Centro de Estudios Científicos, Valdivia, Chile
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
epilepsy; CLCN2 gene; AMP and ATP regulation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ClC-2 belongs to a family of chloride channels that is widely expressed, showing a relatively high level in brain and epithelia (22, 43). In brain, the ClC-2 transcript and protein are present in neurons and astrocytes (16, 38, 40). Using immune electron microscopy, the protein was found in hippocampus, in the soma and dendrites of CA1 and CA3 pyramidal cells, and in some interneuronal cells, but was absent from dentate granular cells. ClC-2 immunostaining was seen close to presumed GABAergic inhibitory synapses. In astrocytes ClC-2 protein has been found at the end feet of astrocytes contacting blood vessels and neurons close to inhibitory synapses (38).
Inwardly rectifying ClC-2-like currents activated by hyperpolarization have been recorded in hippocampal pyramidal cells (40, 41) and in astrocytes (14, 27, 28, 33). In these two cell types the channel might play different roles. In neurons, it is speculated that because of its activation by intracellular Cl, ClC-2 would prevent accumulation of this anion above equilibrium (41, 42). Intracellular accumulation of Cl might take place during high-frequency stimulation due to an increase in extracellular K+ with reversal of KCl cotransport (7, 23). Under these conditions, GABAA receptor activity would become excitatory. It must be pointed out that a mechanism involving ClC-2 in preventing a depolarizing GABA response requires Cl accumulation above equilibrium as a channel is, obviously, not able to extrude ions. According to this reasoning, loss of function mutations of the ClC-2 channel could result in increased excitability in certain neurons.
Hyperpolarization-activated chloride currents are also present in cortical astrocytes incubated with dibutyryl-cAMP, astrocytes cocultured with neurons, or in acute brain slices. These currents were sensitive to physiological changes in extra- and intracellular pH (15, 27), were activated by hyposmotic swelling (13), and were absent in tissues from ClC-2-null mice (28). These currents could have roles in K+ buffering, pH regulation, and volume regulation (45). Changes in any of those functions could alter the neuron microenvironment and promote changes in excitability.
Recently, a genome wide search study identified a susceptibility locus for idiopathic generalized epilepsy (IGE) on chromosome 3q26 (34). This is the location of the CLCN2 gene, encoding for the voltage-dependent ClC-2 chloride channel, thus constituting a plausible epilepsy candidate gene. In fact, three different mutations on this channel were found that cosegregated with heterogeneous types of IGE with an autosomal dominant inheritance (18). Two of the mutations predict a truncated protein and the skipping of exon 3, respectively, and they were shown to exert dominant-negative effects leading to complete loss of channel function. A third, missense mutation, produces an amino acid replacement (G715E) in the COOH terminus, which is associated with a gain of function, allowing the channel to be conductive at reduced intracellular Cl concentration. Loss of function would account for hyperexcitability for the first two mutations. A different effect was hypothesized for the third mutation: intense GABAergic and glutamatergic activation could increase intracellular Cl through GABAA-mediated influx in a glutamate-depolarized situation. During repolarization, Cl might transiently be found above equilibrium. The presence of active G715E, but not of wild-type (WT), channels at low intracellular Cl would result in Cl efflux, depolarization, and hyperexcitability of the postsynaptic membrane. This gain of function was proposed to explain hyperexcitability leading to seizures in heterozygous affected patients (18).
All eukaryotic ClC proteins have a long carboxy-terminal cytoplasmic region that contains two cystathionine-ß-synthase (CBS) domains. These domains, typically 60 residues in length, are present in tandem pairs in a diversity of proteins from archaebacteria to eukaryotes (4). Several functions have been proposed for CBS domains, which range from roles in the oligomerization and allosteric regulation of cystathionine-ß-synthase, to the subcellular localization, trafficking, and gating of ClC channels. The biological importance of these domains, on the other hand, is revealed by point mutations in CBS domains of several unrelated proteins that result in various human inheritable diseases (see Ref. 12, and references cited therein). Recently Scott et al. (37) have found that pairs of CBS sequences derived from different proteins bind derivatives of adenosine and, importantly, mutations in these domains that cause different hereditary diseases impair this binding. A glutathione-S-transferase fusion of the isolated CBS domain pair from ClC-2 binds ATP with a KD of 1 mM, while introduction of the epileptogenic G715E mutation (18) shifted KD to 10.4 mM. The authors suggest a functional role of these CBS domains in sensing the energy status of the cell similar to the enzyme AMP-activated protein kinase (AMPK).
