Can insert into membranes and form chloride ion channels. Channel activity depends on the pH. Membrane insertion seems to be redox-regulated and may occur only under oxydizing conditions. Modulates the activity of RYR2 and inhibits calcium influx.
Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-A resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction.
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
Catalysis of facilitated diffusion of a chloride (by an energy-independent process) involving passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism.
A transcript map was previously constructed in the 1200-kb telomeric region of Xq28. One of the cDNAs, XAP121, displayed homology to a p64 bovine chloride channel and to a human chloride channel (p64CLCP, NCC27) at both the nucleotide and the peptide levels. In addition, all three sequences exhibited homologies to numerous ESTs derived from human, mouse, rat and pig. While the human NCC27 and XAP121 homologs encode small peptides of 241 and 243 amino acids, respectively, the bovine peptide has a length of 437 amino acids. This suggests that the human genes represent a novel and separate class of small chloride channels. Unlike other chloride channels, the NCC27 peptide was recently shown to localize intracellularly in the cytoplasm and nucleus. The NCC27 and XAP121 genes have thus been designated CLIC1 and CLIC2 for chloride intracellular channel genes 1 and 2, respectively. Since a direct association exists between a number of human chloride channel genes and a range of hereditary diseases, CLIC2 possibly represents a candidate for one of the many diseases linked to Xq28. To facilitate defined mutation analyses, we determined the genomic structure of the CLIC2 gene.
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
Catalysis of the reaction: R-X + glutathione = H-X + R-S-glutathione. R may be an aliphatic, aromatic or heterocyclic group; X may be a sulfate, nitrile or halide group.
Negative evidence
1:
Inferred from Direct AssayBHF-UCL
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
Catalysis of the transmembrane transfer of a chloride ion by a voltage-gated channel. A voltage-gated channel is a channel whose open state is dependent on the voltage across the membrane in which it is embedded.
IEAInterPro 2 GO
GO biological process
Negative regulation of ryanodine-sensitive calcium-release channel activitydefinition[GO:0060315]
Any process that decreases the activity of a ryanodine-sensitive calcium-release channel. The ryanodine-sensitive calcium-release channel catalyzes the transmembrane transfer of a calcium ion by a channel that opens when a ryanodine class ligand has been bound by the channel complex or one of its constituent parts.
The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
Any process that activates or increases the rate or extent of binding, the selective interaction of a molecule with one or more specific sites on another molecule.
The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
Regulation of cardiac muscle contraction by regulation of the release of sequestered calcium iondefinition[GO:0010881]
Any process that modulates the frequency, rate or extent of cardiac muscle contraction via the regulation of the release of sequestered calcium ion by sarcoplasmic reticulum into cytosol. The sarcoplasmic reticulum is the endoplasmic reticulum of striated muscle, specialised for the sequestration of calcium ions that are released upon receipt of a signal relayed by the T tubules from the neuromuscular junction.
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
Regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulumdefinition[GO:0010880]
Any process that modulates the rate, frequency or extent of release of sequestered calcium ion into cytosol by the sarcoplasmic reticulum, the process in which the release of sequestered calcium ion by sarcoplasmic reticulum into cytosol occurs via calcium release channels.
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.
The directed movement of substances (such as macromolecules, small molecules, ions) into, out of or within a cell, or between cells, or within a multicellular organism by means of some agent such as a transporter or pore.
A transcript map was previously constructed in the 1200-kb telomeric region of Xq28. One of the cDNAs, XAP121, displayed homology to a p64 bovine chloride channel and to a human chloride channel (p64CLCP, NCC27) at both the nucleotide and the peptide levels. In addition, all three sequences exhibited homologies to numerous ESTs derived from human, mouse, rat and pig. While the human NCC27 and XAP121 homologs encode small peptides of 241 and 243 amino acids, respectively, the bovine peptide has a length of 437 amino acids. This suggests that the human genes represent a novel and separate class of small chloride channels. Unlike other chloride channels, the NCC27 peptide was recently shown to localize intracellularly in the cytoplasm and nucleus. The NCC27 and XAP121 genes have thus been designated CLIC1 and CLIC2 for chloride intracellular channel genes 1 and 2, respectively. Since a direct association exists between a number of human chloride channel genes and a range of hereditary diseases, CLIC2 possibly represents a candidate for one of the many diseases linked to Xq28. To facilitate defined mutation analyses, we determined the genomic structure of the CLIC2 gene.
Protein involved in the transport of ions. Such proteins are usually transmembrane and mediate a movement of ions across cell membranes. Transport may be passive (facilitated diffusion; down the electrochemical gradient), or active (against the electrochemical gradient). Active transport requires energy which may come from light, oxidation reactions, ATP hydrolysis, or cotransport of other ions or molecules.
Protein involved in the transport of a molecule (metabolite, protein, etc), a ion or an electron across cell membranes, inside the cell or in a tissue fluid.
Protein which is part of an anion channel found in the plasma lemma and in intracellular membranes. These channels are permeable for various anions, such as iodide, bromide, but also for nitrates, phosphates and even negatively charged amino acids. They are called chloride channels, because chloride is the most abundant anion and the predominant permeating species in all organisms. They have been classified according to their gating mechanisms, which may depend on changes in the transmembrane electric field (voltage-dependent/gated chloride channels, e.g. ClC family), on a protein kinase/nucleotide mediated mechanism (CFTR), an increase in intracellular calcium (calcium activated chloride channels, e.g. CaCC), cell swelling (volume-regulated anion channels, e.g. VRAC) or binding of a ligand, e.g. glycine or - aminobutyric acid (GABA) activated channels. In contrast with cation channels, they are not involved in the initiation or spread of excitation, but in the regulation of excitability in nerve and muscle. They also participate in many housekeeping processes, such as volume regulation, pH regulation in organelles, electrogenesis and control of synaptic activity. The chloride channels are crucial for transepithelial transport and the control of water flow, and often provide unexpected permeation pathways for a large variety of anions.
Protein which is part of a transmembrane protein complex that forms a hydrophilic channel across the lipid bilayer through which specific inorganic ions can diffuse down their electrochemical gradients. The channels are usually gated and only open in response to a specific stimulus, such as a change in membrane potential (voltage-gated) or the binding of a ligand (ligand-gated channel).
Protein which is a component of a voltage-gated channel. Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types. They probably exist in all life forms.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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