Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

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.     

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.     

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.     

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.     

Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli

The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.     

Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins (Review)

Parchorin, p64 and the related chloride intracellular channel (CLIC) proteins are widely expressed in multicellular organisms and have emerged as candidates for novel, auto-inserting, self-assembling intracellular anion channels involved in a wide variety of fundamental cellular events including regulated secretion, cell division and apoptosis. Although the mammalian phosphoproteins p64 and parchorin (49 and 65K, respectively) have only been indirectly implicated in anion channel activity, two CLIC proteins (CLIC1 and CLIC4, 27 and 29K, respectively) appear to be essential molecular components of anion channels, and CLIC1 can form anion channels in planar lipid bilayers in the absence of other cellular proteins. However, these putative ion channel proteins are controversial because they exist in both soluble and membrane forms, with at least one transmembrane domain. Even more surprisingly, soluble CLICs share the same glutaredoxin fold as soluble omega class glutathione-S-transferases. Working out how these ubiquitous, soluble proteins unfold, insert into membranes and then refold to form integral membrane proteins, and how cells control this potentially dangerous process and make use of the associated ion channels, are challenging prospects. Critical to this future work is the need for better characterization of membrane topology, careful functional analysis of reconstituted and native channels, including their conductances and selectivities, and detailed structure/function studies including targeted mutagenesis to investigate the structure of the putative pore, the role of protein phosphorylation and the role of conserved cysteine residues.     

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the 'PTM' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.     

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.     

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.     

CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel

CLIC1 is a member of the CLIC familyof proteins, which has been shown to demonstrate chloride channelactivity when reconstituted in phospholipid vesicles. CLIC1 exists incells as an integral membrane protein and as a soluble cytoplasmicprotein, implying that CLIC1 might cycle between membrane-inserted andsoluble forms. CLIC1 was purified and detergent was removed, yieldingan aqueous solution of essentially pure protein. Pure CLIC1 was mixedwith vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1confers anion channel activity to preformed membranes that isindistinguishable from the previously reported activity resulting fromreconstitution of CLIC1 into membranes by detergent dialysis. Theactivity is dependent on the amount of CLIC1 added, appears rapidly onmixing of protein and lipid, is inhibited by indanyloxyacetic acid-94,N-ethylmaleimide, and glutathione, is inactivated by heat,and shows sensitivity to pH and to membrane lipid composition. Weconclude that CLIC1 in the absence of detergent spontaneously insertsinto preformed membranes, where it can function as an anion-selective channel.

     

The enigma of the CLIC proteins: Ion channels, redox proteins, enzymes, scaffolding proteins?

Chloride intracellular channel proteins (CLICs) are distinct from most ion channels in that they have both soluble and integral membrane forms. CLICs are highly conserved in chordates, with six vertebrate paralogues. CLIC-like proteins are found in other metazoans. CLICs form channels in artificial bilayers in a process favoured by oxidising conditions and low pH. They are structurally plastic, with CLIC1 adopting two distinct soluble conformations. Phylogenetic and structural data indicate that CLICs are likely to have enzymatic function. The physiological role of CLICs appears to be maintenance of intracellular membranes, which is associated with tubulogenesis but may involve other substructures.     

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 2.2A. 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.     

Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore

Chloride intracellular channels (CLICs) are putative pore-forming glutathione-S-transferase homologs that are thought to insert into cell membranes directly from the cytosol. We incorporated soluble, recombinant human CLIC1 into planar lipid bilayers to investigate the associated ion channels, and noted that channel assembly (unlike membrane insertion) required a specific lipid mixture. The channels formed by reduced CLIC1 were similar to those previously recorded from cells and "tip-dip" bilayers, and specific anti-CLIC1 antibodies inhibited them. However, the amplitudes of the filtered single-channel currents were strictly regulated by the redox potential on the "extracellular" (or "luminal") side of the membrane, with minimal currents under strongly oxidizing conditions. We carried out covalent functional modification and site-directed mutagenesis of this controversial ion channel to test the idea that cysteine 24 is a critical redox-sensitive residue located on the extracellular (or luminal) side of membrane CLIC1 subunits, in a cysteine-proline motif close to the putative channel pore. Our findings support a simple structural hypothesis to explain how CLIC1 oligomers form pores in membranes, and suggest that native channels may be regulated by a novel mechanism involving the formation and reduction of intersubunit disulphide bonds.     

CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels

Despite being synthesized in the cytosol without a leader sequence, the soluble 253-residue mammalian protein CLIC4 (Chloride Intracellular Channel 4, or p64H1), a structural homologue of Omega-type glutathione-S-transferase, autoinserts into membranes to form an integral membrane protein with ion channel activity. A predicted transmembrane domain (TMD) near the N-terminus of CLIC4 could mediate membrane insertion, and contribute to oligomeric pores, with minimal reorganization of the soluble protein structure. We tested this idea by reconstituting recombinant CLIC4 in planar bilayers containing phosphatidyethanolamine, phosphatidylserine and cholesterol, recording ion channels with a maximum conductance of approximately 15 pS in KCl under both oxidizing and reducing conditions. The channels discriminated poorly between anions and cations, incompatible with the current "CLIC" nomenclature, and their conductance was modified by the trans (external or luminal) redox potential, as previously observed for CLIC1. We then reconstituted a truncated version of the protein, limited to the first 61 residues containing the predicted TMD. This included a single trans cysteine residue in the putative pore-forming subunits, at the external entrance to the pore. The truncated protein formed non-selective channels with a reduced conductance, but they retained their trans-redox sensitivity, and could still be blocked or inactivated by trans (not cis) thiol-reative dithiobisnitrobenzoic acid. We suggest that oligomers containing the putative TMD are essential components of the CLIC4 pore. However, the pore is inherently non-selective, and any ionic selectivity in CLIC4 (and other membrane CLICs) may be attributable to other regions of the protein, including the channel vestibules.     

CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells

CLIC4 is a member of the chloride intracellular channel (CLIC) protein family whose principal cellular functions are poorly understood. Recently, we demonstrated that several CLIC proteins, including CLIC4, interact with AKAP350. AKAP350 is concentrated at the Golgi apparatus, centrosome, and midbody and acts as a scaffolding protein for several protein kinases and phosphatases. In this report, we show that endogenous CLIC4 and AKAP350 colocalize at the centrosome and midbody of cultured cells by immunofluorescence microscopy. Unlike AKAP350, CLIC4 is not enriched in the Golgi apparatus but is enriched in mitochondria, actin-based structures at the cell cortex, and the nuclear matrix, indicating that CLIC4-AKAP350 interactions are regulated at specific subcellular sites in vivo. In addition to the centrosome and midbody, CLIC4 colocalizes with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells, suggesting that CLIC4 may play a role in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Biochemical studies show that CLIC4 behaves mainly as a soluble cytosolic protein and can associate with proteins of the microtubule cytoskeleton. The localization of CLIC4 to the cortical actin cytoskeleton and its association with AKAP350 at the centrosome and midbody suggests that CLIC4 may be important for regulating cytoskeletal organization during the cell cycle. These findings lead to the conclusion that CLIC4 and possibly other CLIC proteins have alternate cellular functions that are distinct from their proposed roles as chloride channels.     

From glutathione transferase to pore in a CLIC

Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water-soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S-transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels.     

Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin

Chloride intracellular channels (CLICs) are soluble, signal peptide-less proteins that are distantly related to Omega-type glutathione-S-transferases. Although some CLICs bypass the classical secretory pathway and autoinsert into cell membranes to form ion channels, their cellular roles remain unclear. Many CLICs are strongly associated with cytoskeletal proteins, but the role of these associations is not known. In this study, we incorporated purified, recombinant mammalian CLIC1, CLIC4 and (for the first time) CLIC5 into planar lipid bilayers, and tested the hypothesis that the channels are regulated by actin. CLIC5 formed multiconductance channels that were almost equally permeable to Na(+), K(+) and Cl(-), suggesting that the 'CLIC' nomenclature may need to be revised. CLIC1 and CLIC5, but not CLIC4, were strongly and reversibly inhibited (or inactivated) by 'cytosolic' F-actin in the absence of any other protein. This inhibition effect on channels could be reversed by using cytochalasin to disrupt the F-actin. We suggest that actin-regulated membrane CLICs could modify solute transport at key stages during cellular events such as apoptosis, cell and organelle division and fusion, cell-volume regulation, and cell movement.     

Apolipoproteins L1-6 share key cation channel-regulating residues but have different membrane insertion and ion conductance properties

The human apolipoprotein L gene family encodes the apolipoprotein L1–6 (APOL1–6) proteins, which are effectors of the innate immune response to viruses, bacteria and protozoan parasites. Due to a high degree of similarity between APOL proteins, it is often assumed that they have similar functions to APOL1, which forms cation channels in planar lipid bilayers and membranes resulting in cytolytic activity. However, the channel properties of the remaining APOL proteins have not been reported. Here, we used transient overexpression and a planar lipid bilayer system to study the function of APOL proteins. By measuring lactate dehydrogenase release, we found that APOL1, APOL3, and APOL6 were cytolytic, whereas APOL2, APOL4, and APOL5 were not. Cells expressing APOL1 or APOL3, but not APOL6, developed a distinctive swollen morphology. In planar lipid bilayers, recombinant APOL1 and APOL2 required an acidic environment for the insertion of each protein into the membrane bilayer to form an ion conductance channel. In contrast, recombinant APOL3, APOL4, and APOL5 readily inserted into bilayers to form ion conductance at neutral pH, but required a positive voltage on the side of insertion. Despite these differences in membrane insertion properties, the ion conductances formed by APOL1-4 were similarly pH-dependent and cation-selective, consistent with conservation of the pore-lining region in each protein. Thus, despite structural conservation, the APOL proteins are functionally different. We propose that these proteins interact with different membranes and under different voltage and pH conditions within a cell to effect innate immunity to different microbial pathogens.     

Intracellular Ion Channel CLIC1: Involvement in Microglia-Mediated β-Amyloid Peptide(1-42) Neurotoxicity

Microglia can exacerbate central nervous system disorders, including stroke and chronic progressive neurodegenerative diseases such as Alzheimer disease. Mounting evidence points to ion channels expressed by microglia as contributing to these neuropathologies. The Chloride Intracellular Channel (CLIC) family represents a class of chloride intracellular channel proteins, most of which are localized to intracellular membranes. CLICs are unusual in that they possess both soluble and integral membrane forms. Amyloid β-peptide (Aβ) accumulation in plaques is a hallmark of familial Alzheimer disease. The truncated Aβ_25-35 species was shown previously to increase the expression of CLIC1 chloride conductance in cortical microglia and to provoke microglial neurotoxicity. However, the highly pathogenic and fibrillogenic full-length Aβ_1-42 species was not examined, nor was the potential role of CLIC1 in mediating microglial activation and neurotoxicity by other stimuli (e.g. ligands for the Toll-like receptors). In the present study, we utilized a two chamber Transwell? cell culture system to allow separate treatment of microglia and neurons while examining the effect of pharmacological blockade of CLIC1 in protecting cortical neurons from toxicity caused by Aβ_1-42- and lipopolysaccaride-stimulated microglia. Presentation of Aβ_1-42 to the upper, microglia-containing chamber resulted in a progressive loss of neurons over 3 days. Neuronal cell injury was prevented by the CLIC1 ion channel blockers IAA-94 [(R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] and niflumic acid (2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid) when presented to the upper chamber only. Incubation of microglia with lipopolysaccharide plus interferon-γ led to neuronal cell injury which, however, was insensitive to inhibition by the CLIC1 channel blockers, suggesting a degree of selectivity in agents leading to CLIC1 activation.