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Am J Physiol Cell Physiol 290: C45-C56, 2006. First published July 27, 2005; doi:10.1152/ajpcell.00209.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

Alexandre Hinzpeter,1 Joanna Lipecka,1 Franck Brouillard,1,2 Maryvonne Baudoin-Legros,1 Michal Dadlez,3 Aleksander Edelman,1,2 and Janine Fritsch1,4

1Institut National de la Santé et de la Recherche Médicale Unité 467 and 2Proteomic Core Facilities, Institut Fédératif de Recherche 94, Faculté de Médecine, Université Paris-Descartes, Paris; 3Department of Physics, University of Warsaw, Warsaw, Poland; and 4Institut National de la Santé et de la Recherche Médicale U561, Hôpital Saint Vincent de Paul, Université Paris-Descartes, Paris, France

Submitted 2 May 2005 ; accepted in final form 19 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl–, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl– concentration [Cl–]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl–]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport

CLC-2 is one of the nine mammalian members of the ClC chloride channel family. It is slowly activated by hyperpolarization and can be further activated by extracellular acidification or hypotonic cell swelling (26, 33, 62). ClC-2 is expressed in many tissues, such as the brain, intestine, kidney, stomach, salivary glands (for review, see Ref. 32), and heart (17). Although a clear understanding of its physiological function remains to be determined, a growing body of evidence suggests that ClC-2 can play different roles, depending on the tissue in which it is expressed. Disruption of the ClC-2-encoding gene in the mouse leads to degeneration of male germ cells and photoreceptors, probably resulting from a defect in transepithelial transport across Sertoli cells and the retinal pigment epithelium (7). ClC-2 may participate in fluid secretion in the murine small intestine (27) or in fluid absorption in the colon, as suggested by immunolocalization (8, 36) and functional data (9). On the basis of studies performed with hippocampus cells (57) or transfected dorsal root ganglion neurons (58), ClC-2 has been implicated in the regulation of the effects of GABAA receptor action by controlling intracellular chloride concentration. In humans, mutations in the gene ClCN2 that can be predicted to cause hyperexcitability of GABAergic synapses have recently been found to be associated with idiopathic generalized epilepsies (28).

Most of the knowledge about the gating properties of ClC channels comes from biophysical and mutational analysis of ClC-0 and ClC-1 (for review, see Ref. 32). These studies suggested a homodimeric structure of the channels with two independent protopores, recently confirmed by crystal structures of bacterial ClC homologs (19). ClC-0 gating is controlled by two interdependent processes: a fast gate activated by depolarization, which acts on each protopore, and a slow or common gate activated by hyperpolarization, which controls both pores simultaneously (11, 49). It has been proposed that the side chain of a glutamate residue within the pore serves as the protopore gate (20), but less is known about the molecular nature of the slow gate that might involve a large conformational change as indicated by its strong temperature dependence (50). The fast gate strongly depends on voltage and external Cl– concentration. This particular feature results from the effect of chloride ions on channel gating coupled to translocation of Cl– in the membrane electrical field (12, 51). Recent studies (63, 65) of ClC-2 transiently expressed in human embryonic kidney (HEK-293) have shown that ClC-2 current relaxation could be described by two exponentials, probably reflecting the presence of the two gates (protopore and common gates, by analogy to ClC-0). The ClC-2 conductance (9, 16, 23, 40, 57) and the hyperpolarization-activated slow gates of ClC-0 and mutant ClC-1 (49) depend on intracellular chloride concentration ([Cl–]i), with an increase in [Cl–]i favoring channel opening. It has been suggested that the [Cl–]i and voltage dependence and ClC-2 might arise from a voltage-dependent movement of intracellular chloride in the outward direction.

Studies on endogenous ClC-2 channels or in heterologous expression systems have shown important differences in the voltage sensitivity and activation kinetics of the current. For example, ClC-2 activation kinetics are slower when ClC-2 is expressed in ovocytes (62) compared with HEK-293 cells (13, 43). Activation kinetics of endogenous currents recorded in neurons (14, 57), T84 cells (23), salivary glands (43), or colonocytes (8) also differ, requiring from less than one to several seconds for steady-state activation. These observations suggest that splice variants or unknown protein partners differentially expressed according to cell type may modulate channel kinetics and gating parameters.

Little is known about the existence of ClC channel partners modulating channel gating. The renal human ClC-K channel is the unique example among the ClC channel family that requires a {beta}-subunit for proper function (21). It is reasonable to assume that additional proteins are also involved in the regulation of channel trafficking and function. For example, interaction between ClC-5 and cofilin seems essential for the role of ClC-5 in albumin uptake in the proximal tubule (30). Using the two-hybrid technology to probe the COOH termini of ClC-2, Furukawa et al. (24) detected a direct interaction with protein phosphatase-1. By immobilization of purified intact ClC-2 on a solid phase, Dhani et al. (15) identified an interaction between ClC-2 and the retrograde motor dynein, which is important for the regulation of ClC-2 cell surface expression.

The goal of the present study was to extend the search for possible interacting partners that may modulate ClC-2 function. By means of mild lysis conditions, ClC-2 immunoprecipitation, and mass spectrometry, we found an association between ClC-2 and the two protein chaperones Hsp90 and Hsp70 in HEK-293 cells stably expressing ClC-2. Interaction of native ClC-2 with Hsp90 was also detected in mouse brain. Hsp90 inhibition reduced plasma membrane channel expression without affecting protein stability. Electrophysiological studies demonstrated that Hsp90 inhibition decreased ClC-2 current amplitude and decreased the sensitivity of the channel to intracellular chloride.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Constructs. The cDNA of rat ClC-2 was kindly provided by T. J. Jentsch (Hamburg, Germany). Mutagenesis was performed using recombinant PCR and sequencing. To facilitate immunoprecipitation of a large amount of ClC-2, we decided to engineer a construct containing a FLAG sequence (GAC TAC AAG GAT GAC GAC GAC AAG) encoding for the FLAG epitope (DYKDDDDK). Preliminary experiments were conducted to assess whether the epitope could affect channel expression or activity. Insertion of the epitope was tested in each of the four extracellular loops. Insertion was made between amino acids L132 and N133 (Loop1), K210 and E211 (Loop2), K313 and T314 (Loop3), or K400 and E401 (Loop4). Insertion of the FLAG epitope in Loop1 did not significantly affect expression, localization, conductance, or voltage dependence of the channel. On the other hand, insertion in Loop3 and Loop4 markedly decreased current amplitude, and insertion in Loop2 even abolished ClC-2 current, reflecting modifications of channel activity and/or absence of surface expression. Sequences coding for wild-type (WT)ClC-2 or for ClC-2FLAG (insertion in Loop1) were subcloned in the internal ribosome entry site-enhanced green fluorescent protein plasmid (pIRES-EGFP) (Clontech, Palo Alto, CA), allowing independent expression of the two proteins EGFP and ClC-2. This expression system thus enabled us to visualize transfected cells while keeping the terminal parts of ClC-2 free.