In the present study we partially challenge some of the conclusions of Haug et al. (18) concerning the functional effects of the mutations described, by using patch clamp and confocal microscopy. Additionally, we test the effects of ATP and AMP on ClC-2 electrophysiological properties. We found that intracellular AMP affects the channel kinetic, increasing its rate of activation and deactivation. The G715E missense mutation, located between both CBS domains, significantly decreased the effect of AMP compared with WT channels.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Minigene construction and expression.
A 963-bp fragment of the human genomic CLCN2 sequence, containing exons 2, 3, and 4 and the corresponding introns was cloned into the pCR3.1 vector. The 963-bp genomic sequence was generated from genomic DNA isolated from Caco-2 cells using PCR and specific primers located in exon 2 (sense, 5'-AGATGTATGGCCGGTACACTCAGG-3') and exon 4 (antisense, 5'-TCAGGCTGTCGGTATGTTAGAA-3'). Subsequently, the 11-bp deletion in intron 2 found by Haug et al. (18) (IVS2-14del11 mutation) was performed by PCR, and both constructs were sequenced. The expression of both minigenes, WT and intron211bp, was studied by RT-PCR in the following cells lines: N2a neuroblastoma cells, GT1-7 mouse hypothalamic cells (30), and HEK-293 cells. The cells were transfected with each minigene (1 µg of plasmid DNA) using Lipofectamine (Invitrogen), and 48 h later total RNA was extracted using TRIzol (Invitrogen) and reverse transcription was performed using SuperScript II (Invitrogen). Thirty PCR cycles (15 s at 94°C, 30 s at 58°C, 60 s at 72°C) were performed using cDNA of the transfected cell lines with the sense primer located at the start of exon 2 (5'-ATGTATGGCCGGTACACTCAGG-3'), and the antisense primer in the pCR3.1 vector (5'- TAGAAGGCACAGTCGAGG-3'). PCR reactions were in the linearly increasing phase of amplification under these conditions, as verified by using different number of PCR cycles (2040 cycles). PCR products were viewed on a 1.5% agarose gel by ethidium bromide staining, and the amplicon intensity was quantified using Scion Image software.
Confocal microscopy localization experiments.
HEK-293 cell were transiently transfected with 1 µg of the corresponding hClC-2 WT-GFP, M200sfX231-GFP, or 74117-GFP plasmid on glass coverslips and 24 h after transfection were mounted in a perfusion chamber for observation. The plasma membrane was stained by incubation with 10 µM 1-(3-sulfonatopropyl)-4-[ß[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8-ANNEPS, Molecular Probes) during 2 min. The location of fusion protein was analyzed with a Zeiss model LSM 510 confocal microscope equipped with Ar (488 nm) and HeNe (543 nm) lasers. The objective lens used was c-apocromatic 63x/1.2W corr. The excitation of GFP and di-8-ANNEPS was done simultaneously by the 488 nm laser beam by single-track function. The beam path was set using a 488 nm main dichroic mirror and a 545 nm secondary dichroic mirror. The light emitted for GFP was detected in channel 2 with a 505530 nm band-pass filter and di-8-ANNEPS in channel 1 with a 560615 nm band-pass filter. The image size was 1,024 x 1,024 pixels, and the pinhole setting was 110 (corresponding to 0.87 and 1.0 Airy units for channels 1 and 2, respectively). The images were processed using Huygens Professional deconvolution software.
Electrophysiological methods.
