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EDITORIAL FOCUS
Centro de Estudios Científicos, Valdivia, Chile
CLC-2 IS A CHLORIDE CHANNEL that belongs to the ClC family of anion channels and transporters. ClC-2 is activated by hyperpolarization, leading to strict inward rectification, and is further activated by intracellular chloride and extracellular acidification. Work with recombinant ClC-2 in different expression systems, and the study of ClC-2-like channels in native cells, has not given a unified picture for the behavior of the channel. This is particularly so for the expression of ClC-2 in mammalian cells and in amphibian oocytes, which show time constants for the activation process differing by up to two orders of magnitude. In the present issue of the American Journal of Physiology, Hinzpeter et al. (Ref. 7; see p. C45 in this issue) report the interaction of heat shock proteins Hsp90 and Hsp70 with ClC-2. What is particularly interesting in these novel findings is that Hsp90 is able not only to modulate the trafficking of ClC-2 to the plasma membrane but also to modulate ClC-2 activity by changing its intracellular chloride sensitivity. As discussed by the authors, these effects on gating might contribute to the explanation of some of the seemingly contradictory results in different expression systems and cell types (9).
Hinzpeter et al. immunoprecipitated ClC-2 from overexpressing human embryonic kidney-293 cells and then identified Hsp70 and Hsp90 as coimmunoprecipitating proteins by mass spectrometry. The interaction with Hsp90 is not an overexpression artifact, because these authors were able to pull down ClC-2 with an anti-Hsp90 antibody in brain tissue. Pharmacological manipulation of the activity of Hsp90 suggested that this protein did not alter the total amount of ClC-2 channel protein present in the cells, but affected markedly the relative intracellular to membrane distribution of ClC-2. This in itself points to a regulation of function by the heat shock protein, but a most exciting aspect of Hinzpeter et al.’s work has to do with the effects of manipulating the activity of Hsp90 on the gating of ClC-2. Activation of Hsp90 appears to accelerate the rate of ClC-2 opening by hyperpolarization and to facilitate opening by shifting the V0.5, the voltage required to reach 50% apparent open probability. Both effects, explored through inhibition of Hsp90 with geldanamycin, were dependent on intracellular chloride concentration, being enhanced as the intracellular anion was increased. The effects of Hsp90 overexpression by thermal stress on the kinetics of ClC-2 are the opposite of those of pharmacological inhibition. Hsp90 joins p34cdc2/cyclin B, which regulates ClC-2 expression levels by ubiquitination (18), the SGK1–3 kinases and ubiquitin ligase Nedd4-2, which modulate plasma membrane ClC-2 abundance (13), and protein kinase C, which decreases ClC-2 activity (2, 16). However, the finding by Hinzpeter et al. that an interacting protein can, on the one hand, regulate the abundance of ClC-2 in the membrane, while it alters in a subtle way its gating properties had not been described before. Despite the excitement, a word of caution must be sounded about the possible specificity of the interaction, because Hsp90 can constitute up to 2% of total cell protein under nonstress conditions (15).
The observation that interacting proteins are able to affect ClC-2 gating is of great interest. As pointed out by the authors, it has been a puzzling recurrent result that the voltage dependence and kinetics of ClC-2 can be markedly different when studied in different expression systems. A similar variability has been reported in ClC-2-like currents studied in different native cells or cell lines. This could be due to the expression of different splice variants, but there are only two examples of variants producing small functional changes (3, 4), which are insufficient to account for the observed variability. Interaction with a partner protein differentially expressed in different cell types could provide a more comprehensive explanation for the variability in ClC-2 behavior. ClC-2 can be activated by hyperpolarization, cell swelling, or low extracellular pH. Deletion of a stretch of amino acids at the amino terminus of ClC-2 results in constitutively open channels without any voltage, cell swelling, or pH dependence when Xenopus oocytes are used as the expression system (10). In contrast, the same deletion mutant of ClC-2 when studied in mammalian cells shows hyperpolarization activation and extracellular pH dependence (17). In the light of the results reported by Hinzpeter et al., one explanation of these data could be that the NH2 terminus deletion might alter the interaction of ClC-2 with partner proteins such as Hsp90 specifically in the amphibian oocyte and that a similar interaction is absent or somehow altered in the mammalian cells. Interestingly, some of the oocyte-specific results can be reproduced in mammalian cells recorded by the so-called nystatin-perforated patch-clamp recording technique, which, incidentally, is the approach used by Hinzpeter et al. This might point to a diffusible component essential for the putative protein-protein interaction. Previous work (1) has highlighted the amino end of ClC-2 as a site for interaction with the actin cytoskeleton; whether this might be related to the interaction with Hsp90 has not been investigated.
