Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells

Tonini, Raffaella; Ferroni, A; Valenzuela, Stella M.; Warton, Kristina; Campbell, Terry; Breit, Samuel N.; Mazzanti, Michele

doi:10.1096/fasebj.14.9.1171

Cited by 100 publications

(128 citation statements)

References 13 publications

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“…Several alternative models have been proposed for the structure of the membrane inserted CLIC proteins (8,11). Sequence analyses indicated that Cys-24 to Val-46 might form a transmembrane helix in the channel state (13). Previously, this seemed unlikely due to the GST-like monomer structure (8), which would require an unfolding of the N-domain on membrane insertion.…”

Section: Discussionmentioning

confidence: 99%

“…Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 10 -12). Electrophysiological studies of FLAG epitope-tagged CLIC1 in the plasma membranes of Chinese hamster ovary K1 cells suggest an extracellular N terminus and a cytoplasmic C terminus (13). These experiments imply that in the channel form of the protein, CLIC1, crosses the membrane an odd number of times.…”

mentioning

confidence: 86%

“…4C and Table II). Tip dip electrophysiological studies show that both the monomer and the dimer produce ion channels in artificial bilayers ( channels seen in inside-out patches from Chinese hamster ovary cells transfected with CLIC1 (13). The channel characteristics for the monomer, dimer, and inside-out patches from transfected Chinese hamster ovary cells (10) are as follows: conductance, 28 Ϯ 9, 28 Ϯ 9, and 30 Ϯ 2 picosiemens (Fig.…”

Section: Fig 4 the Dimer Interface Of Oxidized Clic1 Plus Channel Amentioning

confidence: 99%

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The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

Littler

Harrop

Fairlie

et al. 2004

Journal of Biological Chemistry

198

354

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Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24 -Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.Chloride ion channels control a variety of cellular processes that are central to normal function and disease states (1). The CLIC 1 family is a recently identified class of Cl Ϫ channel proteins that consists of seven members (p64, parchorin, CLIC1-5) (2, 3). A conserved C-terminal CLIC module of ϳ240 amino acids is present in each member of the family with several members containing additional, unrelated Nterminal domains. Most CLICs are localized to intracellular membranes and have been linked to functions including apoptosis, pH, and cell cycle regulation (4 -6). The CLIC ion channels are unusual in that they possess both soluble and integral membrane forms (2). In this regard they are similar to some bacterial toxins and several classes of intracellular proteins including Bcl-x L and the annexins (7). Our understanding of how such dual natured proteins enter the membrane is limited by the dearth of high resolution structures for key states in this process.We have recently determined the crystal structure of a soluble monomeric form of CLIC1 (8) and found that it is a structural homologue of the GST superfamily of proteins (9). This soluble form of CLIC1 consists of two domains, the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all ␣-helical C-domain, which is typical of the GST superfamily. CLIC1 contains an intact glutathione-binding site that was shown to covalently bind glutathione via a conserved CLIC cysteine residue, Cys-24. This led to the suggestion that CLIC1 function may be under redox control, possibly via reactive oxygen or nitrogen species.The structure and stoichiometry of the integral membrane form of the CLIC proteins is still unclear. Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 1...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 86%

Section: Fig 4 the Dimer Interface Of Oxidized Clic1 Plus Channel Amentioning

confidence: 99%

See 1 more Smart Citation

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

Littler

Harrop

Fairlie

et al. 2004

Journal of Biological Chemistry

198

354

View full text Add to dashboard Cite

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“…The CLIC family consists of seven proteins (Shanks et al, 2002), with CLICs 1, 4, and 5 known to possess chloride channel activity (Tonini et al, 2000;Tulk et al, 2002;Berryman et al, 2004;Singh and Ashley, 2006). CLIC1 was originally identified in monocytes (Valenzuela et al, 1997) and is able to insert into membranes from the aqueous phase (Tulk et al, 2002;Warton et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

CLIC1 Function Is Required for β-Amyloid-Induced Generation of Reactive Oxygen Species by Microglia

Milton¹,

Abeti²,

Averaimo³

et al. 2008

J. Neurosci.

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129

131

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The Alzheimer's disease (AD) brain is characterized by plaques containing ␤-amyloid (A␤) protein surrounded by astrocytes and reactive microglia. Activation of microglia by A␤ initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase; the resultant oxidative stress is thought to contribute to neurodegeneration in AD. We have previously shown that A␤ upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. We now demonstrate that A␤ promotes the acute translocation of CLIC1 from the cytosol to the plasma membrane of microglia, where it mediates a chloride conductance. Both the A␤ induced Cl Ϫ conductance and ROS generation were prevented by pharmacological inhibition of CLIC1, by replacement of chloride with impermeant anions, by an anti-CLIC1 antibody and by suppression of CLIC1 expression using siRNA. Thus, the CLIC1-mediated Cl Ϫ conductance is required for A␤-induced generation of neurotoxic ROS by microglia. Remarkably, CLIC1 activation is itself dependent on oxidation by ROS derived from the activated NADPH oxidase. We therefore propose that CLIC1 translocation from the cytosol to the plasma membrane, in response to redox modulation by NADPH oxidase-derived ROS, provides a feedforward mechanism that facilitates sustained microglial ROS generation by the NAPDH oxidase.

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“…A schematic diagram for the ionic pathways identified from the CHO cells are shown in Fig. 1 [25][26][27][28][29][30]. The sensor chip allows us to repeatedly expose cultured cells to mercurials and other reagents and to follow the response in real time.…”

Section: Introductionmentioning

confidence: 99%

Dynamic Effects of Hg2+-induced Changes in Cell Volume

Heo¹,

Meng²,

Sachs³

et al. 2008

Cell Biochem Biophys

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Using a microfluidic volume sensor, we studied the dynamic effects of Hg 2+ on hypotonic stressinduced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 μM HgCl 2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl 2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg 2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na + with impermeant NMDG + in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg 2+ -induced excess swelling. The volume decrease in the presence of Hg 2+ was inhibited by tetraethylammonium and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium, blockers of K + and Cl -channels, respectively, suggesting that part of the Hg 2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steadystate volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.

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Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells

Cited by 100 publications

References 13 publications

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

CLIC1 Function Is Required for β-Amyloid-Induced Generation of Reactive Oxygen Species by Microglia

Dynamic Effects of Hg2+-induced Changes in Cell Volume

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