Chloride Intracellular Ion Channel Research Articles

Chloride intracellular ion channel 2 regulates the cellular calcium through modulation of cardiac ryanodine receptor channels

Chloride intracellular ion channel 2 regulates the cellular calcium through modulation of cardiac ryanodine receptor channels

In Vitro Enzymatic Studies Reveal pH and Temperature Sensitive Properties of the CLIC Proteins.

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins' enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment.

Sterol Structural Features' Impact on the Spontaneous Membrane Insertion of CLIC1 into Artificial Lipid Membranes.

Background: A membrane protein interaction with lipids shows distinct specificity in terms of the sterol structure. The structure of the sterol's polar headgroup, steroidal rings, and aliphatic side chains have all been shown to influence protein membrane interactions, including the initial binding and subsequent oligomerization to form functional channels. Previous studies have provided some insights into the regulatory role that cholesterol plays in the spontaneous membrane insertion of the chloride intracellular ion channel protein, CLIC1. However, the manner in which cholesterol interacts with CLIC1 is yet largely unknown. Method: In this study, the CLIC1 interaction with different lipid:sterol monolayers was studied using the Langmuir trough and neutron reflectometry in order to investigate the structural features of cholesterol essential for the spontaneous membrane insertion of the CLIC1 protein. Molecular docking simulations were also performed to study the binding affinities between CLIC1 and the different sterol molecules. Results: This study, for the first time, highlights the vital role of the free sterol 3β-OH group as an essential structural requirement for the interaction of CLIC1 with cholesterol. Furthermore, the presence of additional hydroxyl groups, methylation of the sterol skeleton, and the structure of the sterol alkyl side chain have also been shown to modulate the magnitude of CLIC1 interaction with sterols and hence their spontaneous membrane insertion. This study also reports the ability of CLIC1 to interact with other naturally existing sterol molecules. General Significance: Like the sterol molecules, CLIC proteins are evolutionarily conserved with almost all vertebrates expressing six CLIC proteins (CLIC1-6), and CLIC-like proteins are also present in invertebrates and have also been reported in plants. This discovery of CLIC1 protein interaction with other natural sterols and the sterol structural requirements for CLIC membrane insertion provide key information to explore the feasibility of exploiting these properties for therapeutic and prophylactic purposes.

Cysteine mutagenesis reveals novel structure-function features within the predicted transmembrane domain of chloride intracellular ion channel 1 (CLIC1).

Chloride intracellular ion channels (CLICs) are a unique class of metamorphic proteins that exists as soluble proteins and can auto-insert into the membrane to form an ion channel. The soluble structure of all the CLICs is well established but the structural information on the membrane form is lacking. CLICs are known to have a single putative transmembrane (PTM) region and proteins oligomerize to a tetramer to form a functional channel. CLIC1 is a sensor of oxidative stress, overexpressed in different types of cancer, and involved in diabetes, cell viability, and angiogenesis. The PTM region of CLIC1 encompasses residue 24-46, where cysteine at position 24 is a critical redox-sensitive residue. However, the precise arrangement of the residues in the pore region is not known. Hence, to identify the key residue involved in the gating mechanism and ion selectivity, the putative residues lining the pore region were mutated using site-directed mutagenesis. We incorporated the substituted cysteine accessibility method (SCAM) and recorded the channel activity of the mutants in the whole-cell and inside-out configurations. The ion selectivity was measured by perfusing with different anions (chloride, bromide, fluoride, and phosphate). The open channel probability (Po) is significantly higher in R29C mutant (0.88) compared to WT (0.21), suggesting R29C residue is involved in CLIC1 gating. Similarly, the CLIC1 is highly selective for chloride ions as compared to other anions, and the selective filer is located between V33C and K37C residues. We probed substituted cysteine residues with cadmium and identified that the T40C showed a reduction in Po and amplitude. Our results have delineated the pore lining of the CLIC1 and identified the residues involved in its gating and regulating the chloride selectivity.

Chloride intracellular channel protein 1 expression in normal and atherosclerotic human arteries. Challenges for a new therapeutic target in atheromatosis.