ClC-2 has high expression in the brain and currents that might be mediated by ClC-2 have been detected in glial cells and neurons. It has been suggested that expression of ClC-2 in certain neurons such as pyramidal cells, could be involved in preventing the accumulation of intracellular chloride above electrochemical equilibrium. This is believed to contribute to the prevention of excitation mediated by glycine or GABAA receptors. These are agonist-activated chloride channels, which normally evoke inhibition because they are expressed in neurons that exclude chloride actively. It has been argued that the activation of glycine and GABAA receptors open ClC-2 channels by increasing intracellular chloride and that this prevents excessive chloride accumulation during high-frequency neuronal activity. Some credence for this hypothesis came from experiments showing that infection with ClC-2 could confer GABA-dependent inhibition to dorsal root ganglion neurons showing GABA-evoked excitation (16). It was, therefore, not totally unexpected when, 2 years ago, a report revealed three mutations in CLCN2, the ClC-2 gene, that cosegregated in autosomal-dominant fashion with idiopathic generalized epilepsies (6). Two mutations, predicting a severe truncation of the protein and altered splicing with nearly complete loss of a large transmembrane helix, were predicted to cause dominant negative effects. These results have not been reproduced by others (8, 12). A third, missense mutation (G715E) is of some interest in relation to the present discussion. G715 is found in the cytoplasmic COOH terminus tail between two cystathione 
-synthase CBS domains. The mutation was reported to make the channel less sensitive to internal chloride concentration (6). This would imply that the activity of the channel at very low intracellular chloride is higher in mutant than in nonmutated channels. It could be speculated that this might be expected if the activity of Hsp90 or its effect on ClC-2 were enhanced by the G715 mutation. Although the effect of G715E is controversial (12), it might be worthwhile to explore this possibility by attempting to reproduce the effects of Hsp90 inhibition on wild-type and G715E mutants.
As mentioned above, a further aspect of Hinzpeter et al.'s work has to do with the control of the plasma membrane abundance of ClC-2. Among other effects, Hsp70 and Hsp90 have been shown to interact transiently with the heart potassium channel human ether-à-go-go-related gene (hERG). Inhibition of Hsp90 prevents maturation and reduces hERG currents, and it is believed that this chaperone is essential for the correct folding of the channel (5). The effect of Hsp90 inhibition on ClC-2 membrane abundance appears to be of a different kind, because it involves only a shift between intracellular and membrane ClC-2 without modification of the expression level. This is more reminiscent of a mobilization to the plasma membrane of protein located in intracellular membranes. Little is known about ClC-2 recycling and membrane targeting. A recent study (14) investigated membrane targeting of ClC-2 in polarized epithelial cell lines. ClC-2 was shown to be specifically targeted to the basolateral membrane of various cell lines, and a dileucine motif in the second COOH terminus CBS domain was found to be essential for this sidedness. In addition, mutations of two further dileucine motifs in the first CBS domain led to intracellular retention. It appears, therefore, that CBS domains must play a role in membrane trafficking, but nothing is known about a possible relationship to Hsp90 activity.
It is interesting to speculate how the type of interaction discovered by Hinzpeter et al. between the ClC-2 channel and Hsp90, itself not an integral membrane protein, might exert an effect on the chloride dependence of gating, which should in principle be physically separate from putative regions of interaction. Functional and structural studies demonstrate that chloride channels of the ClC family have a dimeric double-barreled structure, with each monomer contributing an identical pore. Evidence for the presence of slow and fast hyperpolarization-dependent gating processes in ClC-2, that might correspond to individual pore gating, and a common process opening both pores simultaneously has been provided (11, 20). The chloride dependence of ClC-2 is thought to be conferred by interaction of the permeant anion with the selectivity filter within the pore. Recently, however, the role of the COOH-terminal CBS-2 domain of ClC-2 in its voltage gating has been demonstrated. In fact, a single-point mutation in a conserved CBS-2 histidine shifted the V0.5 of ClC-2 to more positive potentials by 
30 mV (19). These results highlight the role of CBS domains in controlling a chloride-dependent gating process that is occurring at the selectivity filter of the channel. It would therefore not be far fetched to picture a ClC-2-Hsp90 protein-protein interaction, perhaps involving the CBS domains, producing gating modulation in addition to membrane trafficking regulation.
Hsp70 and Hsp90 have known roles as protein chaperones and participate in protein folding, control of conformational maturation, and activity of various proteins that include, at least for Hsp90, some membrane receptors and ion channels and transporters. The findings reported by Hinzpeter et al. in this issue of the American Journal of Physiology demonstrate a novel interaction of Hsp proteins with the ClC-2 chloride channel effecting its cellular location and the ability of intracellular chloride to gate the channel. In addition to starting to provide possible explanations for cell-specific diverse behaviors of the channel, the new reported findings show that a single protein interaction can alter both gating and membrane trafficking. It is tempting to speculate that the CBS domains that have been shown to have an importance in regulating subcellular distribution, trafficking, and gating of ClC-2 could be the targets of the action of associated proteins providing a (perhaps tissue specific) fine-tuning of ClC-2 activity.
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