CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels

The International Journal of Biochemistry & Cell Biology 36 (2004) 1599–1612

CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels Philip G. Board∗ , Marjorie Coggan, Sarah Watson, Peter W. Gage, Angela F. Dulhunty Division of Molecular Bioscience, John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia Received 17 November 2003; accepted 14 January 2004

Abstract 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. © 2004 Elsevier Ltd. All rights reserved. Keywords: Ryanodine receptor Ca2+ channels; CLIC proteins; Cytoplasmic Ca2+ regulation; Glutathione transferase

1. Introduction CLIC proteins are a new class of soluble and membrane-bound proteins that have been grouped together on the basis of their sequence similarity. The proteins were named CLIC because the first members of this family to be characterized formed intracellular chloride channels (Heiss & Poustka, 1997). CLIC proteins are found in the membranes of the nucleus, secre∗ Corresponding author. Tel.: +61-2-6125-4714; fax: +61-2-6125-4712. E-mail address: [email protected] (P.G. Board).

tary vesicles, endoplasmic reticulum, trans Golgi vesicles and large dense core vesicles (Chuang, Milner, Zhu, & Sung, 1999; Duncan, Westwood, & Ashley, 1997; Edwards, 1999; Redhead, Sullivan, Koseki, Fujiwara, & Edwards, 1997; Valenzuela et al., 2000): they have been implicated in kidney function, cell division and bone resorption (Landry et al., 1989; Nishizawa, Nagao, Iwatsubo, Forte, & Urushidani, 2000; Schlesinger, Blair, Teitelbaum, & Edwards, 1997; Valenzuela et al., 2000). Although CLIC-1 has been shown to form chloride channels (Tonini et al., 2000; Tulk, Schlesinger, Kapadia, & Edwards, 2000; Valenzuela et al., 2000; Warton et al., 2002), little is

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known about the function of other CLICs (CLIC-2 to CLIC-5) (Cromer, Morton, Board, & Parker, 2002). Indeed, in spite of sequence similarities, it has not been demonstrated that all members of the CLIC family form chloride channels and there is growing evidence in support of other functions. The structure, location and functional characteristics of the recently identified CLIC-2 protein (Heiss & Poustka, 1997) have not been described and are the subject of the present report. We have reported sequence and structural similarity between CLIC-1 and an omega class glutathione transferase (GSTO1-1) (Dulhunty, Gage, Curtis, Chelvanayagam, & Board, 2001a), which has been confirmed by the solution of the crystal structure of CLIC-1 showing that it has a typical GST fold, similar to that of GST-omega (Harrop et al., 2001). We found that GST-omega modulates ryanodine receptor (RyR) calcium release channels and suggested that it may modulate Ca2+ release from intracellular stores during radiation (Dulhunty et al., 2001a). Since RyRs, either alone or in combination with inositol 1,4,5-trisphosphate receptors, are found in the membranes of calcium stores in a variety of cells (including cardiac, skeletal and smooth muscle, neurons, and lymphocytes) it is possible that GSTs and CLICs may prove to be important modulators of intracellular [Ca2+ ]. Consequently these proteins may influence contraction in skeletal and cardiac muscle and other Ca2+ -dependent cell processes, with likely roles in cardiovascular function, respiration, movement, neuronal activity and immunogenesis. Because of the structural and sequence similarities between the GSTs and CLICs, we decided to test whether CLIC-2 modulates RyRs like GST-omega. The possibility that CLIC-2 can modulate ion channels has not previously been investigated. Our aim in this study has been to characterise the expression of CLIC-2 and to study its biochemical and physiological properties. We find that CLIC-2 is structurally similar to CLIC-1 and GSTO1, and is widely distributed in human tissues including heart and skeletal muscle. Although CLIC-2 has little catalytic activity with previously identified glutathione transferase substrates, we find that it is a strong inhibitor of cardiac RyR channels in both lipid bilayers and in cardiac SR vesicles. This is the first reported function of the CLIC-2 protein and supports our hypothesis

