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J. Biol. Chem., Vol. 279, Issue 46, 48449-48456, November 12, 2004

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Characterization of a Novel Interaction between the Secretory Na⁺-K⁺-Cl^- Cotransporter and the Chaperone hsp90^*

Charles F. Simard, Nikolas D. Daigle, Marc J. Bergeron, Geneviève M. Brunet, Luc Caron, Micheline Noël, Valérie Montminy, and Paul Isenring, A Canadian Institute of Health and Research Clinician Scientist II

From the Nephrology Research Group, Department of Medicine, Faculty of Medicine, Laval University, Québec, Canada G1R 2J6

Received for publication, June 23, 2004 , and in revised form, September 1, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The first isoform of the Na⁺-K⁺-Cl^- cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl^-] (Cl^-_i) represents a key signaling mechanism by which NKCC1-induced Cl^- movement is autoregulated and by which Cl^- entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl^-_i-dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na⁺-K⁺-Cl^- cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cation-Cl^- cotransporters (CCCs)¹ are polytopic membrane proteins that couple the movement of Cl^- to that of Na⁺ and/or K⁺ ions in or out of cells (1–4). Seven such proteins have been identified to date; the Na⁺-K⁺-Cl^- cotransporters (NKCCs) isoform 1 and 2 (5, 6), the Na⁺-Cl^- cotransporter isoform 1 (7), and the K⁺-Cl^- cotransporters isoform 1–4 (8–11). Other proteins that are homologous to the CCCs have also been identified (12); they are termed orphan CCCs because their ionic substrates have not been identified to date.

The NKCC1 is considered a housekeeping carrier that is responsible for the bumetanide-sensitive component of Na⁺-K⁺-Cl^- cotransport across cellular membranes (1–4). One of its main functions is to regulate cell volume (V_cell) and intracellular Cl^- concentration (Cl^-_i). Because of its basolateral localization in polarized cells, this carrier also promotes fluid and electrolyte secretion through a variety of epithelia by cooperating with apically disposed ion transport systems including Cl^- channels (13–17).

Several lines of evidence suggest that NKCC1 activity is increased by phosphorylation of its cytosolic termini (see Fig. 1) in response to Cl^-_i reduction or cell shrinkage. To this effect, recent studies have reported binding of proline-alanine-rich stress-related kinase (PASK) to NKCC1 and provided strong evidence that this interaction leads to phosphorylation-dependent activation of Na⁺-K⁺-Cl^- cotransport (18–20). Along the same line, NKCC1 has been found to associate with mitogen-activated protein kinase/p38 and undergo c-Jun NH₂-terminal kinase-mediated phosphorylation in gel kinase assays (20, 21). To date, however, the mechanisms by which changes in cell volume (V_cell) or Cl^-_i (Cl^-_i) alter these phosphorylation-dependent events are unknown.

View larger version (50K): [in this window] [in a new window]   FIG. 1. Hydropathy plot model of huNKCC1. The symbols represent amino acid residues. The C1t and C3t baits are shown in gray. This model was drawn using the PLOT program (B. Forbush, personal communication).

Piechotta et al. (20) have recently shown that PASK is able to activate NKCC1 mutants lacking the capacity to associate with the enzyme. Based on these findings, they hypothesized that this kinase is a scaffolding protein for other kinases, and thus that it may play its role by coordinating regulatory activities at phosphoacceptor sites. However, PASK does not fit the usual profile of a scaffolding protein, exhibiting intrinsic kinase activity and sharing no homology with chaperones, cochaperones, or adaptor molecules. In this regard, hsp90 or calnexin would represent alternative candidates considering, moreover, that they have been implicated in a variety of ion transport processes (27–30).

In this study, we have used the yeast two-hybrid system to uncover additional regulatory proteins that may interact with the C terminus of NKCC1. We have identified a sequence that belongs to hsp90 and showed that its inhibition by geldanamycin is accompanied by a loss of NKCC1 activation in response to low Cl^-_i. Provided that such an effect is not caused by other geldanamycin-related events such as the unfolded protein response (31), and given the ability of hsp90 to associate with multiple cytosolic and integral membrane proteins, it is tempting to postulate that this chaperone plays a more generalized role in Cl^-_i and V_cell regulation by coordinating the activity or the bioprocessing of multiple ion transport systems during changes in the extracellular environment.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemicals, Reagents, Kits, and Cell Strains—Unless mentioned otherwise, chemicals, reagents, or kits were from several suppliers. They included the following antibodies (Abs): 1) a mouse anti-hemagglutinin (HA) monoclonal Ab (Calbiochem, number MM5–101R), 2) a goat anti-LexA Ab (Santa Cruz Biotechnology, number SC-1726), 3) a mouse anti-hsp90 monoclonal Ab (BD Transduction Laboratories, number 610418), 4) the mouse anti-Squalus acanthias (sa) NKCC1 J3 monoclonal Ab (32), 5) a mouse horseradish peroxidase-coupled IgG (Amersham Biosciences, number NXA931), 6) a goat horseradish peroxidase-coupled IgG (Roche Applied Science, number 1301977), and 7) the goat Alexa Fluor® 594-conjugated (Molecular Probes, number A11005). Vectors and cDNA constructs were propagated in XL1-blue cells (Stratagene). Some of these sequences were also introduced in EGY48 yeasts (Clontech) as described below.