Background and Aims: Chloride intracellular ion channel protein 1 (CLIC1) is involved in inflammation from various disease and oxidative stress endothelial cells disfunction. Its role in atherosclerosis is scarcely suggested in the literature, being restricted to experimental induced atherosclerosis in mice. Its involvement in human atheroma plaque formation is completely unknown in this moment. AIM.To study CLIC1 expression in normal human fetal and adult aorta, on early steps of atheroma plaque development and also in complicated atherosclerotic lesions.

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.

Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface-bilayers

Profiling ion flux through human intracellular chloride ion channels using live-cell based techniques, such as patch-clamp electrophysiology, is laborious and time-consuming. The integration of scalable microfluidic systems with automatable protocols based on droplet-interface-bilayers (DIBs) within which ion channels are incorporated circumvents several limitations associated with live-cell measurements and facilitates testing in controllable in vitro conditions. Here, we have designed and tested novel microfluidic layouts for the formation of arrays of DIBs in parallel and developed the first example of a miniaturised, DIB-based, fluorescence assays for Cl− fluxing, allowing the investigation of the functional properties of the human chloride intracellular ion channel 1 (CLIC1). The microfluidic protocols relied on passive geometries for droplet pairing and DIB formation. Using recombinantly expressed CLIC1, we identified the best conditions to maximise protein integration into a lipid bilayer and the oligomerisation of the protein into functional ion channels. Finally, CLIC1 ion channel functionality was assessed relative to α-Haemolysin into microfluidic DIBs using the same Cl− fluxing assay.

A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction

BackgroundSterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. MethodWe have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. ResultsUnlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane.Conclusion: In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. General significanceMembers of the CLIC family of proteins demonstrate unusual structural and dual functional properties – as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting activity and a novel regulatory role for cholesterol in such a process.

X-ray and Neutron Reflectivity Study Shows That CLIC1 Undergoes Cholesterol-Dependent Structural Reorganization in Lipid Monolayers.

CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6-7 nm2 with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer.

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.

The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1’s acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1’s membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1’s ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.

Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air-Water Interface.

CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels.

Elucidating the Mechanism for Sterol Regulation of Chloride Intracellular Ion Channel Protein Interactions with Lipid Membranes

Sterols have been reported to modulate conformation and function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family. These largely soluble proteins possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. To date, the structure of their membrane-bound form and factors influencing their auto-insertion remains largely unknown. We have performed Langmuir-film, x-ray, and neutron reflectivity experiments to study the interaction of wild-type or mutant versions of the protein CLIC1 with monolayers prepared from 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) alone, or mixtures of this lipid with either one of the following sterols: cholesterol, β-sitosterol, ergosterol, hydroxyecdysone or cholestane. In lipid monolayers lacking sterols, CLIC1 did not insert. However significant membrane insertion occurred when CLIC1 was added to membranes containing cholesterol. Substitution of membrane cholesterol with different sterols, not only increased CLIC1's membrane interaction but also increased its rate of insertion. However, CLIC1 showed no insertion into monolayers containing cholestane, which lacked the intact sterol 3β-OH group. Unlike CLIC1-wild-type and C24A-CLIC1 mutant protein, G18A and G22A mutants did not insert in monolayers containing cholesterol. These findings for the first time demonstrate that the spontaneous membrane insertion of CLIC1 is dependent on the presence of cholesterol in membranes, along with the presence of an intact 3β-OH group within the sterol structure, which is critical for CLIC1-membrane interactions. Our novel findings also extend to the identification of a cholesterol-binding domain-GXXXG motif within CLIC1 that facilitates the protein's membrane interaction and insertion.