that CLIC-2 is a contributor to intracellular Ca2+ homeostasis. 2. Experimental procedures 2.1. Expression and purification of recombinant CLIC-2 Human CLIC-2 was expressed in E. coli with a residual N-terminal poly-His tag that facilitated the purification of the recombinant protein. The coding sequence was amplified from the EST clone AI129485 and ligated between the BamHI and PstI sites in the pQE-30 vector (Qiagen, Clifton Hills, Australia) and transformed into E. coli M15[pREP4] host cells. The primers used for the amplification were CLIC2ExSBF (5 -ctccgcggtggatccggcctgcggcccggcact) and CLIC-2ExPR (5 -ttgcatgctgcagcctgtaagagctctcct) and contained BamHI and PstI sites, respectively. The resulting cDNA clone was re-sequenced to exclude amplification errors and was termed pQECLIC-2. A culture of pQECLIC-2 in M15[pREP4] cells was grown over night in the presence of 0.1 mM isopropyl thio-␤-d-galactoside and processed by the methods described by Whittington et al. (1999). The recombinant protein was purified by immobilised metal affinity chromatography with Ni-agarose as previously described for other His-tagged GSTs (Whittington et al., 1999) with the exception that buffer A (50 mM sodium phosphate/300 mM NaCl) was adjusted to pH 7 and the purified enzyme was dialysed against 5 mM Hepes or 50 mM Hepes plus 10% glycerol, pH 7. For some preparations the recombinant CLIC-2 was further purified by gel filtration on a Pharmacia fast protein liquid chromatography Superose 12 column equilibrated with 50 mM Hepes, 10% glycerol pH 7.0. In some experiments a non-His-tagged preparation of CLIC-2 was prepared. CLIC-2 was expressed with His-tagged ubiquitin fused at the N-terminal. Subsequently the ubiquitin was removed by digestion with a ubiquitin specific protease (Baker, Smith, Marano, McKee, & Board, 1994). The purity of preparations was assessed by SDS-PAGE gels stained with Coomassie Blue R250 (Laemmli, 1970) and the apparent molecular size was assessed

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by comparison with a range of proteins (Mr 14,400–97,000 Da) in a low molecular weight calibration kit (Amersham Pharmacia Biotech Uppsala Sweden). 2.2. Northern blotting A Northern blot containing mRNA from multiple human tissues was obtained from Panomics Inc. (Redwood City, CA). The membrane was hybridized with a 32 P-labelled CLIC-2 cDNA probe using the protocol and reagents supplied by the manufacturer. After autoradiography the membrane was rehybridized with a 32 P-labelled ␤-actin probe to evaluate track loading. 2.3. Quaternary structure of CLIC-2 Size exclusion chromatography was conducted on a Pharmacia fast protein liquid chromatography Superose 12 column equilibrated with 300 mM NaCl, 2 mM ␤-mercaptoethanol, 50 mM Hepes pH 7.5 and run at a flow rate of 0.5 ml/min. Standards included thyroglobulin (670 kDa), bovine ␥-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and Vitamin B12 (1.35 kDa). 2.4. Molecular modelling The high sequence identity between CLIC-2 and CLIC-1 was sufficient to develop a molecular model of CLIC-2. The model was constructed by the use of the SWISS-Model program (Schwede, Kopp, Guex, & Peitsch, 2003) using the crystal structures of human CLIC-1 (1K0MA, 1K0MB, 1K0OA, 1K0NA, 1K0NB) as templates. 2.5. Enzyme assays GST activity with a range of substrates (Table 1) was determined as described (Whittington et al., 1999). Thiol transferase activity was measured with hydroxyethyl disulfide as a substrate (Axelsson, Eriksson, & Mannervik, 1978) and glutathione-dependent dehydroascorbate reductase activity was determined spectrophotometrically with dehydroascorbate prepared immediately before use (Wells, Xu, & Washburn, 1995).

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Table 1 Activity of recombinant CLIC-2 and GSTO1-1 with various substrates Substrate/activity

t-Butyl hydroperoxide Cumene hydroperoxide Thioltransferase

Specific activity (␮mol/(min mg)) CLIC-2

GSTO1-1a

0.03 ± 0.020 0.16 ± 0.050 ND

ND ND 2.92 ± 0.120

All values are the mean ± standard deviation of at least three determinations. ND: not detectable. a Data from Board et al. (2000).

2.6. CLIC-2 antibody Anti-serum was raised in a New Zealand white rabbit using purified His-tagged CLIC-2 with Freund’s adjuvant and a standard immunization protocol. 2.7. Lipid bilayers and solution Bilayers were formed and SR vesicles incorporated as previously described (Dulhunty et al., 2001a). SR vesicles (10 ␮g/ml), prepared from sheep heart (Laver et al., 1995), were added to the cis chamber using cis and trans solutions containing (mM): 20, CsCl; 1.0, CaCl2 ; 10N tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES) (pH 7.4 adjusted with CsOH) as well as either cis/trans (mM) 230/230 CsCH3 O3 S plus 500/0 mannitol or 230/30 CsCH3 O3 S. Channel activity was recorded with symmetrical 250/250 mM Cs+ . Potentials are expressed as Vcis − Vtrans (i.e.Vcytoplasm − Vlumen ). 2.8. Recording and analysis of single-channel data The methods we used to record and analyse single-channel data have been described previously (Dulhunty et al., 2001a). Channel activity was recorded at +40 and −40 mV to determine whether or not the actions of CLIC-2 were voltage-dependent. The bilayer potential was switched from one potential to the other every 30 s or more throughout the experiment. Estimates of channel activity were based on the mean current (I ) flowing through the channels, the channel open probability, mean open time and mean closed times measured using the software package