Cells. HEK-293 cells were cultured in DMEM supplemented with 10% serum (GIBCO, Paisley, UK) at 37°C, 5% CO2 atmosphere. Cells were transfected with linearized pIRES ClC-2, pIRES ClC-2FLAG, or with empty plasmid using Lipofectamine (Invitrogen, Carlsbad, CA). Stably expressing cells were selected by adding 500 µg/ml G418 (Invitrogen) in the media. Isolated clones were tested for fluorescence, protein expression, and ClC-2 current.

Antibodies and drugs. Two polyclonal anti-ClC-2 antibodies were used: one raised against an NH2-terminal peptide (residues 1–90 of human ClC-2, H-90) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the second, homemade (36), named pAb137, raised against a carboxy-terminal peptide (residues 847–862). The anti-FLAG M2 antibodies coupled to agarose beads were purchased from Sigma-Aldrich (St. Louis, MO). Anti-GFP antibodies were from Santa Cruz Biotechnology and anti-Hsp90 (Spa-835) was from Stressgen (Victoria, BC, Canada). Geldanamycin (GA) was bought from Invitrogen (San Diego, CA) and radicicol was obtained from Sigma-Aldrich.

Immunoprecipitation and silver staining. To identify coimmunoprecipitated proteins, cells stably expressing WTClC-2 or ClC-2FLAG were lysed on ice with lysis buffer containing 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 0.1% Igepal (buffer A) complemented with anti-protease tablets (Roche Mini, Mannheim, Germany) and centrifuged at 24,000 g for 60 min at 4°C. Soluble protein concentration was measured using the RCDC assay from Bio-Rad (Hercules, CA). Equal amounts of protein (200 mg) were precleared with 50 µg of a nonrelevant antibody coupled to agarose beads for 60 min before 50 µg of anti-FLAG antibodies were added and then incubated overnight. Beads were pelleted at 3,000 g, washed six times with lysis buffer, and resuspended in 2x Laemmli buffer before SDS-PAGE separation. The mass spectrometry-compatible silver staining was used for mass spectrometry (MS) experiments (54). Briefly, gels were fixed in a 45% ethanol-5% acetic acid solution, sensitized with 0.02% sodium thiosulfate, and impregnated with 0.1% AgNO3. Staining was developed with 0.04% formaldehyde-2% Na2CO3, and the reaction was stopped with 1% acetic acid.

Mass spectrometry. Differentially expressed protein bands were excised, and proteins of interest were digested with trypsin (sequencing grade; Promega, Madison, WI), while still in the gel (5). Peptides eluted from protein bands after tryptic digestion were separated by HPLC (RP-18 column, 75 mM, Promega) and then analyzed using ESI-MS on a Q-TOF (Micromass) mass spectrometer working in the regime of data-dependent MS to MS/MS switch. Proteins were identified using Mascot and PeptIdent software, available online at http://www.matrixscience.com and http://www.expasy.org/tools/peptident.html, respectively.

Hsp90/ClC-2 coimmunoprecipitation. Coimmunoprecipitation experiments were performed using cells stably expressing WTClC-2 lysed with buffer A. Lysis buffer was complemented with 10 mM sodium molybdate because it has been demonstrated to stabilize Hsp90/client protein complexes (60). Protein samples were precleared with agarose beads coupled to a nonrelevant antibody, and concentration was measured using the RCDC Bio-Rad assay.

ClC-2 was immunoprecipitated from 1 mg of total extracts with 10 µg of anti-ClC-2 antibody (pAb137). Hsp90 was immunoprecipitated from 1 mg of total proteins with 5 µg of anti-Hsp90 antibodies. Nonimmunized IgGs were used as a negative control. Immunoprecipitates were resuspended in 2x Laemmli buffer, heated for 1 min at 95°C, complemented with 2 volumes of 1x Laemmli buffer containing 3.5 M urea and incubated at 37°C for 1 h. Samples were separated by SDS-PAGE (7%) in the presence of 5 M urea before being transferred onto nitrocellulose membranes. Membranes were blocked for 1 h in a solution containing 5% nonfat milk and 0.1% Tween 20 in Tris-buffered saline and then incubated overnight at 4°C with anti-ClC-2 or anti-Hsp90 antibodies in the blocking solution. Protein bands were visualized with an enhanced chemiluminescence detection system (Amersham Biosciences, Little Chalfont, UK).

Normal 57BL6 mice (Charles River) were briefly anesthetized with halothane and decapitated. The experiments were carried out under license no. 7514 of the Veterinary Department of the French Ministry of Agriculture (Decret 87-848, 19 October 1987). Brains were rapidly removed, disrupted in a Potter, and lysed in buffer A complemented with 10 mM sodium molybdate at 4°C during 3 h. Samples were centrifuged twice for 1 h at 25,000 g to remove the nonsoluble fraction. Agarose beads were used to preclear the sample, and the protein concentration was measured using the RCDC assay. Anti-Hsp90 (6 µg) or nonspecific rat IgG were added to equal amounts of proteins (10 mg) to precipitate Hsp90. Precipitates were washed five times in lysis buffer, and detection of ClC-2 was performed as described above.