Vectors—Subcloning, expression of reporter genes, or DNA amplification was carried out with the following (empty or insert-bearing) vectors: 1) pGilda (Clontech), which contains 5' to 3', the vasopressin promoter, a sequence encoding the LexA DNA-binding domain, a multiple cloning site, a His transformation marker, and an ampicillin resistance gene (AMP^R); 2) pB42AD (Clontech), which contains the P_GAL1 promoter, a sequence encoding the activating domain of a transcription factor and the HA epitope, a multiple cloning site, a Trp transformation marker and an AMP^R; 3) p8op-LacZ (Clontech), which contains LexA operators (ops) that can interact with the pB42AD-derived activating domain, two reporter genes (Leu and LacZ) that are under the control of LexA, a Ura transformation marker, and an AMP^R; 5) pJB20, an expression vector that contains the strong cytomegalovirus promoter, a geneticin resistance gene and an AMP^R; 6) vector pBluescript (Stratagene), which contains a multiple cloning site and an AMP^R.

NKCC1 Constructs—Two NKCC1 baits were used for the two-hybrid screen (Fig. 1). One consists of a 567-bp (base pair) fragment that encodes the proximal putative cytosolic C terminus of huNKCC1 from residue 9 after the 12th transmembrane domain to residue 197, and the other consists of a 252-bp fragment that encodes the distal C terminus of huNKCC1 from residue 1128–1212. These two fragments were first obtained from an available huNKCC1/vector pBluescript (33) through 30 cycles of PCR using the high fidelity PCR master kit (Roche Applied Science) with an XmaI- or EcoRI-capped forward primer and an NcoI- or BamHI-capped reverse primer (see Table II). The PCR fragments (called C1t and C3t) were cloned in pGilda generating C1t/pGilda and C3t/pGilda, which should encode LexA-C1t and LexA-C3t fusion proteins in yeasts.

Yeast Two-hybrid Screen—EGY48 yeasts (Y) were first transformed with the construct p8op-lacZ and seeded on -Ura plates generating the strain Yop. These cells were transformed once more with C1t/pGilda or C3t/pGilda and seeded on -Ura-His plates generating YopC1t or YopC3t. The latter strains were subsequently tested for expression of the hybrid protein by Western blotting (see below and Fig. 2), and they were also tested for autonomous reporter gene activation on -Leu plates containing 80 µg/ml X-galactosidase.

View larger version (82K): [in this window] [in a new window]   FIG. 2. Western analyses of yeasts transformed with C1t, C3t, or hsp901–245/pB42AD. These studies were performed as described recently (55). Briefly, yeast proteins were extracted with a yeast lysis solution (8 M urea, 5% SDS, 40 mM Tris-HCl, pH 6.8, 0.1 mM EDTA, and 125 µM -mercaptoethanol) to which glass beads and protease inhibitors were added. The analyses were performed with 50–100 µg of proteins using the anti-LexA Ab or the anti-HA Ab.

Two procedures were carried out to confirm the specificity of the identified interactions. First, Y cells were transformed with a single prey/pB42AD and tested for expression of a hybrid protein by Western blotting (see below and Fig. 2) after selection of colonies on -Trp plates. Second, Yop cells were cotransformed with pairs of plasmids (single prey + empty pGilda versus single prey + LAM/pGilda²) and tested for reporter gene activation on -Ura-His-Trp-Leu + X-galactosidase plates.

Heterologous Expression of NKCC1—Stable cell lines generated in previous studies were used to study the hsp90-NKCC1 interaction outside of the yeast two-hybrid system (12). They consist of HEK-293 cells transfected with pJB20_M and HEK-293 cells transfected with saNKCC1/pJB20_M or huNKCC1/pJB20_M. For all of the studies, cell lines were sub-cultured onto polylysine pre-coated plates and grown to confluence in Dulbecco's modified Eagle's medium + 10% fetal bovine serum.

