ◽  
Yogesh Singh ◽  
Jamshed Warsi ◽  
Carlos Munoz ◽  
Florian Lang
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Cell Volume Regulation ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Wild Type ◽  
Constitutively Active

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

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 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.

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.

Associations between transports of alanine and cations across cell membrane in rat hepatocytes

1986 ◽  
Vol 251 (5) ◽  
pp. G575-G584 ◽  
Author(s):  
L. O. Kristensen
Keyword(s):  
Amino Acid ◽  
Cell Membrane ◽  
Cell Volume ◽  
Driving Force ◽  
Rat Hepatocytes ◽  
The Body ◽  
Cell Swelling ◽  
Molar Ratio ◽  
Regulatory Processes ◽  
Intracellular Content

Alanine transport across the liver cell membrane is a regulated key process in the amino acid metabolism of the body. The majority of alanine influx in hepatocytes is Na+ dependent and is stimulated by intracellular negativity. The molar ratio between cotransported Na+ and alanine is 1:1. Alanine efflux is stimulated by intracellular Na+, whereas the role of the membrane potential is unclear. The transmembrane Na+ electrochemical gradient seems to be the exclusive driving force for cellular alanine accumulation. At a physiological Na+ gradient, intracellular alanine can exceed the extracellular concentration about 20-fold, but metabolism will exert a conspicuous sink effect. Na+-coupled uptake of alanine appears to be a challenge that triggers a sequence of regulatory events: increased cellular Na+ leads to an increase in active Na+-K+-pumping and thus in K+ influx; influx of alanine and cations tends to increase the cellular content of osmotically active substances implying a tendency to water uptake; cell swelling, even when modest, induces an increase in the permeability of a conductive pathway for K+ leading to net efflux of K+ (with accompanying anions) and cellular hyperpolarization. Net efflux of K+ prevents excessive cell volume increase during amino acid accumulation, whereas hyperpolarization tends to support the driving force for alanine influx (and anion efflux). The pathway for K+ efflux needs further characterization, but it may involve single-file diffusion with Ca2+ as an activator. This model suggests that cell volume regulatory processes mainly serve to compensate for changes in intracellular content of ions and metabolites during activation of specialized cellular processes.

scholarly journals Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

2018 ◽  
Vol 50 (4) ◽  
pp. 1460-1473 ◽  
Author(s):  
Michael Kittl ◽  
Heidemarie Dobias ◽  
Marlena Beyreis ◽  
Tobias Kiesslich ◽  
Christian Mayr ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Amino Acid ◽  
Cell Membrane ◽  
Cell Volume ◽  
Cell Membrane Potential ◽  
Microglial Cells ◽  
Cell Swelling ◽  
Electrophysiological Recordings ◽  
A Cell

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

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