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Channel2 (developed by Professor P.W. Gage and M. Smith). Mean current measurements include data from bilayers containing one, two or three channels, while open probability, mean open time and mean closed times were measured only from single-channel recordings. The measurements of channel activity were obtained from 30 to 60 s segments of channel activity at +40 and −40 mV, under control conditions, 6–8 min after addition of CLIC-2 and 2–3 min after addition of antibody or after perfusion of the cis solution. Solutions were exchanged by perfusion using back-to-back syringes. The efficiency of exchange, determined spectrophotometrically at 710 nm, by dilution of 1 mM Antipyrylazo III (in distilled H2 O) during perfusion with different volumes of distilled water. Perfusion with 11 ml of solution (∼6 volumes) gave ∼3000-fold dilution and perfusion with 6 ml (∼4 volumes) gave 10–20-fold dilutions. 2.9. Ca2+ release from SR vesicles Methods used have been described previously (Dulhunty et al., 1999). SR vesicles (100 ␮g/ml), prepared from sheep heart (Laver et al., 1995), were added to a solution containing (mM): 100, H2 PO4 (pH = 7); 4, MgCl2 ; 1, Na2 ATP; 0.5, antipyrylazo III. The method previously used with skeletal SR vesicles was modified for cardiac SR by (1) adding an ATP regenerating system—phospho(enol)pyruvate (5 mM) and pyruvate kinase (25 ␮g/ml)—to the solution (2) a period of 3 min was allowed for loading after each addition of Ca2+ . Extravesicular [Ca2+ ] was monitored at 710 nm. Vesicles were loaded with Ca2+ by adding four aliquots of CaCl2 , each initially increasing the extravesicular Ca2+ concentration by 7.5 ␮M. Time (3 min) was allowed between each addition of Ca2+ for uptake to reduce the Ca2+ concentration to resting levels. Then thapsigargin (200 nM) was added to block the Ca2+ ATPase and then 20 ␮M Ca2+ to initiate Ca2+ -induced Ca2+ release. Ca2+ release with thapsigargin was subtracted from the initial rates of release of with Ca2+ -induced Ca2+ release. Addition of ruthenium red (4–5 ␮M) confirmed that Ca2+ release was through the RyR. The Ca2+ ionophore A23187 (3 ␮g/ml) was added to measure the Ca2+ remaining in the vesicles at the end of each experiment. Experiments were performed with either vehicle alone

(control) or vehicle plus 29 ␮M CLIC-2 (CLIC-2), added before the first Ca2+ loading step. The rate of Ca2+ uptake during the 4th Ca2+ load was measured (i.e. uptake 9 min after addition of buffer or buffer plus CLIC-2). 2.10. Statistical analysis of single-channel data Results from 5 to 10 bilayers were measured depending on variability. Data is given as mean±1S.E.M. and tested for significance (P < 0.05) with an independent or paired Student t-test, or a non-parametric “sign” test as appropriate. 3. Results 3.1. The CLIC-2 protein 3.1.1. Protein sequence The cDNA amplified from the EST clone AI129485, contained an open reading frame that encoded a peptide of 243 amino acids identical in sequence to the previously reported CLIC-2 cDNA (Heiss & Poustka, 1997). The encoded protein has a predicted molecular mass of 27.811 kDa. The deduced sequence of CLIC-2 is aligned with the sequences of CLIC-1 and GSTO1 in Fig. 1. CLIC-1 and CLIC-2 show 58.8% amino acid sequence identity and in contrast CLIC-2 and GSTO1 show 18.6% identity. In this alignment there are 34 residues that are conserved between all three sequences, including a glycine and a cis-proline that are thought to be conserved throughout the glutathione transferase family for structural reasons (Cromer et al., 2002; Dulhunty et al., 2001a). Since CLIC-1 has been shown to be a member of the glutathione transferase structural family, the high degree of similarity between the CLIC-1 and CLIC-2 sequences suggests that CLIC-2 is also a member of the GST structural family (Dulhunty et al., 1999; Harrop et al., 2001). We used the coordinates of the crystal structure of CLIC-1 as a template for the construction of a molecular model of CLIC-2 (Fig. 2). Given the high level of sequence similarity between CLIC-1 and CLIC-2 it is not surprising that the model superimposes well on the CLIC-1 structure. In contrast to the rest of the model, the loop between helix 5 and helix 6 (Leu155-Arg170) shows a relatively high B

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Fig. 1. A multiple alignment of the amino acid sequences of CLIC-1 CLIC-2 and GSTO1. Secondary structure elements are based on the crystal structure of CLIC-1 (Harrop et al., 2001). Above the line arrows represent strands and grey bars represent helices. Conserved residues are highlighted in grey. Residues that are conserved throughout the GST structural family are indicated by an asterisk above the line. The conserved active site motif is underlined. Sequences are available from SwissProt with the following accession numbers: CLIC-1, O00299; CLIC-2, O15247 and GSTO1, P78417.