Pretreatment of cells. Two types of treatment were used to investigate the possible functional consequences of Hsp90/ClC-2 interaction before protein analysis, immunolabeling or patch-clamp measurements: 1) a 30-min to 24-h incubation of the cells with 10 µM GA or 200 nM radicicol, and 2) a 1-h incubation of the cells at 43°C, followed by a 6-h recovery period at 37°C.

Whole cell and membrane protein analysis. For whole cell protein analysis, 40 µg of total protein extracts were solubilized in RIPA buffer and resuspended in 2x Laemmli buffer containing 7 M urea. Samples were heated at 37°C for 1 h before being separated by SDS-PAGE as above. The amount of ClC-2 was estimated by Western blot analysis, and GFP detection was used to verify equal protein loading.

To follow surface expression of ClC-2, membrane proteins were biotinylated with sulfosuccinimidyl-6-(biotin-amido)-hexanoate (EZ-Link sulfo-NHS-biotin; Pierce, Rockford, IL) before lysis. Samples were then precleared with Sepharose beads for 1 h at 4°C. Biotinylated proteins were purified with monomeric avidin beads (Pierce, Rockford, IL), washed five times in lysis buffer and resuspended in 2x Laemmli buffer as above. ClC-2 expression was analyzed by Western blot and GFP was probed in the same membranes to assess the absence of intracellular proteins in the sample.

Immunofluorescence and confocal microscopy. ClC-2 immunostaining was performed on HEK-293 cells stably expressing WTClC-2. Cells were grown on poly-D-lysine-precoated coverslips, fixed with cold methanol, and permeabilized with 0.1% Triton in PBS. Coverslips were blocked with 1% bovine serum albumin in PBS before incubation with the primary ClC-2 antibody (Santa Cruz; final dilution 1:125 in PBS-Triton). After washing and blockage with 5% goat serum in PBS, coverslips were incubated with the Alexa Fluor 594 secondary antibody (Molecular Probes) at a dilution of 1:1,000. Coverslips were subsequently mounted on glass slides with Vectashield mounting medium (Vector Laboratories) and photomicrographs were taken with a Zeiss confocal microscope.

Whole cell patch-clamp recordings. Stably transfected cells were plated in 35-mm cell culture plastic petri dishes that were mounted on the stage of an inverted microscope. Patch-clamp experiments were performed at room temperature with an Axopatch 200A amplifier controlled by a computer via a CED 1401 interface (CED, Cambridge, UK). The bath was grounded via an agar bridge. Pipettes were pulled from hard glass (Kimax 51) using a Sutter micropipette puller. Current recordings were performed using the nystatin-perforated patch-clamp configuration that gave stable recordings over long time courses. Nystatin stock solution (50 mg/ml) was prepared daily in DMSO. Aliquots were diluted (1:250) in the internal solutions, which were then sonicated for at least 30 s. The bath solution contained (in mM) 150 NaCl, 2 CaCl2, 1 MgCl2, 10 HEPES-Na+, and 35 sucrose, pH 7.4, adjusted with NaOH. The pipette solution (135 mM Cl–) contained (in mM) 131 NaCl, 2 MgCl2, 10 HEPES, pH 7.3, adjusted with NaOH. Lower Cl– solutions were prepared by equimolar replacement of NaCl with Na+ gluconate (for the 100 mM Cl– solution) or by replacement of NaCl with 35 mM Na2SO4 and the appropriate concentration of Na+ gluconate (for the 64, 35, and 15 mM Cl– solutions). Access resistance (Ra) gradually declined after the formation of an on-cell patch, and recordings were started when Ra decreased to <20 M{Omega}, usually 15–20 min after patch formation. Mean Ra was 14.2 ± 3.6 M{Omega} (n = 108). Accurate control of internal chloride concentration was verified by measuring the reversal potentials of the currents elicited by voltage ramps applied at the end of a –120-mV hyperpolarizing voltage jump.

Changes in liquid junction potential were calculated (4) and taken into account when necessary.

Currents were recorded by application of regular voltage pulses of desired length and amplitude from a holding potential of 0 mV with an interval of at least 60 s to allow current deactivation.

Time courses for activation and deactivation were described by fitting a mono- or double-exponential plus a constant term in equations of the form I(t) = Af exp(–t/{tau}f) + As exp(–t/–{tau}s) + A0, where I(t) is current as a function of time, Af, As, and A0 are fast, slow, and time-independent constants, respectively, and {tau}f and {tau}s are the fast and slow time constants.

The relative open probability as a function of voltage was estimated from measurements of the initial currents at 40 mV after each negative test voltage jump. The conductance values were adjusted by using a Boltzmann distribution of the form G = Go + Gmax/[1 + exp(V – V0.5)/k], where G, Go, and Gmax are conductance as a function of voltage, residual conductance, and maximal conductance (extrapolated), respectively. V0.5 is the voltage at which 50% activation occurs, and k is the slope factor.

All measured values are presented as means ± SE. The significance of differences between means was determined using an unpaired t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of ClC-2 interacting proteins. To facilitate immunoprecipitation of a large amount of ClC-2, we inserted a FLAG tag sequence in the first extracellular loop of the protein. The FLAG tag sequence has previously been shown to bind with high affinity to the anti-FLAG M2 monoclonal antibody (29). Insertion of this epitope did not modify either the expression or the activity of the channel (not shown). This epitope also presented the advantage of keeping free the long cytoplasmic regions of the protein, which are possible targets for interacting proteins.

ClC-2 was immunoprecipitated from 200 mg of proteins obtained from 5 x 107 HEK-293 cells stably expressing ClC-2FLAG or WTClC-2 as a control. As shown in Fig. 1A (lane 1), four prominent bands with respective molecular masses of 90, 200, and two at >250 kDa were specifically (with respect to lane 2) immunoprecipitated with the anti-FLAG antibody. Another less abundant band with an apparent mass of 70 kDa was also differentially precipitated from the ClC-2-expressing cells. Experiments were repeated three times and produced similar protein profiles.



