Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The study identifies Hsp90 as a molecular chaperone for KATP channels. Inhibition of Hsp90 function reduces, whereas overexpression of Hsp90 enhances, channel expression at the cell surface. Hsp90 facilitates channel biogenesis by targeting the SUR1 subunit. Up-regulation of Hsp90 also enhances expression of some SUR1 mutants with folding defects.


INTRODUCTION
ATP-sensitive potassium (K ATP ) channels in pancreatic ␤-cells, by virtue of their sensitivities to intracellular nucleotides ATP and ADP, serve as molecular linkers between cell metabolism and cell excitability, thus mediating glucoseregulated insulin secretion (Aguilar-Bryan and Bryan, 1999;Nichols, 2006). The ␤-cell K ATP channel is an octameric complex of four inward rectifier potassium channel (Kir6.2) subunits and four sulfonylurea receptor 1 (SUR1) subunits (Aguilar-Bryan and Bryan, 1999;Nichols, 2006). Mutations in the genes ABCC8 encoding SUR1 or KCNJ11 encoding Kir6.2 that uncouple channel activity from glucose metabolism underlie congenital forms of hyperinsulinism and diabetes (Aguilar-Bryan and Bryan, 1999;Ashcroft, 2005;Flanagan et al., 2009). Studies characterizing disease-associated K ATP channel mutations have indicated that many mutations, especially those identified in congenital hyperinsulinism, cause channel dysfunction by disrupting folding or assembly of channel proteins in the endoplasmic reticulum (ER) and thereby subsequent trafficking to the plasma membrane (Cartier et al., 2001;Taschenberger et al., 2002;Ashcroft, 2005;Lin et al., 2006;Yan et al., 2007). Despite the importance of channel biogenesis and surface expression in ␤-cell function, little is known about how these processes are governed at the molecular level.
Generation of K ATP channels in the ER requires correct folding and coassembly of two different channel subunits with distinct structures. Whereas Kir6.2 is a relatively simple protein with only two-transmembrane helices and cytosolic N-and C-terminal domains, SUR1 contains 17 transmembrane helices divided into three transmembrane domains TMD0, TMD1, and TMD2, and two cytoplasmic nucleotide binding domains, NBD1 and NBD2 (Neagoe and Schwappach, 2005). We have shown previously that normal biogenesis of K ATP channels is an inefficient process; only ϳ20% newly synthesized channel subunits mature into functional channel complex, whereas the majority is degraded by the ubiquitin-proteasome pathway (Yan et al., 2004. With regard to channel assembly, studies have shown that a tripeptide ER retention/retrieval motif RKR present in both SUR1 and Kir6.2 prevents unassembled or partially assembled channel proteins from exiting the ER (Zerangue et al., 1999). Under physiological conditions where both subunits are coexpressed, assembly of SUR1 and Kir6.2 into octameric complexes masks the RKR signals to allow channel forward trafficking to the plasma membrane. Deletion or mutation of the RKR peptide to AAA also allows individual subunits to reach the cell surface by bypassing a critical ER qualitycontrol checkpoint (Tucker et al., 1997;Zerangue et al., 1999).
Molecular chaperones represent a heterogeneous group of conserved cellular proteins that assist the folding of newly This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 -02-0116) on April 28, 2010. translated proteins. Recent studies suggest that manipulation of molecular chaperones could be used to treat human disease caused by protein misfolding (Welch, 2004;Balch et al., 2008;Powers et al., 2009;Yang et al., 2009). A prominent example is the cystic fibrosis transmembrane conductance regulator (CFTR) mutant ⌬F508, which accounts for most of the cystic fibrosis cases (Rowe et al., 2005). CFTR is structurally homologous to SUR1, both proteins belonging to the ATP-binding cassette (ABC) transporter protein family (Higgins, 1995). The molecular chaperone network monitoring CFTR biogenesis has been extensively studied and shown to involve the ER luminal chaperone calnexin and the cytosolic heat-shock protein (Hsp)40/70/90 chaperone complex (Loo et al., 1998;Wang et al., 2006). By contrast, virtually nothing is known about the molecular chaperones that participate in folding and degradation of K ATP channels. In addition, there is a lack of understanding of how biogenesis of heteromeric protein complexes such as K ATP channels is regulated. This lack of knowledge hinders progress on exploiting molecular chaperones for treatment of disease caused by misfolding of K ATP channels.
To address these issues, we have taken a proteomics approach in which epitope-tagged SUR1 and Kir6.2 are expressed in the insulinoma cell line INS-1. Proteins associated with the channel subunits were isolated by affinity purification and identified by tandem mass spectrometry. Here, we show that K ATP channel subunits are associated with components of the Hsp90 chaperone complex in the ER, including Hsp40, heat-shock complex (Hsc)70, and Hsp90. The Hsp90 chaperone network has been reported to affect the biogenesis/maturation efficiency of several membrane proteins, including the aforementioned ABC transporter CFTR and the voltage-gated potassium channel human ether-a-gogo-related gene (hERG; Kv11.1). However, other membrane proteins with similar complex topology such as the P-glycoprotein and the multidrug resistance-associated protein (MRP), also ABC transporters, and other Kv channels, including Kv1.5 and Kv2.1, are not affected by perturbation of Hsp90 function (Loo et al., 1998;Ficker et al., 2003). We therefore sought to determine whether the Hsp90 chaperone complex plays a role in K ATP channel processing and maturation. We demonstrate that inhibition of Hsp90 reduces K ATP channel expression at the cell surface, whereas overexpression of Hsp90 increases the number of K ATP channels in the plasma membrane. Importantly, we found that the Hsp90 complex interacts preferentially with the SUR1 subunit. Moreover, although perturbation of Hsp90 significantly alters cell surface levels of a SUR1 expressed alone, it has little effect on those of a Kir6.2 expressed alone. Our results indicate that the Hsp90 chaperone regulates biogenesis efficiency of K ATP channels by targeting the SUR1 but not the Kir6.2 subunit.