Factor suggesting that this region of the model is less reliable. It is notable that this loop also differs significantly from the GSTO1 structure (Board et al., 2000). The modelled CLIC-2 structure has characteristics of a typical member of the glutathione transferase structural family. The molecule has two domains, the N-terminal has a ␤␣␤␣␤␤-thioredoxin-like fold and the C-terminal domain is comprised entirely of alpha helices. CLIC-2 has an active site CPFC motif that is very similar to the CPYC and CPSC motifs found in human glutaredoxins 1 and 2 (Gladyshev et al., 2001). The CPF component of this motif is conserved in both GSTO1 and CLIC-1. This motif is positioned

at the amino terminal end of helix 1 as it is in the glutaredoxins (Fig. 2). This suggested that CLIC-2 may exhibit thioltransferase activity that is a characteristic of glutaredoxins and is also exhibited at a low level by GSTO1-1 (Board et al., 2000; Gladyshev et al., 2001). 3.1.2. CLIC-2 expression Northern blots of RNA extracted from a range of human tissues showed the presence of two equally abundant hybridizing species of approximately 1.45 and 2.37 kb (Fig. 3). A third smaller (0.8 kb) less abundant band was detectable in the lung, spleen and possibly

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Fig. 2. A homology model of CLIC-2 based on an alignment of CLIC-1 and CLIC-2 and the atomic coordinates of CLIC-1 (1K0MA, 1K0MB, 1K0OA, 1K0NA, 1K0NB). The CLIC-2 (white) model is superimposed on the CLIC-1 (black) structure (1K0MA). The position of Cys-30 and Cys-33 that contribute to the glutaredoxin-like active site motif are indicated. A loop with a high B Factor between leucine155 and arginine 170 is shown.

the testis. Further studies will be required to determine if these represent different gene products or splice variants of CLIC-2. The highest levels of expression appear to occur in the spleen and lung however the relative levels of expression are difficult to interpret as the commercial Northern blot used in this study does not exhibit equal loading of mRNA from each tissue as

Fig. 3. Tissue distribution of CLIC-2 mRNA expression. The blotted RNA was initially hybridized with the CLIC-2 cDNA (A) then rehybridized with a ␤ actin cDNA (B) to evaluate RNA loading.

Fig. 4. SDS-PAGE of purified recombinant CLIC-2. Lane 1, molecular mass markers. Lane 2, 20 ␮gm purified recombinant CLIC-2.

indicated by hybridization with a ␤actin cDNA. There was clear expression of both mRNA species in a range of other tissues including heart and skeletal muscle. There are differences in the relative levels of expression of GSTO1 and CLIC-2 in different tissues. The relatively high expression of CLIC-2 in the lung contrasts with the low level of lung GSTO1 mRNA we noted in a previous study (Board et al., 2000). 3.2. Biochemical characterisation of CLIC-2 3.2.1. Recombinant CLIC-2 Recombinant CLIC-2 with an amino terminal 6xHis tag was readily expressed in E. coli and purified by immobilized metal ion affinity chromatography on Ni-agarose. The purified recombinant CLIC-2 prepared by this procedure had a relative molecular mass of 34.9 kDa on SDS-PAGE and was normally better than 98% pure (Fig. 4). This size is notably larger than the molecular mass of 27.811 kDa calculated for the native protein, and 29.079 kDa calculated for the His tagged form expressed here. It should be noted that GSTO1 showed a similar increase in apparent molecular mass during SDS-PAGE (Board et al., 2000). Gel filtration studies revealed that under the conditions used here, CLIC-2 elutes in a volume that corresponds to a molecular mass of 36 ± 1.7 Da.