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

Ion Channel Proteins that Spontaneously Insert into Lipid Bilayer Membranes: An Impedance Spectroscopy Study Employing Tethered Membranes

The Chloride Intracellular Ion Channel (CLIC) protein family, the Annexins, the bacterial toxins Hemolysin (AH), Streptolysin, Perfringolysin Listeriolysin, Pneumonlysin, Ivanolysin and Colicins all possess unusual mechanisms for inserting into cellular and/or host membranes. They are representatives of proteins that spontaneously insert into membranes, by-passing the route for the incorporation of most integral membrane proteins. For example, the soluble form of the 240-amino acid polypeptide CLIC1 is known to exist in at least two conformations due to a large rearrangement of its amino terminus under the influence of oxidation. Oxidation promotes its binding to and insertion into lipid bilayers, forming a chloride selective ion channel. Spontaneous membrane insertion also occurs for the 293-amino acid polypeptide, AH which is secreted as a water-soluble monomer, and when in contact with a membrane inserts and forms heptameric pores. The 36-amino acid peptide (pHLIP) which is a truncated sequence of the C-helix of the integral membrane protein bacteriorhodopsin also inserts into lipid bilayers at low pH. In the case of the 316-amino acid polypeptide Annexin B12, reversible insertion into membranes can also occur at acidic pH. In the present study, we report the functional ion channel activity of a number of spontaneously inserting proteins into Tethered Membranes via a novel impedance spectroscopy assay. We report on conditions that promote insertion and determine the ease of reversible dissociation of protein from the membrane. Also reported will be the kinetics of both the insertion and elimination of the protein based on the functional conductance changes of the membrane.

Metamorphic Response of CLIC1 Chloride Intracellular Ion Channel upon Interaction with the Membrane

The Chloride Intracellular Channel (CLIC) family can be characterized as ‘metamorphic’proteins; namely, capable of reversibly shifting between two or more different-folded conformations. The CLICs are expressed as soluble proteins but can also auto-insert into the membrane to form active ion channels. Such a conformational transition must involve large-scale structural rearrangement to confer favourable interactions with the membrane. To date, little is known about the process and cellular triggers for CLIC membrane insertion; although a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural rearrangement from a monomer to a dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis in solution facilitates CLIC1 membrane insertion is unclear. We have sought to characterize the structural response of CLIC1 upon interaction with the membrane. A novel labeling scheme for CLIC1 was devised to enable site-directed-labeling of single native cysteines with either fluorophores or spin labels. Fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) revealed that the interaction of CLIC1 with the membrane results in a large-scale inter-domain movement. The two phases for membrane interaction, i.e. association followed by insertion, were also monitored by probing the local environment of an N-terminal transmembrane tryptophan residue (Trp35) using fluorescent quenching. A sucrose-loaded-vesicle sedimentation assay was also used to quantify membrane binding. Our results suggest oxidation of CLIC1 monomer in the presence of the membrane promotes CLIC1 binding and insertion. Our current model for the structural transitions and environmental triggers of CLIC1 membrane-induced metamorphosis will be discussed.

Blockade of chloride intracellular ion channel 1 stimulates Aβ phagocytosis

In amyloid-beta (Abeta)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-alpha and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Abeta phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Abeta-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, Abeta25-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of Abeta phagocytosis, we treated BV-2 cells with biotinylated Abeta1-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased Abeta phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Abeta1-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Abeta1-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Abeta, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Abeta phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.

Trimeric structure of the wild soluble chloride intracellular ion channel CLIC4 observed in crystals

The crystal structure of a wild type of the human soluble chloride intracellular ion channel CLIC4 (wCLIC4) has been determined at a resolution of 2.2 Å. The structure shows a homotrimer in an asymmetric unit, which is first observed in CLICs. The assembly of the trimer takes a unique triple interaction mode between three monomers with a hydrogen-bond network and hydrophobic contacts. Through such complicated interactions, the homotrimer of wCLIC4 is firmly stabilized. The structure shows an oligomeric mode with a unique assembly mechanism by which the oligomerization of CLIC4 can be performed without any intramolecular disulfide bond formation. It indicated a possibility that CLIC4 may take a unique structural organization distinct from CLIC1 for docking with lipid bilayers. In addition, the structure shows distinct conformational states of the h2 region for respective monomers of the trimer, which reveal an intrinsic conformational susceptibility for this significant region in the structural transition.