HEK-293 cells were grown and transiently transfected with expression plasmids for the hClC-2 constructs and H3-CD8 (1 µg/0.3 µg) to identify effectively transfected cells as described previously (6). In coexpression experiments the plasmid constructs were transfected in 1:1 ratio (0.5 µg each). Experiments were performed on cells in 35-mm cell-culture plastic Petri dishes mounted directly on the microscope stage. The bath solution contained (in mM) 140 NaCl, 2 CaCl2, 1 MgCl2, 22 sucrose, and 10 HEPES, pH 7.4, adjusted with Tris. The pipette solution (35 mM Cl) contained (in mM) 100 sodium gluconate, 33 CsCl, 1 MgCl2, 2 EGTA, 1 trisodium ATP, and 10 HEPES, pH 7.4, adjusted with Tris. Low-Cl solution (10 mM) was prepared by replacement of CsCl with sodium gluconate. In some experiments, ATP was replaced with 2 mM AMP. In experiments lacking nucleotides the MgCl2 concentration used was 0.25 mM or 1 mM, the calculated free Mg2+ concentrations for pipette solutions containing 1 mM ATP and 2 mM AMP, respectively. Liquid junction potentials were calculated (3), and appropriate corrections were applied. Standard whole cell patch-clamp recordings were performed as described elsewhere (9) using an Axopatch 200B (Axon Instruments) or an EPC-7 (List, Germany) amplifier. The bath was grounded via an agar-150 mM KCl bridge. Patch-clamp pipettes had resistances of 23 M
. The voltage pulse generator and analysis programs were from Axon Instruments. When giving trains of pulses, an interval of 60 s or 90 s between pulses was left at the holding potential to allow for complete current deactivation. In some experiments, a ramp taking the voltage from 130 to 30 mV in 50 ms was given after each pulse to test for selectivity conservation. Experiments where a change in Erev was seen were discarded. The currents generated by transfection were neither observed in untransfected cells nor in cells transfected with the
H3-CD8 plasmid alone. There was no detectable functional difference between constructs encoding WT or mutant ClC-2 channels and their respective fusion proteins with GFP.
Time courses for current activation and deactivation were described by fitting a double exponential plus a constant term equation (6) of the form:
![]() | (1) |
![]() | (2) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
It is intriguing that the 74117 mRNA has been found to be present in peripheral blood cells of both patients and healthy controls, albeit at a lower level in the latter (18). We reasoned that perhaps a cell-specific splicing might favor the
74117 mRNA in cells of neural origin. We constructed two minigenes as illustrated in the scheme in Fig. 1E, one containing a normal intron 2 and one carrying the 11-bp deletion. These were transfected into N2a neuroblastoma cells, GT1-7 mouse hypothalamic cells, and HEK-293 cells, and their processing was assessed by amplifying the RNA products by RT-PCR. The results are shown in the gel for N2a cells. Both minigenes generated the RNAs of the expected three-exon size (495 bp), suggesting that the splicing machinery recognized the genomic structure of these constructs. A product of the expected, exon-3-skipped size was also observed (365 bp). Sequencing of the amplicons confirmed their identities with RNAs stretches corresponding to exon 2/3/4 and exon 2/4. Using a semiquantitative RT-PCR approach, we demonstrated that 495- and 365-bp products were generated in similar ratios independently of which minigene was used in the experiment. In N2a cells the fraction of exon 3 skipping was 0.38 ± 0.03 and 0.31 ± 0.03 (means ± SE, n = 7) for minigenes WT and intron2
11bp, respectively. This difference did not reach statistical significance by t-test (P = 0.1). Experiments with GT1-7 and HEK-293 cells produced similar results (data not shown).