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This apparent size is in close agreement with the size determined by denaturing SDS-PAGE and suggests that CLIC-2 is a monomer under the conditions it was studied here. 3.2.2. Enzyme assays The capacity of CLIC-2 to catalyze glutathionedependent reactions was tested with a range of compounds that have been shown to be substrates for other GSTs. CLIC-2 was inactive with most of the substrates including 1-chloro-2,4-dinitrobenzene; 1,2-dichloronitrobenzene; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; 1,2-epoxy-3-(4-nitrophenoxy) propane; 4-phenylbut-3-en-2-one; ethacrynic acid; p-nitrobenzyl chloride; p-nitrophenylacetate; menaphthyl sulphate; trans-nonenal; trans,trans-nona-2,4-dienal; Dehydro ascorbate. Perhaps significantly, there was no detectable thioltransferase activity which is a distinct feature of glutaredoxin and GSTO1-1 which share a very similar active site motif with CLIC-2. In contrast, CLIC-2 had weak glutathione peroxidase activity with cumene hydroperoxide or t-butyl hydroperoxide (Table 1). 3.3. Functional properties of CLIC-2 3.3.1. CLIC-2 inhibits cardiac RyR channels CLIC-2 strongly inhibited native cardiac RyR channels that were activated by 10 ␮M cis Ca2+ . When CLIC-2 was added to the cytoplasmic (cis) side of the channels there was a substantial decrease in single-channel activity as illustrated in the brief trace in Fig. 5A (left) and in the all points histograms for a 30 s recording (Fig. 5A, right). The fall in activity was fully reversed when CLIC-2 was washed out of the cis chamber. Relative mean current through cardiac RyR channels during exposure to CLIC-2 (expressed either as a His-tagged fusion protein (hatched bars) or as a ubiquitin fusion protein with ubiquitin subsequently removed (grey bars)) is plotted in Fig. 5B. The average results show that (a) both the His-tagged CLIC-2 and untagged CLIC-2 caused a similar significant reduction in channel activity, (b) the changes in channel activity were similar at positive and negative potentials and (c) the reduction in activity could be reversed by perfusion of the cis chamber with control solution. The decrease in activity occurred 6–8 min after adding

Fig. 5. CLIC-2 reversibly inhibits cardiac RyR activity. Channel activity was recorded in symmetrical 250 mM Cs+ solutions with 10 ␮M Ca2+ in the cis solution. (A, left panel) Current records showing the effect of CLIC-2. The potential was +40 mV and channel opening is upward from the closed level (C, solid line), to the maximum single-channel conductance (O, dashed line). The upper record shows control activity, the middle record shows activity after addition of 3 ␮M CLIC-2 to the cis chamber, and the lower record shows recovery after perfusion of the cis chamber. (A, right panel) All points histograms from 60 s of channel activity (two 30 s segments of activity at +40 mV). (B) The histograms show the average relative mean current at −40 mV (left) and +40 mV (right) under control conditions (control), during exposure to 1–8 ␮M CLIC-2 (CLIC-2) and after washout of CLIC-2 (wash). The cross-hatched bars show data for His-tagged protein (n = 9), and the grey bars show data for protein expressed as a ubiquitin fusion protein, with ubiquitin subsequently cleaved (n = 6) (methods). Clearly, the His tag does not interfere with the action of the CLIC-2 protein. The vertical bars show ±1S.E.M. The asterisks indicate data that is significantly different from the preceding condition.

CLIC-2 to the bilayer solution, while reversal occurred within 1 min of removing CLIC-2 from the solution. The effects of CLIC-2 were specific and unrelated to the 50 mM Hepes, 10% glycerol (pH 7) buffer used

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junction with a significant increase in the mean closed time. The changes in each parameter were reversed when CLIC-2 was washed from the cis chamber by perfusion. 3.4. An antibody to CLIC-2 abolished the inhibitory effect of the protein

Fig. 6. The concentration-dependence of CLIC-2 inhibition. Average mean current is shown as a function of CLIC-2 concentration. The symbols show the average data from at least five experiments at each concentration of CLIC-2, with data at +40 and −40 mV pooled. CLIC-2 was added to the cis chamber and mean current measured from 60 s of activity recorded 6–8 min after CLIC-2 addition. Since the effect of CLIC-2 was the same at positive and negative potentials, data from both potentials have been grouped in the average relative mean currents.

as vehicle for the protein. Neither addition of vehicle alone, nor its perfusion from the cis chamber, altered channel activity (n = 6). The concentration-dependence of the effect of CLIC-2 on RyR channels is shown in Fig. 6. The IC50 for the CLIC-2-induced reduction in the mean current flowing through the channels was ∼750 nM. single-channel analysis of data from a subset of 17 experiments in which bilayers contain only one active channel, showed a significant reduction in the average open probability (Fig. 7). This fall in P0 was due to a significant reduction in the mean open time in con-