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  		Fig. 1. Identification of proteins coimmunoprecipitated with ClC-2. A: ClC-2FLAG was immunoprecipitated with anti-FLAG antibodies from stably transfected human embryonic kidney (HEK)-293 cells (lane 1). As a control, the same procedure was performed using cells stably transfected with the wild-type (WT) channel (lane 2). The specific bands in lane 1 and their counterparts in lane 2 were excised and identified by MS-MS mass spectrometry. Their identities are given at left. B, left: Western blot analysis of ClC-2 after immunoprecipitation of Hsp90 from HEK-293 cells stably expressing ClC-2 WT (lane 1) or the empty plasmid (lane 2). Precipitated proteins were probed with H-90 anti-ClC-2 (1:1,000 dilution); right, Western blot analysis of Hsp90 after immunoprecipitation of ClC-2 from the same cells with PAb137 (lane 1) and from control immunoprecipitates obtained with nonrelevant rabbit IgGs (lane 2). C: Western blot analysis of ClC-2 after immunoprecipitation of Hsp90 from 10 mg of mouse brain homogenates (lane 1). Control experiments were performed using nonrelevant rat IgGs (lane 2). Total cell extracts (40 µg) from mouse brain proteins were probed with ClC-2 antibody PAb137 (lane 3).

 
The five bands indicated by arrows in Fig. 1 were identified by mass spectrometry. Samples at the same position in the control lane were also analyzed. ClC-2 was present in the bands at 70 kDa (68 peptides, score 2,103), 90 kDa (74 peptides, score 2,283), 200 kDa (77 peptides, score 2,278), and in both bands >250 kDa (56 peptides, score 2,092; and 68 peptides; total score 2,215), suggesting that the quaternary structure of the protein was maintained. The presence of ClC-2 at 70 kDa could be due to partial proteolysis of the protein. In addition, Hsp70 (12 peptides, score 767), Hsp90{alpha} (6 peptides, score 320) and Hsp90{beta} (6 peptides, score 234) were identified as coimmunoprecipitated proteins in samples at 70 and 90 kDa. The access numbers were gi 228578 for ClC-2, gi 12653415 for Hsp70, gi 123678 for Hsp90{alpha}, and gi 72222 for Hsp90{beta}. All identified proteins were absent in the control lane.

Coimmunoprecipitation of Hsp90 with ClC-2. In this study, we focused on the interaction between ClC-2 and Hsp90. Hsp90 is an abundant cytosolic protein (accounting for 1 to 2% of cytosolic proteins in unstressed eukaryotic cells) that functions as a molecular chaperone facilitating the folding of a variety of proteins (44).

To rule out the possibility that the interaction of Hsp90 with ClC-2 detected by mass spectrometry could be caused by the presence of the FLAG epitope, we performed immunoprecipitation of WTClC-2 using the carboxy-terminal ClC-2 antibody. Interaction between the two proteins was also tested by reverse immunoprecipitation using the anti-Hsp90 antibody. HEK-293 cells stably expressing WTClC-2 or the empty plasmid as a control were lysed using buffer A containing 10 mM sodium molybdate. Samples were thoroughly precleared with nonrelevant antibodies coupled to agarose beads, and the immunoprecipitated partner was probed using the appropriate antibody. As shown in Fig. 1B, left, ClC-2 was detected in anti-Hsp90 immunoprecipitates (lane 1, n = 3) but not in immunoprecipitates obtained with nonspecific IgG (lane 2). Conversely, Hsp90 could be detected in the anti-ClC-2 immunoprecipitates from WTClC-2-expressing cells (Fig. 1B, right, lane 1, n = 3) but not from cells transfected with the empty plasmid (lane 2).

To determine whether this interaction existed in vivo, the same experiments were repeated using mouse brain homogenates known to express high levels of ClC-2. Protein extracts were incubated with the anti-Hsp90 antibody, and immunoprecipitated proteins were analyzed using Western blotting with anti-ClC-2 IgGs. ClC-2 was detected as a band migrating at ~85 kDa (Fig. 1C, lane 1) in this tissue, whereas the signal was absent when protein extracts were incubated with a nonrelevant antibody (lane 2). Similar electrophoretic migration was observed using Western blot analysis of total protein extracts (lane 3). These results thus suggest that Hsp90 interacts with endogenous ClC-2 in mouse brain.

Inhibition of Hsp90 decreases channel cell surface abundance without affecting protein expression. Hsp90 activity depends on binding and hydrolysis of ATP to the NH2-terminal domain coupled to a conformational cycle involving opening and closing of a dimeric molecular clamp (47). The two structurally unrelated antibiotics, GA and radicicol, specifically inhibit Hsp90 activity by competing with ATP on its binding site (53, 59). This inhibition most often increases ubiquitination and proteasomal degradation of Hsp90 client proteins (6). To investigate the possible effect of Hsp90 inhibition on ClC-2 expression, cells were treated during 24 h with 10 µM GA or 200 nM radicicol. Total proteins were analyzed using immunoblast analysis with anti-ClC-2 antibodies, whereas the GFP protein amount was used as an index of protein loading (Fig. 2A). Inhibition of Hsp90 activity did not significantly affect the amount of ClC-2 in the total cell extracts (n = 3).



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  		Fig. 2. Geldanamycin (GA) and radicicol do not modify total ClC-2 expression but decrease the abundance of ClC-2 at the cell surface. A: HEK-293 cells stably expressing WTClC-2 were treated for 24 h with 10 µM GA or 200 nM radicicol. Whole cell extracts (40 µg) were probed with the H-90 antibody. Blots were also probed with anti-green fluorescent protein (GFP) to assess protein loading. B: ClC-2 staining with H-90 anti ClC-2 in HEK-293 cells stably expressing WTClC-2 before or after a 5-h incubation with GA. Arrows indicate cell surface ClC-2. Scale bar represents 10 µm. C: ClC-2 expression at the cell surface was estimated after biotinylation of membrane proteins. ClC-2 was absent from nontransfected cells, and its expression decreased when cells were treated with GA. Blots were probed with anti-GFP to verify that cytoplasmic proteins were absent in the membrane fractions. Bottom, anti-GFP Western blot analysis was performed on the total cell extracts before membrane protein purification. D: total ClC-2 signal (bands at 200 and 90 kDa) was normalized to the amount of GFP.