It has been claimed that both M200fsX231 and 74117 mutants of ClC-2 reach the membrane to exert dominant-negative effects that inhibit the activity of WT ClC-2 markedly (18). In view of our results, we reexamined the issue of the localization of these mutants and compared it to that of WT ClC-2. GFP fusion protein distribution was compared with that of the membrane dye di-8-ANNEPS. Figure 2, AC, shows WT ClC-2-GFP, di-8-ANNEPS and merged images, respectively. ClC-2-GFP (Fig. 2A, green) had a wide intracellular distribution and what appeared as discrete peripheral labeling that might correspond to the plasma membrane. Comparison with di-8-ANNEPS distribution (Fig. 2B, red) and, particularly, examination of the merged image (Fig. 2C) revealed discrete areas of superposition (yellow). The distributions of M200fsX231-GFP and
74117-GFP fusion proteins are shown in Fig. 2, DF and GI, respectively. The fluorescence from M200fsX231-GFP and
74117-GFP was observed throughout the cell interior, and no superposition with the membrane dye di-8-ANNEPS was detected.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The M200fsX231 mutant of ClC-2 is predicted to produce a severely truncated protein. Possible fates for this putative protein include its elimination by quality control checkpoints during its biogenesis and trafficking, remaining in intracellular compartments, and reaching the plasma membrane. Haug et al. (18) studied this point by transfection of fluorescent-labeled fusion proteins and confocal microscopy. Their interpretation of the fluorescence data was that the mutant proteins reached the plasma membrane. It is difficult to distinguish, however, true membrane localization with the presence of intracellular fluorescence near the plasma membrane. We have used here a double-labeling approach with ClC-2-GFP channel fusion proteins and di-8-ANNEPS, a specific marker for the plasma membrane. The study of WT ClC-2 reveals a complex localization pattern. There is abundant intracellular protein in vesicular structures. In addition, there is plasma membrane labeling, revealed by superposition with di-8-ANNEPS, which suggests that ClC-2 is localized in discrete membrane areas. The distribution of ClC-2-GFP protein in transiently transfected HEK-293 cells is similar to that seen by immunofluorescence in COS7 fibroblasts, which express ClC-2 endogenously. The distribution in COS7 cells was seen to correspond to plasma membrane, early endosome, and perinuclear localization (8). This type of distribution is consistent with the results observed here. In addition, preliminary experiments (not shown) also identify a vesicular distribution of ClC-2-GFP in an early endosomal compartment.
By contrast with the subcellular distribution of WT-GFP, that of the M200fsX231-GFP fusion protein was entirely intracellular and there was no obvious superposition with di-8-ANNEPS fluorescence. It is possible that a dominant-negative effect might arise by M200fsX231 altering the trafficking of WT proteins, preventing their access to the membrane. Functional experiments, however, reveal no hint of such an effect, as coexpression of WT-GFP and M200fsX231 produced robust currents in our hands. It is interesting to note that truncation mutations of ClC-1, also a dimeric chloride channel, have always been found to be associated with recessive myotonia (22). The analysis of the alternatively spliced protein 74117 leads to very similar conclusions. Cotransfection of the mutant cDNA together with the WT channel cDNA produced sizable currents, with no evidence of a dominant-negative effect of the mutant. In addition, transfection of HEK-293 cells with cDNA encoding a
74117-GFP fusion protein gave a purely intracellular, reticular in appearance, distribution for the GFP fluorescence.
The experiments of coexpression of M200fsX231 and 74117 mutants with WT channels were performed with the same amount of total cDNA as those of expression of WT alone. This implies that the amount of WT cDNA was halved. The currents obtained, although lower than those seen with twice the amount of WT cDNA, were higher than 50%. We believe that this is caused by the lack of linearity between amount of cDNA used in transfection and activity of expressed protein (unpublished observations).
A puzzling finding concerning the IVS2-14del11 mutant is that the alternatively spliced mRNA that was predicted by Haug et al. (18) to arise from its expression has been found both in patients and healthy controls. Using a minigene approach, we find that regardless of the presence or absence of the deletion, an exon 3-skipped mRNA is produced, in addition to the normally spliced mRNA. The same result was obtained in N2a neuroblastoma cells, GT1-7 mouse hypothalamic neurons, and HEK-293 cells. Unlike the result obtained with human samples, we find no difference in the proportion of exon-skipped to normally spliced mRNA in any of the cell lines transfected with minigenes mimicking mutated or WT genes. It is not possible to state whether the results obtained in experiments with minigenes are representative of the splicing mechanisms taking place in vivo. The fact that the correct splicing did take place, however, argues for similar mechanisms applying to the minigene products in the cell lines as with the RNA arising from the entire gene. On this basis we would have to conclude that there is no difference in the way that mutated and WT gene products are treated by the cellular splicing machinery. We have no simple explanation for the discrepancy between previous results and our minigene experiments. Our results suggest, nevertheless, that there will be no difference in ClC-2 channel expression between normal individuals and those carrying mutation IVS2-14del11.