A specific effect of CLIC-2 on cardiac RyR activity was indicated by the fact that, after inhibition of channel activity by CLIC-2, the addition of a polyclonal antibody to CLIC-2 to the cis solution restored activity to control levels. The single-channel records and all points histograms (Fig. 8A–D) as well as the average data (Fig. 8E) show a significant reduction in channel activity in the presence of CLIC-2, at positive and negative potentials, and a significant reversal of the decrease after addition of the antibody. On average, activity did not then change significantly when CLIC-2 and antibody were washed from the cis solution by perfusion. The antibody did not have a direct effect on the RyR because when the antibody was added in the absence of CLIC-2, there was no significant change in channel activity (Fig. 8F). There was a small increase in activity in 4 of 5 channels at −40 mV when the antibody was washed out of solution—the increase in activity in the average data was not significant. In 33 of 44 experiments, channel activity was depressed to less than 50% of the control activity with CLIC-2 concentrations 1 ␮M. However, activity in nine experiments fell by less than 50%, and activity

Fig. 7. Effects of CLIC-2 on single-channel characteristics. single-channel analysis was performed on records from 17 experiments in which one channel only was open at any time in the bilayer. Records were obtained with 10 ␮M cis Ca2+ and 6–8 min after exposure to CLIC-2 at 2–12 ␮M. Similar results were obtained at +40 and −40 mV and data from the two potentials was included in the averages. Addition of CLIC-2 caused a significant fall in (A) open probability, and (B) mean open time and a significant increase in (C) mean closed time. All changes were fully reversed by CLIC-2 washout. The vertical bars in (A–C) indicate ±1S.E.M. The asterisks indicate values that were significantly different from the preceding measurement.

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Fig. 8. The effects of CLIC-2 are reversed by an anti-CLIC-2 antibody. Left panel: Current recorded +40 mV from a bilayer containing three RyR channels under control conditions (A), after inhibition by 3 ␮M CLIC-2 (B) and subsequent addition of 5 ␮l of serum with anti-CLIC-2 antibody (C) and after washing the CLIC-2 and antibody out of the cis chamber (D). Channel opening is upward from the closed level (C, solid line) to the maximum open single-channel conductance (O1 , first dashed line), and to the summed maximum conductances of the two channels (O2 , second dashed line). The channels spend substantially longer periods in the closed state in the presence of CLIC-2. The right panel shows all points histograms for 30 s of continuous activity under each condition. (E) Average normalised mean current from 10 experiments at −40 mV (left) and +40 mV (right) under control conditions (cont), in the presence of 3–8 ␮M CLIC-2 (CLIC-2), with CLIC-2 plus 5 ␮l of serum containing anti-CLIC-2 antibody (AB) and after perfusion of the cis chamber (wash). Asterisks show significant differences from the preceding condition. (F) Average normalised mean current from five experiments at −40 mV (left) and +40 mV (right) under control conditions (cont), with 5 ␮l of anti-CLIC-2 antibody (AB) and after perfusion of the cis chamber (wash).

increased in two experiments. This variability did not depend on the initial activity of the channel nor on the preparation of either cardiac SR or of CLIC protein. We have obtained preliminary evidence that, unlike GSTO1-1 (Dulhunty et al., 2001a), CLIC-2 inhibits

skeletal muscle RyR channels also (n = 4 experiments). This suggests that there may be significant differences in the interactions between the different CLIC and CLIC-like proteins and the different RyR isoforms.

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Table 2 Effects of CLIC-2 on the total uptake (column 2) and release (column 3) of Ca2+ and on rates of Ca2+ uptake (column 4) and Ca2+ -induced Ca2+ release (column 5) from cardiac SR vesicles

Control Buffer CLIC-2

Total uptake (nmol)

Total release (nmol)

Ca2+ uptake (nmol/(mg min))

CICR (nmol/(mg min))

59.9 ± 1.3 (4) 57.2 ± 1.3 (2) 58.1 ± 1.0 (6)

58.8 ± 0.5 (4) 58.5 ± 0.7 (2) 63 ± 0.5 (6)

473 ± 21 (5) 500 ± 23 (10) 554 ± 39∗ (9)

66.7 ± 2.8 (5) 64.2 ± 2.6 (10) 49.7 ± 1.4∗ (9)

Control values were obtained after addition of water at a volume equivalent to that added with 36 ␮M CLIC-2. Buffer with addition of Hepes buffer (50 mM Hepes, 200 mM NaCl, pH 5.7) alone at an equivalent volume to that added with 36 ␮M CLIC-2. CLIC-2 values were obtained after adding 18–36 ␮M CLIC-2 in Hepes buffer. The asterisks indicate values obtained with CLIC-2 that are significantly different from the values obtained with buffer alone. The amount of Ca2+ loaded in the absence and presence of CLIC-2 was measured in two ways; total uptake was the amount of Ca2+ taken up during the initial loading phase of the experiment (with a total addition of 60 nmol of Ca2+ ), while total release was the Ca2+ released during the remained of the experiment (leak in thapsigragin, plus CICR, plus ionophore-induced release). Load indicated by total release tended to be greater than normal in the presence of CLIC-2. Values for n ≥ 4 are the mean ± S.E.M., values for n = 2 are mean ± S.D.