 
Because interaction of Hsp90 with client proteins may influence protein trafficking (45), we further investigated whether Hsp90 inhibition could modify the distribution of ClC-2 using an immunolabeling approach. As shown in Fig. 2B, the pattern of ClC-2 staining was quite similar between untreated and treated cells, except that membrane staining was less visible. However, the faint and nonuniform membrane staining in control cells did not allow an accurate comparison of the amount of ClC-2 at the membrane. A more quantitative analysis was performed using cell surface proteins isolated with the biotinylation procedure. Protein loading was estimated by GFP Western blot analysis of total extracts, and biotinylated ClC-2 was normalized to GFP amounts. Treatment by GA for 30 min or 3 h (Fig. 2C) significantly decreased the overall amount of ClC-2 present in the bands at 200 and 90 kDa compared with control cells, by 26.4 ± 4%, n = 3, and 38.7 ± 9%, n = 6, respectively (Fig. 2D).

Functional assay for ClC-2/Hsp 90 interaction: effect of GA and radicicol on ClC-2 current. We performed whole cell patch-clamp studies to investigate whether there was any functional evidence for ClC-2/Hsp90 interaction in HEK-293 cells stably expressing ClC-2. As previously described (13, 43), HEK-293 cells transfected with EGFP alone exhibited no voltage-dependent current with the solutions used. In ClC-2- expressing cells, hyperpolarizing voltage jumps elicited typical time-dependent inward currents whose amplitude remained stable in control conditions during tens of minutes. Application of 10 µM GA resulted in a gradual decrease of ClC-2 amplitude, which reached the steady state within 30 min. Shown in Fig. 3A are examples of current traces at –120 mV recorded before and after 30-min application of GA using a pipette solution containing 64 mM Cl–. The current decrease was often preceded (in ~80% of the cells studied) by a small transient current increase (1.29 ± 0.08-fold). The same effects were observed during application of 200 nM radicicol. The rapid transient current increase was not further investigated in the present study. At –120 mV, with 64 mM internal Cl–, current amplitude was decreased by 45.3 ± 3.6% (n = 10) and by 40.0 ± 1.2% (n = 7) in response to GA and radicicol, respectively. Results from dose-response experiments (Fig. 3B) showed that the maximal inhibitory effects of the two inhibitors were obtained within the same range of concentrations needed for inhibition of ATP hydrolysis (52). We observed that the extent of current inhibition varied according to the chloride concentration in the pipette solution. Current recordings before and after GA treatment, at 15 and 135 mM internal Cl– are shown in Fig. 3, C and D, respectively. At 15 mM internal Cl–, current amplitude decreased by 36.9 ± 4.3% (n = 8) and by 72.22 ± 3.8% (n = 7) at 135 mM internal Cl–.



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  		Fig. 3. Effect of Hsp90 inhibitors on ClC-2 current. A: typical slow-activating ClC-2 currents recorded before and 30 min after the addition of 10 µM GA in the bath solution. Currents were recorded with a pipette solution containing 64 mM intracellular Cl– concentration ([Cl–]i) during a 5-s voltage jump from a holding potential of 0 to –120 mV, followed by a +40-mV step. B: percentages of current inhibition at various concentrations of Hsp90 inhibitors. GA and radicicol reduced ClC-2 currents by 45.3 ± 3.6% and 40.0 ± 1.2% at the maximal doses tested, respectively. Error bars indicate means ± SE from a minimum of 7 independent experiments. C and D: current traces recorded at –120 mV before and after 30-min application of GA, with pipette solutions containing 15 and 135 mM [Cl–]i, respectively.

 
Effect of GA on chloride dependence of ClC-2 gating. Current inhibition could result from a decrease of the channel conductance, a decrease of its open probability (Po), or a decrease of the amount of ClC-2 channels at the plasma membrane. Because inhibition of Hsp90 by GA diminished membrane expression of ClC-2 (Fig. 2, C and D), part of the current inhibition was probably linked to a decrease in the amount of ClC-2 channels at the plasma membrane. However, the increased current inhibition observed at high [Cl–]i suggested that GA could additionally modulate [Cl–]i-dependent gating parameters. Activation of ClC-2 channels is known to depend on both the membrane potential and intracellular chloride concentration (9, 16, 23, 28, 40, 57). Figure 4A shows families of current traces recorded in untreated or GA-treated cells at various internal [Cl–]. The threshold of current relaxation became less negative when [Cl–]i increased. At 135 mM internal Cl–, sizable current relaxations were recorded at –40 mV and a fraction of the channel was already activated at the resting potential as evidenced by the presence of instantaneous currents when stepping the test voltages. This time-independent component could not be ascribed to the activation of a different class of channels, because it could be inhibited by 100 µM ZnCl2 (a known blocker of ClC channels; see Refs. 10, 14, 35) in the bath solution (by 82.3 ± 2.3%, n = 4). Activation by changes in cell volume could also have been ruled out because the same profile of current recording was obtained when the osmolarity of the extracellular medium was increased with sucrose (not shown).