Pretreatment of HEK-293 Cells—The cell lines were subjected to two treatments before protein analyses, influx assays, or immunofluorescence (IF) studies (1). A 20-min incubation to 37 °C or 42 °C temperatures followed by a 4- or 24-h recovery period in Dulbecco's modified Eagle's medium + 10% serum (2). A 30- or 60-min incubation in a tracer-free hypotonic low Cl^- medium (161 mosM, 4 mM Cl^-) or in a tracer-free regular medium (322 mosM, 144 mM Cl^-); both solutions were supplemented with 10 µM ouabain (see Table I). In some of the studies, 1 µg/ml geldanamycin was added during the recovery period and in the preincubation media. Here, the hypotonic low Cl^--medium is used to activate NKCC1 as it has been shown to reduce Cl^-_i more than an isotonic Cl^- medium without leading to swelling- or shrinkage-induced changes in NKCC1 activity; indeed, cotransporters expressed in HEK-293 cells are only weakly sensitive to V_cell in the absence of Cl^-_i (1–5, 26, 33–38).

Subsequent to the preincubation in a low Cl^- medium or a regular medium, HEK-293 cells were reincubated for 2 min in a regular medium containing 1 to 2 µCi/ml ⁸⁶RbCl + 10 µM ouabain ± 250 µM bumetanide. Influxes were terminated by the addition of a tracer-free high K⁺ medium containing 250 µM bumetanide and 10 µM ouabain followed by several rinses in the same solution. Cells were then solubilized in 2% SDS and ⁸⁶Rb was detected by liquid -scintillation counting using the TopCountNXT microplate counter (Packard Instrument Co.).

For each condition tested, absolute counts (F_A) among 4–8 wells were averaged and converted into flux rates (F_R) by normalizing F_A to the [⁸⁵Rb] and [⁸⁶Rb] used in the flux medium and to the protein content of individual wells (F_R = F_A x [⁸⁵Rb]/[⁸⁶Rb] x influx time in min x protein content in µg). F_R among two to three experiments were then reaveraged and expressed as means ± standard error (S.E.). When appropriate, differences between normalized flux rates were analyzed by Student's two tail t tests, and the null hypothesis was rejected for p values < 0.05.

Protein Analyses—Cytosolic and membrane proteins were extracted from various cell strains in lysis buffers containing protease inhibitors (see figure legends for composition of solutions). For some experiments, proteins were also immunoprecipitated from precleared cell lysates (20 µg/µl of proteins) with different Abs (anti-hsp90 or anti-saNKCC1) through 1-h incubations at 4 °C followed by a 30-min incubation at 4 °C with 30 µl/ml of 10% Sepharose A (Amersham Biosciences). Bound antigens were microcentrifuged afterward at 4 °C (15, 000 x g), washed four times in a high salt solution (500 mM Tris, pH 8.0, 200 mM NaCl, 0.05% Tween) and released from Sepharose pellets through 2-min incubations at 100 °C in protein sample buffer.

Proteins solubilized or immunoprecipitated from membrane extracts were separated on SDS-polyacrylamide Tricine gels (6 µg or 10% of an immunoprecipitation reaction/lane), transferred to Immobilon-P nylon blots (Millipore) and revealed by chemiluminescence (Amersham Biosciences) after sequential incubation of the blots with a primary and secondary Ab. Whole-cell extract proteins were quantified with the DC protein assay (Bio-Rad), whereas protein bands separated by gel electrophoresis were quantified numerically with the National Institutes of Health program Scion Image Beat 4.0.2. When appropriate, differences between band intensities were also analyzed by Student's two tail t tests.

Cellular Distribution of NKCC1 and hsp90—IF studies of pJB20_M- or saNKCC1-transfected HEK-293 cells were conducted as described previously (12). Briefly, cells were grown on poly-D-lysine precoated coverslips, fixed in 4% paraformaldehyde, permeabilized at -20 °C with methanol/acetone, and incubated with Abs (J3 or anti-hsp90 followed by a fluorescent secondary anti-IgG). Coverslips were subsequently mounted on glass slides with Aqua-mount® (Lerner Laboratories), and micrographs were taken with a Bio-Rad MRC 1024 confocal microscope.