3.5. Effects of CLIC-2 on Ca2+ accumulation and release in cardiac SR vesicles Ca2+ uptake and Ca2+ release from cardiac SR vesicles were examined to assess the effect of CLIC-2 on RyR activity in a more physiological situation. There was a significant 10% increase in the rate of net Ca2+ uptake in the presence of CLIC-2 (Table 2), as expected if CLIC-2 inhibits Ca2+ currents through the RyR. Although the rate of Ca2+ uptake increased, the amount of Ca2+ loaded (indicated by both the total accumulation (A) and total release (B)) were not significantly altered by the protein. Ca2+ -induced Ca2+ release, measured after adding thapsigargin to block the Ca2+ , Mg2+ -ATPase, was significantly depressed by 30% in the presence of CLIC-2 (Table 2).

4. Discussion In this paper, we have reported a tentative structure of CLIC-2, analysed its potential enzyme activity and show for the first time that a CLIC protein can modulate RyR channel activity. The structural and enzyme activity differences between the GSTs and CLIC proteins raised the question of whether a CLIC protein could modulate the cardiac RyR channel. The fact that RyR activity was inhibited by the CLIC protein suggests that the binding site on the CLIC/GST for the RyR complex must be located in a region of the proteins with strong structural similarity. CLIC-2 is one of only a few endogenous inhibitors of the cardiac

RyR that have been described and, although our evidence is indirect, it supports a role for the protein in preventing excess Ca2+ leak from the SR in the heart. 4.1. CLIC-2 is closely related to CLIC-1 and more distantly related to GSTO1 The sequence similarity between CLIC-1 and CLIC-2 suggests that CLIC-2 is a member of the GST structural family and the development of a plausible homology model strongly supports that notion. The CLIC proteins clearly have many features in common with the Omega class GST including a similar subunit size and conservation of the CPF motif in the active site region. The quaternary structure is one feature of the CLIC proteins that differs significantly from other members of the cytosolic glutathione transferase structural family. Members of the alpha, mu, pi, theta, sigma, zeta and omega classes have all been shown to be dimers. In contrast, CLIC-1 has been shown to be a monomer by gel filtration and by crystallographic studies (Harrop et al., 2001). The gel filtration data obtained in the present study suggests that CLIC-2 is also a monomer. 4.2. Expression and purification of CLIC-2 The studies described here demonstrate that recombinant CLIC-2 can be readily expressed in E coli. Some members of the GST structural family bind to immobilized glutathione and can be efficiently purified by affinity chromatography on glutathione agarose.

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In our studies CLIC-2 failed to bind to glutathione agarose thus preventing its purification by that method. In this case, we used glutathione agarose where the glutathione was linked to the agarose through the sulphur of glutathione. This would have precluded potential binding via the formation of a disulfide bond through an active site cysteine. The inclusion of an N-terminal poly His tag allowed the purification of recombinant CLIC-2 by immobilized metal ion affinity chromatography. While it is possible that the nine additional residues fused to the amino terminal may alter its functional properties, this has not been a significant problem for other GSTs that we have studied by this methodology (Tan & Board, 1996; Tan, Chelvanayagam, Parker, & Board, 1996; Whittington et al., 1999). Clearly, the presence of the His tag did not alter the ability of CLIC-2 to inhibit cardiac RyR channels. 4.3. Enzyme activity of CLIC-2 It is particularly notable that the position and sequence of the CXXC active site motif is well conserved between CLIC-2 and the glutaredoxins (Gladyshev et al., 2001). As thioltransferase activity is the predominant enzymatic activity exhibited by the glutaredoxins, it is surprising that this activity was not associated with recombinant CLIC-2. In previous studies we found that GSTO1-1 has low thioltransferase activity despite the fact that its active site contains only one cysteine. In contrast, we found that CLIC-2 has low glutathione peroxidase activity that is a property of several other members of the GST structural family such as GSTA2-2 and GSTT2-2. 4.4. Tissue distribution of CLIC-2 Northern blot analysis showed expression of CLIC-2 mRNA in a range of tissues with highest levels in the spleen and lung. GSTO1 was also found to be expressed in a wide range of tissues but there are clear differences in the relative levels of expression of GSTO1 and CLIC-2 particularly in the lung which expresses only low levels of GSTO1 mRNA (Board et al., 2000). There was distinct expression of CLIC-2 in heart and skeletal muscle which is particularly relevant to its role in the modulation of RyRs.