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  		Fig. 4. [Cl–]i dependence of ClC-2 current. A: representative families of current traces recorded with internal solutions containing 15, 100, and 135 mM [Cl–]i in untreated cells and in cells preincubated for 3 h with 10 µM GA. Currents were measured in response to voltage jumps elicited from 0 to –160 mV in 20-mV steps, followed by a +40-mV step. The duration of the pulses at 100 and 135 mM [Cl–]i was decreased at the most negative voltages to prevent changes in [Cl–]i and consequently in the reversal potential of the current. For illustration purposes, the beginning of the tail currents at +40 mV was set at the same time. B: open probability (Po)/V curves at 15 mM and 135 mM [Cl–]i in control conditions {bullet} and {blacktriangledown}, respectively). C: Po/V curves at 15 and 135 mM [Cl–]i after a 3-h incubation with 10 µM GA ({circ} and {triangledown}, respectively). For B and C, solid curves were drawn according to a Boltzmann equation and show fits to all points on the graph. The apparent Po as a function of voltage was calculated by measuring the current at the beginning of the pulse at +40 mV after the various conditioning prepulses. GA did not affect the Po/V curve at 15 mM [Cl–]i but decreased the rightward shift observed when [Cl–]i was increased. The fits obtained gave mean values ± SE of the slope factors (k, mV) of –21.6 ± 1.1 (at 15 mM [Cl–]i, n = 7) and –30.38 ± 1.2 (at 135 mM [Cl–]i, n = 11) for control cells and –24.6 ± 1.0 (at 15 mM [Cl–]i, n = 13) and –29.98 ± 1.22 (at 135 mM [Cl–]i, n = 13) for GA-treated cells. D: dependence of voltage at 50% activation (V0.5) on internal Cl–. V0.5 values in order of increasing [Cl–]i for untreated or (treated cells) were –124.9 ± 1.8 mV (–128.2 ± 2.4 mV), –116.5 ± 2.7 mV (–121.8 ± 3.2 mV), –104.0 ± 2.1 mV (–116.0 ± 2.7 mV), –90.6 ± 2.1 mV (–109.2 ± 2.9 mV), and –78.1 ± 4.1 mV (–104.8 ± 4.9 mV). Results are means ± SE of 7 to 14 cells for each concentration tested.

 
The relative Po of the channels as a function of voltage was estimated from the single Boltzmann fit of the initial currents at the +40 mV postpulse (Fig. 4, B and C). Similarly to previous reports (28, 40), increasing [Cl–]i in untreated cells resulted in a shift of the activation curve to more depolarized potentials (Fig. 4B). V0.5 at 135 mM internal Cl– was shifted by about 46 mV in the rightward direction compared with that at 15 mM. Treatment of the cells with GA did not significantly affect the Po/V relationship at the lowest [Cl–]i but attenuated the rightward shift induced by increasing [Cl–]i without major changes in the slope factors (Fig. 4C). The values for V0.5 at different [Cl–]i are plotted in Fig. 4D. The effect of GA was chloride dependent, so that the slope of V0.5 vs. [Cl–]i became less steep with respect to that for untreated cells. In the presence of GA, opening of the channels required more negative potentials for [Cl–]i >15 mM. The change in the slope of V0.5 vs. [Cl–]i thus suggests that GA did not simply increase the intrinsic energy difference between the open and closed states but also altered parameters involved in the chloride sensitivity of current activation. Because at the same time GA reduced the number of channels at the membrane, the possibility existed that decreased ClC-2 expression was in fact responsible for the reduced chloride sensitivity of the channel in the presence of the drug. The GA-induced decrease of the instantaneous currents at 135 mM internal Cl– was indeed reminiscent of the positive correlation found between the fraction of noninactivating ClC-0 channels and the amount of expression of these channels in oocytes (50). When the fraction of noninactivating current (Ao) estimated from the fits of the current traces at +40 mV was plotted as a function of the peak current amplitude (which reflects the number of open channels at the membrane), we observed a positive correlation between Ao values and the intensity of maximum currents (Fig. 5A). GA treatment reduced Ao by half (from 40.3 ± 3.8% to 20.9 ± 3.8%) while reducing maximum current amplitude at +40 mV. Comparable results were obtained in the presence of radicicol (not shown). The results shown in Fig. 5A thus suggest that the decrease of the fractional amplitude of Ao induced by GA is related to the decrease of maximal current amplitude induced by the drugs. However, a plot of the V0.5 values as a function of peak current amplitude (Fig. 5B) did not show any correlation, indicating that the changes in V0.5 induced by GA were not related to the decrease in the number of the channels at the membrane.



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  		Fig. 5. Analysis of the fraction of noninactivating channels (A0) and of V0.5 values at 135 mM internal chloride as a function of the amount of ClC-2 expression in control cells ({blacktriangledown}) or GA-treated cells ({triangledown}). A: correlation of A0 estimated from the fits of current deactivation with maximum current at +40 mV after a voltage jump at –120 mV. B: absence of correlation of V0.5 values with maximum current amplitude.

 
Effect of GA on current activation and deactivation kinetics. In previous studies (13, 63), ClC-2 current relaxations have been described by two exponentials, probably reflecting the presence of two gates. We thus examined whether GA affected both components equally or preferentially one of the two components of current activation independently of its effect on instantaneous currents at 135 mM internal Cl–.

Activation time constants measured at –120 mV as a function of [Cl–]i between 15 and 100 mM, are plotted in Fig. 6. In control conditions, the time dependence of current activation at 15 and 35 mM internal Cl– was well fit by a single-exponential time function, whereas at higher [Cl–]i, current relaxation was better described by a double-exponential model. As previously reported (13, 63), the two time constants differed ~10-fold (named fast and slow hereafter). Increasing [Cl–]i enhanced the rate of current activation. This faster rate resulted not only from changes in the time constants, which were reduced between 64 and 100 mM [Cl–]i (Fig. 6A), but also from the increase of the weight of the fast component from undetectable at 15 and 35 mM [Cl–]i to ~20% at the highest [Cl–]i (Fig. 6, B–D, open bars). GA treatment prevented the chloride-dependent decrease of the two time constants of current activation (Fig. 6A) without major changes in the relative proportions of the two components (Fig. 6, B–D, solid bars).