Sequence Analyses—The bp content of cloned inserts was determined by automated sequencing used plasmid-derived primers (Table II). Blast searches, sequence alignments and structure predictions were carried out with DNAStar (Lasergene), the PLOT program (Biff Forbush), and online programs available through NCBI, EMBL-EBI, and the BCM Search Launcher Web pages.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Two-hybrid Screen—The cell strains used for this experiment are called YopC1t and YoptC3t. They consist of p8op-lacZ-transformed EGY48 yeasts retransformed with the plasmid C1t/pGilda, which encodes the proximal huNKCC1 cytosolic C terminus, or with C3t/pGilda, which encodes the distal region of this domain. Transformation of YopC1t with a human heart library/pB42AD and selection of these cells on -Ura-His-Trp plates led to the formation of over 1 million colonies. Of these, 41 grew on -Ura-His-Trp-Leu plates and 32 expressed above-background -gal activity on -Ura-His- Trp-Leu + X-galactosidase plates. Transformation of YopC3t cells with a human brain library/pB42AD and selection of these cells as above led to the formation of 3.2 million colonies. Of these, 54 survived the quadruple selection step and 34 expressed above-background -gal activity.

Confirmation studies in the yeast system revealed that the identified interactions were specific (not shown). Interestingly, inserts found in the prey plasmids of -gal positive -Leu-resistant colonies corresponded to the coding regions of 12 different proteins. Seventeen of the inserts (1 from YopC1t and 16 from YopC3t) had nucleotide sequences that encode part of hsp90 (residues 1–243 for the YopC1t-derived prey and 1–245 for the 16 YopC3t-derived preys). Despite the high number of hsp90 preys uncovered (25% of the interactors), we observed that a least six of them had a unique 5'-untranslated region.

In Fig. 2, Western analyses of Yop cells transformed with C1t/pGilda, C3t/pGilda, and hsp90_1–245/pB42AD showed that the expected hybrid proteins probed with an anti-LexA or anti-HA Ab are produced. Indeed, bands at the adequate molecular size mark are visualized in all lanes. These results indicate that Yop cells transformed with hsp90 + C1t or hsp90 + C3t did not become -gal positive and -Leu-resistant in the absence of bait or prey expression, confirming further that the hsp90-NKCC1 interaction is specific in the yeast system.

Biochemical Characterization of HEK-293 Cells Before and After a Thermal Stress under Anisotonic Conditions—These studies were carried out to determine the effect of a thermal stress on hsp90 and NKCC1 synthesis in a mammalian cell line and to confirm the hsp90-NKCC1 interaction outside of the yeast system. The cell lines used included pJB20_M-transfected HEK-293 cells, which are known to express huNKCC1, and saNKCC1-transfected HEK-293 cells, which express saNKCC1 in addition to huNKCC1. The latter were preferred over huNKCC1-transfected cells because of the availability of an anti-saNKCC1 monoclonal Ab (called J3) that is a highly sensitive and specific (32). It should be noted, moreover, that saNKCC1 and huNKCC1 share over 74% identity in amino acid sequence and that they appear to be regulated similarly (25, 37). For these studies, cell lines were maintained in a regular medium (see Table I) prior to harvesting. Results are shown in Fig. 3 using representative blots (n = 3–4 for each condition).

View larger version (82K): [in this window] [in a new window]   FIG. 3. Protein analyses of HEK-293 cells maintained in a regular medium before and after a thermal stress. Representative blots among three to four for each condition are used to illustrate the results. Here, proteins were extracted with a mammalian cell lysis solution (see ref. 12) to which glass beads and protease inhibitors were added. Upper panel, in the first three lanes, whole HEK-293 cell lysates were assayed by Western analyses using the anti-hsp90 Ab. In lanes 4, 5, and 6, whole lysates were incubated with J3 and immunoprecipitated proteins were assayed by Western analyses using the anti-hsp90 Ab. In lanes 7, 8, and 9, the same experiments were repeated without the primary Ab. For lanes 3, 6, and 9, HEK-293 cells were also subjected to a 20-min heat shock treatment followed by a 24-h recovery period. Lower panel, these studies were carried out as in the upper panel except that the detecting Ab was J3 and the immunoprecipitating Ab was anti-hsp90. The quantity of proteins loaded per lane was identical, and the exposure time following the chemiluminescence assay was 1 min.

The effect of a 20-min heat treatment was also tested by Western analyses of HEK-293 whole cell extracts. After such a treatment, a 2.7-fold increase (p = 0.03) in hsp90-specific signal is noted (compare the example in Fig. 3, lanes 3 and 2 of the upper panel), confirming that this cell line possesses heat shock-inducible pathways. For saNKCC1, on the other hand, the heat stress does not lead to significant changes in the J3 signal (see corresponding lanes in the lower panel), indicating that these heat shock-dependent pathways probably have little if no effects on the net turnover rates of NKCC1 proteins.