A further question that applies to both M200fsX231 and 74117 is whether the severely truncated or deleted proteins produced survive the quality control mechanisms at the endoplasmic reticulum and beyond. These quality control mechanisms are known to deal with many conditions that result in altered, nonnative protein conformation, leading to retention in the endoplasmic reticulum followed by translocation to the cytosol and degradation (44).
The third mutation associated with IGE is a missense mutation, G715E, affecting an amino acid located in the long intracellular carboxy terminus of the channel between the two CBS domains. Haug et al. (18) have proposed that this mutation provokes a change in the channel gating, making it less sensitive to internal chloride concentration. This would imply that the activity of the channel at very low intracellular Cl is higher in the mutant than in nonmutated channels. We have examined this point, but our results show no differences regarding voltage dependence of gating and kinetic parameters between WT and G715E channels. Moreover, we could not detect any difference in Cl dependence.
It might be interesting to point out that a COOH terminus endoplasmic reticulum export signal (FCYENE) was used in the experiments reported by Haug et al. (18). This export signal has been reported to profoundly alter the steady-state distribution of channels in the plasma membrane, increasing the number of proteins on the cell surface (26). We cannot discard that this difference might be at the source of the discrepancy between our results and those reported before (18).
Two different gating processes, a fast and a slow gate, have been proposed for the voltage-dependent opening of ClC-2 (47). These are analogous to those first proposed for ClC-0, the voltage-dependent Cl channel of Torpedo electroplax (31). Slow and fast gating of ClC-2 are promoted by hyperpolarization. Fast gating depends additionally on intracellular Cl, an effect that is reflected in a shift of the V0.5 to more a depolarized potential with increasing intracellular concentration. This intracellular Cl dependence of gating is a property of the pore, more specifically of competition between Cl and a glutamic acid residue side chain for an outermost Cl-binding site in the selectivity filter (11). Neutralization of this residue by mutation in ClC-2 abolishes all Cl dependence of the channel (32). It is, therefore, difficult to envisage how a point mutation far from the pore might alter the sensitivity to intracellular Cl. The slow gate, however, seems to be independent of intracellular chloride (32). Regarding this point there is a recent interesting suggestion that CBS domains could play a functional role in the common slow gate (12). Two CBS domains are present in the carboxy terminus of all eukaryotic ClC channel subunits. Point mutations within them cause several hereditary diseases in humans. Examples in the ClC family are ClC-1-related myotonia, Dent disease caused by ClC-5 mutations, and Bartter disease arising by mutation of ClC-Kb (24, 25, 39). Although the physiological role of CBS domains is still unknown, truncations removing parts of the distal CBS domain of ClC channels abolished functional expression in heterologous systems (20, 36).
Interestingly, Scott et al. (37) recently demonstrated that the isolated CBS domain pair from ClC-2 binds ATP and that the affinity decreases one order of magnitude with introduction of G715E mutation. We tested whether there was an electrophysiological correlate of this effect. Our results demonstrate that adenine nucleotides are able to modify the kinetics of the ClC-2 channel. The typical slow activation of the channel is accelerated when intracellular AMP replaces ATP. Both the slow and fast voltage-dependent opening processes are significantly accelerated by AMP replacement. This was observed in the WT and to a lesser degree in the G715E mutant. However, when WT and G715E were coexpressed, the effect of AMP for ATP replacement on opening kinetics was smaller and did not reach statistical significance. To explain this unexpected result, we would have to assume that the dimer made by the association of one WT and one G715E channel would be completely unresponsive to nucleotide replacement, thus producing a dominant-negative effect. As the contribution of the heterodimer to function is expected to be 50%, this would account for the result obtained.