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  		Fig. 6. Effect of GA on the gating properties of ClC-2 at various [Cl–]i. A: time constants of the fast ({triangledown}) and slow ({blacktriangledown}) components of current activation measured at –120 mV as a function of [Cl–]i for untreated cells or GA-treated cells ({triangledown} and {circ}). B–D: fractional amplitudes of the fast (Af), slow (As), and time-independent (A0) components of current activation at –120 mV, with internal solutions containing 15, 64, and 100 mM [Cl–]i. Open bars, untreated cells; filled bars, GA-treated cells. E: time constants for the fit of current deactivation at +40 mV from a conditioning test pulse at –120 mV as a function of [Cl–]i (same symbols as in A). F–H, fractional amplitudes of Af, As, A0) components of current deactivation at 15, 64, and 100 mM [Cl–]i. Open bars, untreated cells; filled bars, GA-treated cells. All data are means ± SE of at least 7 cells.

 
Deactivation at positive potentials could well be described by two exponential time courses even at low chloride concentrations (Fig. 6E), implying that current deactivation was not strictly the reverse process of current activation. Increasing [Cl–]i did not markedly change the time constants of deactivation between 15 and 100 mM [Cl–]i (Fig. 6E) or the weight of the two components (Fig. 6, F–H). Deactivation was less affected than activation by GA. The slow time constant of deactivation was slightly decreased (Fig. 6E), whereas the fast component remained unchanged.

Effect of a thermal stress on ClC-2 currents. Thermal stress that increases Hsp90 synthesis often enhances immunodetection of Hsp90 partners and induces an effect opposite to that of Hsp90 inhibitors. The effect of 1-h heat treatment to 43°C was first investigated by immunoblot analysis of the amount of Hsp90 in whole cell extracts during recovery at 37°C. As shown in Fig. 7 A, the Hsp90 signal increased progressively during the first hours of recovery, and a twofold increase (n = 2) was detected after 6-h recovery at 37°C (n = 2). This increase was not accompanied by significant changes of ClC-2 in whole cell extracts or in the amount of ClC-2 at the membrane (not shown).



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  		Fig. 7. Effect of a thermal stress on ClC-2 current gating. A: Western blot analysis of Hsp90 in whole cell lysates as a function of recovery time at 37°C after a 1-h heat shock treatment at 43°C. B: typical current traces elicited by a voltage step from 0 to –120 mV and then returning to +40 mV with a pipette solution containing 35 mM [Cl–]i, in control conditions and after a 1-h heat shock at 43°C, followed by a 6-h recovery period at 37°C. Activation currents were best fit by a monoexponential equation. C: time constants of current activation for untreated (open bars, n = 10) or heat shock-treated cells (filled bars, n = 7). D: time constants of current deactivation at +40 mV. Deactivation currents were best fit by a biexponential equation with two time constants. Open bars represent data for untreated cells (n = 13) and filled bars represent data for stressed cells (n = 7). *P < 0.0001, significantly different (Student's t-test).

 
To determine whether heat shock treatment could affect ClC-2 gating parameters, we measured ClC-2 using a 35 mM Cl– pipette solution. Figure 7B shows currents recorded during a hyperpolarizing jump to –120 mV in basal conditions or after a 1-h thermal stress, followed by 6 h of recovery. As shown on the current traces, heat shock treatment increased the macroscopic rate of current activation by decreasing about twofold the slow time constant (Fig. 7C) without significantly changing the rate of current deactivation (Fig. 7D). These effects were accompanied by a modest shift of V0.5 from –116.5 ± 2.7 mV (n = 12) to –106.8 ± 2.7 mV (n = 7) that reached statistical significance (P = 0.038).


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In the present study, we have identified two heat shock proteins, Hsp70 and Hsp90, that coimmunoprecipitate with ClC-2 stably expressed in HEK-293 cells, and we provide evidence for a similar association in mouse brain. Pharmacological experiments designed to disrupt the association between Hsp90 and the channel protein showed that Hsp90 does not affect ClC-2 stability but favors cell surface expression. Patch-clamp experiments demonstrated that interaction between both proteins upregulates the chloride conductance by shifting the voltage dependence of channel opening to less negative potentials and by increasing the sensitivity of the channel to [Cl–]i.

Hsp70 is described to play an important role in protein folding, quality control, and membrane translocation processes (56). Hsp90 may also have a role in nascent chain folding but is best known to control the conformational maturation and the activity of a variety of proteins within multichaperone complexes composed of Hsp70, immunophilins, p23, and Hsp (44). Hsp90 client proteins include steroid hormone receptors, transcription factors, various protein kinases, and phosphatases (44). Apart from signaling proteins, Hsp90 has also been shown to interact with several plasma membrane receptors (1, 61, 64) and ion channels (22, 37).

Coimmunoprecipitation of Hsp70 along with Hsp90 suggests an interaction with ClC-2 in a large complex, which probably involves cochaperone partners. We have not yet performed more investigations to test for the presence of such partners. Because the expression of cochaperones may differ among cells and tissues, it would be interesting to analyze whether differentially represented cochaperones could contribute to the variability of the gating properties of ClC-2 between endogenous channels and channels expressed in ovocytes or cell lines.

The molecular basis underlying the interaction between Hsp90 and ClC-2, either direct or indirect, remains to be determined. Some chaperone client proteins contain a tetratricopeptide motif, which binds to an acceptor site in the COOH terminus of Hsp90 (48); but such a motif is absent from the ClC-2 sequence. In any case, little is known regarding the mechanism by which Hsp90 binds to substrates in the absence of tetratricopeptide motifs (46). It must be recognized that an indirect interaction through an unknown ClC-2 partner cannot be excluded. Among others, a possible candidate is dynein, which has already been demonstrated to associate with ClC-2 (15) and is also known as an Hsp90 client protein in other systems (25, 39). However, we could not identify dynein in any band size analyzed. The contribution of dynein could also be questioned from the inhibitory effects of GA and radicicol on cell surface ClC-2 expression. The role of Hsp90 and immunophilins in protein trafficking has been particularly well examined for the glucocorticoid receptor and the tumor suppressor p53 (45), requiring assembly of these proteins with the motor protein dynein. Dhani et al. (15) reported that pharmacological inhibition of dynein increased the expression of ClC-2 at the plasma membrane. Hence, the possible disruption of Hsp90/dynein interaction by GA might produce the same effect, which can be in agreement with the transient increase of current amplitude induced by the two Hsp90 inhibitors but not with the reduced amount of ClC-2 at the plasma membrane after prolonged exposure. ClC-2 trafficking to the plasma membrane has also been shown to be modulated by anterograde or retrograde vesicular transport involving phosphatidylinositol 3-kinase (2), the glucocorticoid-inducible kinases SGKs and the ubiquitin ligase Nedd4-2 (42). Because Hsp90 has been shown to be involved in endothelial nitric oxide synthase translocation to the apical membrane in a phosphatidylinositol 3-kinase-dependent manner (41), it would be interesting to investigate whether a similar mechanism exists for ClC-2 and Hsp90.