The hsp90-NKCC1 interaction identified with the two-hybrid screen was retested in HEK-293 cells through coimmunoprecipitation (coIP) studies and results are also illustrated in Fig. 3, lanes 4, 5, 7, and 8. Quite convincingly, signals of the appropriate sizes can be seen when either J3 or anti-hsp90 is used for immunoprecipitation and when the proteins are derived from saNKCC1-transfected cells (lane 5) but not when they are derived from pJB20_M-transfected cells or the primary Ab is omitted (lanes 4, 7, and 8). Interestingly, it is also seen that the migration pattern of the band in lane 5 is different from that in lane 3. We can therefore conclude from these results that hsp90 does associate with saNKCC1 in HEK-293 cells, perhaps with the immature form of the carrier, and that a heat treatment is not required for this association to occur.

The effect of a 42 °C thermal stress on immunoprecipitable partners is illustrated in Fig. 3, lanes 6 and 5 (see upper and lower panels). Under such circumstances, specific increases in signal intensity are observed for both partners during the recovery period. These increases are in fact 2.6-fold for hsp90 (p = 0.01) and 2.8-fold for NKCC1 (p = 0.02), and they appeared specific as well based on the absence of signals when the primary Ab was omitted (see lane 9). In light of the differences that are observed between lanes 3 and 2 in the upper panel, these findings suggest that the heat-induced rise in immunoprecipitable partners are caused by changes in hsp90 expression.

Biochemical Characterization of HEK-293 Cells Preincubated in a Low Cl^- Medium—These studies were carried out as above to determine whether a reduction in Cl^-_i could have an effect on hsp90 and NKCC1 synthesis or hsp90-NKCC1 heteroformation. For these studies, cells were incubated in the low Cl^- medium (Table I) for 0, 30, or 60 min prior to harvesting. Results are shown in Fig. 4 using again representative blots (n = 3 for each condition).

View larger version (64K): [in this window] [in a new window]   FIG. 4. Protein analyses of HEK-293 cells preincubated in a low Cl- medium regular medium before and after a thermal stress. These studies were carried out as in Fig. 3 and representative blots (among three for each condition tested) are also used to illustrate the results. Here, however, cells were transferred from the tissue culture medium to a low Cl- medium and incubated for 0, 30, or 60 min prior to harvesting. A, Western analyses of whole cell extracts. B, Western analyses of immunoprecipitated proteins.

Influx Assays—The purpose of these studies was to determine whether a functional interaction exists between hsp90 and NKCC1. Bumetanide-sensitive ⁸⁶Rb influx measurements were carried out in mock-, huNKCC1- and saNKCC1-transfected HEK-293 cells subjected to various treatments (42 °C thermal stress versus sham stress, incubation in regular versus low Cl^- medium, and presence or absence of the hsp90 inhibitor geldanamycin). Results are summarized in Fig. 5, 6, and 7.

View larger version (23K): [in this window] [in a new window]   FIG. 5. 86Rb influx in pjB20M-, huNKCC1-, and saNKCC1-transfected cells after incubation in a regular medium or in a low Cl--medium. Cells were preincubated in a regular or low Cl- medium during 60 min and assayed for 86Rb influx in a regular medium ± 250 µM bumetanide. The data are shown as averages ± S.E. of two to four flux rows from two to three experiments. Composition of media used for these studies is shown in Table I. *, for a given cell line, the difference in 86Rb influx between the two conditions is statistically significant (p < 0.01).

View larger version (29K): [in this window] [in a new window]   FIG. 6. Effect of a heat shock treatment and/or geldanamycin on 86Rb influx in pjB20M-, huNKCC1-, and saNKCC1-transfected cells after incubation in a regular medium. Cells were first subjected to various pretreatments as detailed under "Experimental Procedures." They were subsequently incubated during 60 min in a regular medium and assayed for 86Rb influx in the same medium ± 250 µM bumetanide. The data are shown as averages ± S.E. of two to four flux rows from two to three experiments. *, for a given cell line, the difference in 86Rb influx between this condition and all of the other conditions is statistically significant (p < 0.01). , for a given cell line, the difference in 86Rb influx between the + geldanamycin (GEL) condition and the 0 heat shock (HS)/GEL condition is statistically significant (p < 0.01).