What is the physiological meaning of this finding? It is tempting to speculate that the channel, through its CBS domains, is sensing changes in the energetic state of the cell by detection of AMP level. The AMP level is thought to be a key signal to regulate various enzymes under metabolic stress (17). The best studied of these is AMPK, which possesses four CBS domains in tandem in its -subunit. The enzyme is allosterically regulated by AMP, which binds to CBS domain pairs. Human disease-causing mutations impair AMP binding (1). Fusion protein studies suggest that each CBS pair forms a binding site and that there is strong positive cooperativity between them (37). It has been proposed that CBS domains in ClC-2 might fulfill a similar role and that mutation G715E impairs nucleotide binding (37). The effects of AMP on the kinetics of opening of ClC-2 observed here might be related to this postulated effect. As G715 is not in, but between CBS domains, perhaps the mutation alters the three-dimensional structure necessary for the postulated CBS1/CBS2 interaction (12). This might explain the decreased AMP effect seen in the mutant. To explain the even feebler effect of AMP for ATP replacement in the mixed WT and G715E experiment, we have speculated that the heterodimer is unresponsive to intracellular nucleotides. We have attempted to confirm this interpretation by using WT-WT and WT-G715E concatemers constructed as described previously for ClC-2 (46). Surprisingly, neither concatemer was responsive to nucleotide replacement (results not shown). This might be expected if an interaction between the CBS pairs from each monomer were necessary for optimal nucleotide binding/sensing. The concatemerization process, which tethers the COOH terminus of one protein to a short NH2 terminus of another, might destroy this putative interaction.
What would be the consequences of mutations of ClC-2 associated with IGE? This point can only be speculated upon, given the fact that the function of normal ClC-2 in the brain has not been elucidated. In neurons ClC-2 is postulated to prevent paradoxical GABAAR-mediated excitatory actions (42), while a role in extracellular electrolyte homeostasis is a proposed function in astrocytes (38). A dysfunction of either of these processes could lead to disturbances in neuronal excitability and could explain the epileptic phenotype. However, lack of epilepsy in the knockout ClC-2 mouse (5) makes it difficult to postulate a straightforward explanation for the relationship between the mutations and a pathophysiological mechanism. Haug et al. (18) have proposed dominant-negative effects for mutations M200fsX231 and 74117 of ClC-2. Although we find these two mutants to lack function, we do not find any evidence for dominant-negative effects in cotransfection experiments. In addition, our minigene experiments suggest that the
74117 product would arise equally well from a WT or a IVS2-14del11 gene. We have no explanation for this discrepancy. On the basis of our results, we would have to postulate a mechanism of haploinsufficiency to explain the phenotype through a decrease in the number of copies of active channels in the case the mutation producing M200fsX231. Our findings with the G715E mutation highlight the potential importance of the COOH terminus of the protein, including the CBS domains, in an unexpected mechanism of channel regulation. A failure in the G715E mutant channel to respond with accelerated kinetics to a situation of cellular stress depleting ATP and elevating AMP could be a potential pathophysiological mechanism. These situations of high energy consumption might arise in conditions leading to hyperexcitability that might become pathological in individuals carrying the mutation. The effect observed, however, is far from being deleterious, and we are not able to propose a solid pathophysiological mechanism for a possible action. A more detailed delineation of the function played by the kinetic change brought about by AMP in ClC-2 will be required for a full interpretation of these results. It must be pointed out that G715E mutation has only been found in a small family and with only one sib not carrying the mutation and being phenotypically normal. More genetic data will be needed to establish whether this is a genuine mutation or a benign polymorphism.
Finally, our results indicate that there is no simple relationship between ClC-2 mutations and the IGE pathophysiology. This might be due to the fact that we still do not know what the function of the channel is in the normal brain. On the other hand, the analysis of the pathophysiology of the disease-associated mutations is now being complicated by the increasing realization of the influence of modifier genes in the phenotype of human inherited disease. Although some traits are still recognized to be inherited in a monogenic fashion, exceptions to this rule are now being reported (2). The difficulty in generating a genotype-phenotype relation in the case of alterations in CLCN2 might be a consequence of such complexity. The true physiological and cellular nature of the defect might have to await a deeper understanding of the genetic background upon which the defects are expressed.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: L. P. Cid, Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Valdivia, Chile (pcid{at}cecs.cl).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|