We found no evidence of ClC-2 degradation after Hsp90 inhibition, implying that association between the two proteins does not involve the well-established role of Hsp90 as a chaperone implicated in client protein maturation. The functional consequences of pharmacological inhibition of Hsp90 or heat shock treatment are rather reminiscent of the role for tyrosine phosphorylated Hsp90 as a repressor of the purinergic P2X7 receptor function (1) or as a modulator of the NKCC transporter (55) without modifications of the expression level of these two proteins.

Functional experiments demonstrated that GA and radicicol decreased current amplitude. ClC-2 activity can be modulated by phosphorylation or dephosphorylation events (23) and more directly by p34cdc2/cyclin B and the protein phosphatase PP1 (24). The question thus arose whether the reduction of current amplitude involved changes in the channel phosphorylation state through a modulation of kinase and phosphatase activities by Hsp90 inhibitors. However, none of the treatments tested here (heat shock, GA, or radicicol) significantly altered channel phosphorylation level (data not shown), thus excluding overlapping effects via altered channel phosphorylation.

As stated in the introduction, the activation kinetics of ClC-2 channels are highly variable from one cell type to another. Even by using the same cellular context, our results are not in complete agreement with previous results that suggested independence of the slow gate to [Cl–]i (40, 65), whereas we detected a slight decrease of the slow time constant between 64 and 100 mM [Cl–]. Also, instantaneous currents at high [Cl–]i were negligible (40), and two current activation components could be discriminated at low [Cl–]i (15 and 35 mM) (13, 63) vs. one in the present study. We checked whether the latter difference could be linked to the experimental protocols, conventional (used by others) or nystatin-perforated whole cell patch-clamp recordings (this work). Recordings performed with the two methods indicated that current activation displayed a monoexponential time course when using pipette solutions with low [Cl–]i. Because at the same [Cl–]i we could consistently extract two gating processes from deactivation currents, it is reasonable to assume that the weight of the fast process was too weak during activation to be detected in our experiments. A possible explanation for these differences is that the mode of ClC-2 expression, transient in the previous studies or stable in the present study, may influence basal regulation of channel gating.

GA treatment shifted the Po/V curve to more hyperpolarizing potentials in a [Cl–]i-dependent manner, leading to a V0.5/[Cl–]i relation less steep in the presence of GA compared with that under control conditions. Both the slow and fast activation time constants were affected by GA and became less Cl–-dependent above 64 mM [Cl–]i. It should be noted that at the low chloride concentration tested (35 mM), heat shock treatment resulted in more significant changes in V0.5 and activation kinetics than GA treatment. The reason for this different sensitivity is unclear, but this observation suggests that at physiological chloride concentrations both an increase of ClC-2 channels at the membrane and an increased rate of channel opening may contribute to an enhanced ClC-2 current after cellular stress. On the other hand, the shift in the Popen/V curve induced by GA does not seem to be related to the decreased expression of the channels at the membrane (Fig. 5B). Because GA treatment did not affect either the voltage dependence of current activation at low chloride concentrations or the apparent gating charge whatever the [Cl–]i, a likely explanation for the decreased sensitivity to [Cl–]i was a change in the apparent affinity for chloride binding. Considering that gating of the channel probably involves conformational changes of the protein (65) and that Hsp90 usually helps conformational changes of its partners, it is tempting to speculate that the chaperone favors an optimal channel conformation for ion binding and/or movement through the pore. An alternative hypothesis involving a more direct effect of the drugs on channel gating cannot, however, be ruled out. Additional studies such as analysis of mutant ClC-2 channels lacking fast gating and exhibiting a much lower internal chloride sensitivity (40) would be required to determine more precisely the effect of GA on channel gating. It is also evident that the hypothesis of a conformational change can be raised only if a direct interaction exists between the two proteins. Despite these concerns, our observations have brought to light a new regulatory element that may be important for channel activity and for the [Cl–]i sensitivity of channel gating thought to provide a mechanism for the coupling of Cl– fluxes across apical and basolateral membranes in epithelia (9). Within this context, it is interesting to note that the recently reported interaction between Hsp90 and the cotransporter NKCC may play an important role for the [Cl–]i dependence of the transporter (55). Modulation of ClC-2 function by Hsp90 may be of pathophysiological importance linking Hsp90 stimulators such as elevated temperature, oxidative stress, or ischemia to channel activation. It is noteworthy that recovery of barrier function in ischemia-injured ileum requires chloride secretion through ClC-2 channels (38). It has also been suggested that in the heart, an increase in ClC-2 conductance could be proarrhytmic under some pathological conditions such as ischemia and hypoxia (18, 34). Moreover, oxidation has been shown to potentiate the activation of ClC-2 channels by cell swelling in Plasmodium falciparum-infected erythrocytes (31). Although oxidants have been shown to exert a rapid and possible direct effect on ClC-2 expressed in Xenopus oocytes, it cannot be excluded that ClC-2 activation in infected erythrocytes may additionally involve recruitment of the host chaperones to the plasma membrane (3). Along with the cystic fibrosis transmembrane conductance regulator and NKCC1, ClC-2 thus represents the third class of chloride transport system modulated by Hsp90, suggesting a key role for the chaperone in the regulation of cell volume and epithelial secretory or absorptive functions.


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This work was supported by European Community Grant QLRT2001-1335 and a grant from Vaincre la Mucoviscidose.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Edelman, INSERM, Unité 467, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France (e-mail: edelman{at}necker.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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