View larger version (29K): [in this window] [in a new window]   FIG. 7. Effect of a heat shock treatment and/or geldanamycin on 86Rb influx in pjB20M-, huNKCC1-, and saNKCC1-transfected cells after incubation in a low Cl--medium. Cells were first subjected to various pretreatments as in Fig. 6. They were subsequently incubated during 60 min in a low Cl- medium and assayed for 86Rb influx in a regular medium ± 250 µM bumetanide. The data are shown as averages ± S.E. of two to four flux rows from two to three experiments. *, for a given cell line, the difference in 86Rb influx between the + geldanamycin (GEL) conditions and the no GEL conditions is statistically significant (p < 0.01). , the difference in 86Rb influx between this condition and the 0 heat shock (HS)/GEL condition is statistically significant (p < 0.01).

With a thermal stress administered 4 h prior to the flux assay, the component of ion transport mediated by endogenous or heterologous NKCC1s decreases significantly (Fig. 6, bars 2 versus 1, 6 versus 5, and 10 versus 9). Remarkably, this decrease is prevented by the addition of geldanamycin during and after the stress (bars 3 versus 2, 7 versus 6, and 11 versus 10). The role played by hsp90 under such conditions is further suggested by the effect of geldanamycin on non heat-treated cells. In such a case, ⁸⁶Rb influx increases 1.4-fold (bars 8 versus 5 and 12 versus 9) for the two transfected cells. Taken together, these results suggest that constitutively active NKCC1s are down-regulated when hsp90 is functional in the cellular environment.

When heat-treated cells were assayed after incubation in low Cl^- medium (Fig. 7), the changes in carrier activity were very different from those observed after incubation in regular medium. As shown in A, for example, there is no reduction in bumetanide-sensitive ⁸⁶Rb influx (bars 2 versus 1, 6 versus 5, and 10 versus 9) compared with Fig. 6. Interestingly, when the component of so-called constitutive Na⁺-K⁺-Cl^- cotransport is subtracted from the low Cl^--induced influx, a slight increase in ion transport is even apparent (Fig. 7B, bars 6 versus 5 and 10 versus 9).

In Fig. 7A, it can also be noted that the effect of geldanamycin is different from that observed in Fig. 6. For instance, pretreatment of cells with this antibiotic brings about a large decrease in carrier-mediated ⁸⁶Rb influx; this is at least the case for NKCC1-transfected cells (Fig. 7A, lanes 7 and 8 versus 5 and 6, and 11 and 12 versus 9 and 10). Again, this change is much more pronounced when constitutive Na⁺-K⁺-Cl^- cotransport is subtracted from the low Cl^--induced influx. From these results, hence, we can conclude that the activation of NKCC1 in a low Cl^- medium is influenced by the ATPase activity of hsp90.

IF of HEK-293 Cells—As shown in Fig. 8 (panels A–H), the distribution of saNKCC1 in HEK-293 cells is unchanged by a heat shock treatment, the addition of geldanamycin for 16 h, or a reduction in Cl^-_i. Indeed, a strong signal can be seen at the cell surface for all of the conditions tested; as is usually the case of membrane proteins expressed in HEK-293 cells, there is a fair amount of intracellular staining as well. In these studies, the distribution of hsp90 is also seen to be similar among the various conditions tested (data not shown). Here, however, the signal is predominantly cytosolic as illustrated in panel H for one of the conditions (see legend).

View larger version (31K): [in this window] [in a new window]   FIG. 8. Effect of a heat shock treatment and/or geldanamycin on the cellular distribution of saNKCC1. Cells were also subjected to various pretreatments. A–H, cells were first maintained at 37 °C (first column, panels A and E), heat-treated at 42 °C (second column, panels B and F), pretreated with geldanamycin (GEL) for 16 h (third column, panels C and G), or heat-treated + pretreated with geldanamycin (fourth column, panels D and H). They were subsequently incubated in a regular medium (upper row, panels A–D) or in a low Cl- medium (bottom row, panels E–H) during 60 min and assayed by IF. I, IF of mock-transfected cells using the anti-hsp90 Ab (cells were treated as in A). J, IF of mock-transfected cells using the J3 Ab (cells were also treated as in A). All micrographs are from representative fields. HS, heat shock.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although we have not identified the residues involved in the hsp90-NKCC1 association, the two hybrid studies suggest that there are at least two contact sites in each of the partners. In the cotransporter, one of these sites presumably belongs to C1t and the other to C3t. In hsp90, on the other hand, both sites appear to be in the N terminus as all of the preys identified lack bps 738–2199 of the hsp90 open reading frame. This particular localization for NKCC1 binding sites in hsp90 is of particular interest considering that the N terminus of chaperones possesses ATPase activity and homodimerization domains (40). Additional studies such as pull-down assays and mutagenic analyses will be required to determine more precisely which regions in hsp90 and in the cytosolic C terminus of NKCC1 mediate the interaction.