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4.5. RyR modulation by CLIC-2 Although it is widely distributed in human tissues, no function of CLIC-2 has been demonstrated. As noted above we did not find evidence for a significant level of enzymatic activity. However, CLIC-2 inhibited cardiac RyR activity in the same way as GSTO1-1. It is likely that the CLIC and CLIC-like proteins such as GSTO1-1 can subserve a variety of functions, as do many other important RyR regulators including Ca2+ and Mg2+ , ATP and calmodulin (Meissner, 1994), FK-506 binding proteins (FKBP12 and FKBP12.6) (Ahern, Junankar, & Dulhunty, 1994; Brillantes et al., 1994; Marx et al., 2000), homer (Feng et al., 2002) and GST-omega (Dulhunty et al., 2001a). The multiple functions of these essential regulators may allow the RyR channel to respond in synchrony with other cell processes during conditions such as oxidative stress, ischaemia, and fatigue. Important questions are what factors influence CLIC-2 expression and the other cell processes to which CLIC-2 contributes. CLIC-2 required several minutes to interact with RyR channels. The time course was similar, however, to the time course of oxidation/reduction reactions (Haarmann, Fink, & Dulhunty, 1999; Marengo, Hidalgo, & Bull, 1998), phosphorylation (Dulhunty et al., 2001b), calsequestrin dissociation (Beard, Sakowska, Dulhunty, & Laver, 2002) and FKBP12 association/dissociation (Marx, Ondrias, & Marks, 1998; Marx et al., 2001). The rates of these reactions possibly reflect the size and complex geometry of the RyR protein (Radermacher et al., 1994). 4.6. Variability in RyR-2 responsiveness to CLIC-2 A small fraction of cardiac RyR channels were not inhibited by CLIC-2, or were less strongly inhibited than the majority of channels. The degree of inhibition may depend on the presence of a co-protein, or perhaps the phosphorylation or oxidation state of the channel if these are not homogeneous. There are several examples of the actions of associated proteins depending on the presence of other associated proteins. Inhibition by calsequestrin requires triadin/junctin (Beard et al., 2002); activation by the DHPR II-III loop or loop fragments requires FKBP12 (Dulhunty et al., 1999; O’Reilly et al., 2002); and inhibition of skeletal RyRs by a peptide corresponding

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to a sequence in the C-terminal part of the DHPR is interrupted by calmodulin (Sencer et al., 2001). 4.7. Site of CLIC-2 interaction with RyRs Since the experiments were done with native RyR channels incorporated into bilayers with their associated proteins, it is not clear whether CLIC-2 reacts directly with the RyR or with other components of the RyR complex. The fact that CLIC-2 depressed Ca2+ -induced Ca2+ release from SR vesicles indicates that the protein can inhibit Ca2+ release even if all RyR channels do not respond in an identical fashion. The need for a higher concentration of protein required to influence Ca2+ release from SR, compared with that required to effect channels in bilayers is commonly reported (Dulhunty et al., 1999; Lu, Xu, & Meissner, 1994). Cytoplasmic concentrations of CLIC proteins have not been measured. Rowe, Nieves, and Listowsky (1997) reported concentrations of individual GSTs in human heart ranging from 0.3 to 5.2 ␮g/mg of protein and values as high as 50 ␮g/mg in other tissues. This suggests concentrations of GSTs of around 10 ␮M under normal conditions. Nothing is known about the concentrations of GST/CLIC proteins during oxidative stress. However, some GST genes are induced during oxidative stress and the concentration of GST/CLICs may be elevated by 2–3-fold under these conditions (Hayes & Pulford, 1995). 4.8. The physiological significance of CLIC-2 actions The fact that CLIC-2 can modulate cardiac and skeletal RyR channels suggests that the protein may interact with these isoforms of the RyR in brain, lymphocytes, smooth muscle and other tissues in which the RyR regulates cytoplasmic Ca2+ stores. The action of CLIC-2 in depressing RyR channel activity suggests indirectly that it could be effective in preventing or reducing Ca2+ overload in conditions such as ischaemia, and in slowing apoptotic processes.

Acknowledgements The authors acknowledge financial support from Pfizer. We are grateful to Ms. Suzy Pace and Joan

Stivala for assistance with the preparation of skeletal and cardiac sarcoplasmic reticulum vesicles, and to Ms. Louise Cengia for performing the experiments on Ca2+ release from SR vesicles.

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