The existence of two contact regions in NKCC1 points toward the possibility that hsp90 binds to the unfolded form of the carrier, perhaps on the endoplasmic reticulum surface during early biogenesis. This possibility is further suggested by the coIP studies, which showed that hsp90-immunoprecipated NKCC1 migrates predominantly as an immature 130-kDa band on SDS gels (see lanes 5 and 6 in the bottom panel of Fig. 3), whereas non-immunoprecipitated NKCC1 migrates as a broader 130–175-kDa band (see lane 2 and 3 in the same bottom panel). For both the human and the shark NKCC1, the higher molecular size band has been shown to represent the mature glycosylated form of the protein (5, 26, 39). Whether it could also include ubiquitinated NKCC1 or other types of post-translationally modified carrier has not been determined yet.

Whereas hsp90 has been shown to play an important role in early biogenesis for several types of integral membrane proteins including CFTR (28, 41), it has also been found to associate with proteins that have acquired a more advanced folded state in the cellular environment. The glucorcorticoid receptor is one of the best characterized examples of a mature protein that can interact with hsp90 (42–44). In the current work, additional findings indicate that this chaperone could also interact with NKCC1 during later stages in the biosynthetic pathway. For instance, the coIP studies did show that a fraction of hsp90-immunoprecipated NKCC1 migrates as a mature band on SDS gels. In addition, the expression or distribution of saNKCC1 was found to be unaltered by geldanamycin, a finding that appears inconsistent with a scenario where hsp90 would bind to the unfolded carrier exclusively. In this regard, however, it should be mentioned that chemiluminescence-based assays or regular IF studies may have not allowed detecting minor changes in saNKCC1-specific signals given that they offer relatively limited quantitative accuracy and that they were carried out in a cell line that overexpresses the transporter.

Despite the concerns that are raised here in regard to the biochemical characterizations of saNKCC1, it is clear that the effect of geldanamycin on cotransport function is more important than that on expression or distribution of the carrier. In addition, geldanamycin was shown to produce its action in a Cl^-_i-dependent manner, increasing Na⁺-K⁺-Cl^- cotransport slightly in the regular medium but abrogating carrier activation in the low Cl^- medium. Because the regulation of CCC proteins is Cl^-_i-dependent as well, and because hsp90 usually exerts its effects by altering the conformation of proteins to which it binds (40, 42–44), it is tempting to postulate that geldanamycin affected cotransporter function in this work by inhibiting the action of NKCC1-bound hsp90. Based on the coIP, IF, and functional studies discussed above, this possibility could imply that NKCC1s are under tonic inhibition from the action of prebound hsp90 when Cl^-_i is normal, whereas they are activated as a result of this interaction when Cl^-_i is lowered.

If the formation of hsp90-NKCC1 heterocomplexes plays an important role in Cl^-_i-dependent activation of Na⁺-K⁺-Cl^- cotransport, what would be the mechanisms by which the effect of Cl^-_i is transduced? Among various possibilities, a reduction in Cl^-_i could allow hsp90 to fold NKCC1 in an optimal conformation co- or post-translationally, enhancing its transfer in the endoplasmic reticulum, its delivery or maintenance at the cell surface, or its association with various regulatory enzymes. The latter scenario is particularly appealing given that kinases and phosphatases are often considered the most prevalent client proteins of chaperones (40, 44) and that these enzymes are also involved in Cl^-_i-dependent regulation of NKCC1 (1, 4, 25, 37). To this effect, it will be of interest to determine whether PASK-mediated phosphorylation of NKCC1 in HEK-293 cells is still possible in the presence of geldanamycin.

It is important to recognize that the antibiotic geldanamycin may not only prevent binding of hsp90 to NKCC1 but also that of hsp90 or other hsp90-related chaperones to various client proteins (45). Consequently, treatment of HEK-293 cells with this agent will probably trigger a number of downstream cellular events such as those related to the unfolded protein response, which results from the accumulation of misfolded proteins in the endoplasmic reticulum, or those related to changes in the activity of various chaperones (31, 45, 46). These cellular events, which have been shown to include, for instance, down-regulation of phosphaditylinositol 3-kinase or of nuclear factor kappa-B (47, 48), could thus account (at least to some extent) for the functional effects that were observed in the current study. As the role of these intermediates in NKCC1 regulation is currently unknown, it is not possible to exclude with certainty that geldanamycin-mediated changes in Na⁺-K⁺-Cl^- cotransport are in fact unrelated to the hsp90-NKCC1 interaction.

Heat treatments are known to affect cell volume and pH regulation in isotonic as well as anisotonic conditions (27, 29, 30). Commonly observed responses to these treatments include a decrease in pH_i and V_cell as well as an augmented capacity to recover from an acid load and to maintain V_cell under hypertonic conditions (adaptive response); this process by which cells subjected to shrinkage return to a normal volume has been called regulatory volume increase (4). Whereas the Na⁺/H⁺ and/or HCO₃^-/Cl^- exchangers have been implicated in the adaptive responses (27, 29, 30), the role of various ion transport systems in the early heat-induced changes have received modest attention. Because inhibition of NKCC1 has been shown to decrease V_cell under anisotonic conditions (49), the effect of a thermal stress on Na⁺-K⁺-Cl^- cotransport could therefore contribute to the early morphological changes that are observed during changes in ambient temperatures.

The regulatory mechanisms that affect the activity and sub-cellular localization of NKCC1 have been shown to differ among various cell types. For instance, the effect of cell shrinkage on Na⁺-K⁺-Cl^- cotransport is stimulatory in Xenopus laevis oocytes, dog tracheal epithelial cells, and T84 cells, whereas it is inhibitory in HEK-293 cells and shark rectal gland epithelial cells (2, 34, 50–53). Hence, the findings reported here may not apply, at least integrally, to NKCC1 in all cell types. Based on these comments, it will be interesting to determine whether the effect of geldanamycin on cotransporter activity is also V_cell-dependent. It appears that T84 cells, a colonic cell line that expresses robust Na⁺-K⁺-Cl^- cotransport activity (52), would represent a good system to address this question.

In conclusion, we have characterized a novel interaction between hsp90 and NKCC1, demonstrating for the first time that chaperones are probably important binding partners for members of the CCC family. In the case of NKCC1, this interaction could be the interface of the Cl^-_i-dependent signaling mechanism that allows mammalian cells to maintain their water and ion content under normal as well as paraphysiological conditions. These findings are particularly worthy of note in view of the importance given to hsp90 as a potential molecular target in abnormal proliferative states, which are also influenced by the activity of the secretory Na⁺-K⁺-Cl^- cotransporter.

	FOOTNOTES

 
^* This work was supported by grants from the Canadian Institute of Health and Research (MT-15405) and the Kidney Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: L'Hôtel-Dieu de Québec Research Center, 10 Rue McMahon (Rm. 3852), Québec, Canada G1R 2J6. Tel.: 418-691-5151 (15477); Fax: 418-691-5787; E-mail: paul.isenring{at}crhdq.ulaval.ca.

¹ The abbreviations used are: CCC, cation-Cl^- cotransporter; NKCC, Na⁺-K⁺-Cl^- cotransporter; Cl^-_i, intracellular Cl^-; PASK, proline-arginine-rich stress-related kinase; hu, human; Ab, antibody; HA, hemagglutinin; sa, Squalus acanthias; AMP^R, ampicillin resistance gene; op, operator; Y, EGY48 yeasts; -gal, -galactosidase; IF, immunofluorescence; coIP, coimmunoprecipitation; HEK, human embryonic kidney; C1t, proximal segment of the putative cytosolic C terminus of huNKCC1; C3t, distal segment of the putative cytosolic C terminus of huNKCC1; Yop, EGY48 yeasts transformed with the p8op-lacZ reporter plasmid; V_cell, cell volume; Tricine, N-[2-hydroxy-1,1-bis(hydroxy-methyl)ethyl]glycine.

² LAM, which stands for human lamin C, has been reported to interact with no other proteins (54).

³ Bumetanide-sensitive ⁸⁶Rb influxes observed under such conditions are slightly higher than those reported in previous studies (3, 35, 38). These results may be caused by differences in the experimental design that was used for the flux assays. Compared with the other studies, for instance, incubation in the influx medium was increased from 1–2 min; although carrier activity is linear between 0.5 and 3.0 min in this system, the x intercept is slightly above 0. Another difference is that ouabain was added throughout the preincubation and influx periods instead of during the influx period only.

	ACKNOWLEDGMENTS

 
We thank Dr. Jacques Landry, Dr. Jacques Huot, and Dr. Biff Forbush for reagents.

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