Expression Constructs
Wild-type hamster SUR1 or FLAG epitope (DYKDDDDK)-tagged SUR1 (referred to as fSUR1) cDNAs were cloned in the pECE plasmid. Wild-type rat Kir6.2 or HA epitope (YPYDVPDYA)-tagged Kir6.2 cDNAs were cloned into pcDNA3, as described previously (Cartier et al., 2001). Construction of adenovirus carrying wild-type (WT) fSUR1 or WT Kir6.2 or hemagglutinin (HA)-tagged WT Kir6.2 cDNA was as described previously (Lin et al., 2008). The FLAG-tag for SUR1 was placed at the extracellular N terminus of the protein. The HA-tag for Kir6.2 was inserted between amino acid 100 and 101 in the extracellular domain; an extra nine amino acids, DLYAYMEKG, was added between amino acid 98 and 99 to aid accessibility of the epitope. We have shown in previous studies that these epitope tags do not affect the trafficking or function of the channels (Cartier et al., 2001;Lin et al., 2005). The fSUR1 recombinant adenovirus was made using a modified pShuttle plasmid (AdEasy kit; Stratagene, La Jolla, CA) containing a tetracycline-inducible promoter. Recombinant viruses were amplified in human embryonic kidney 293 cells and purified according to the manufacturer's instructions.

Virus Infection
INS-1 cells clone 832/13 (kindly provided by Dr. Christopher Newgard, Duke University, Durham, NC) were plated in 10-cm plates and cultured for 24 h in RPMI 1640 medium with 11.1 mM d-glucose (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, and 50 M ␤-mercaptoethanol (Hohmeier et al., 1997). For Kir6.2 expression, recombinant adenoviruses containing Kir6.2 or HA-Kir6.2 with desired titers were used as described previously (Lin et al., , 2008. For fSUR1 expression, cells were coinfected with equal amounts of two recombinant adenoviruses, one adenovirus encoding a tetracycline-inhibited transactivator (tTA) and the other adenovirus a tTA-regulated gene expressing fSUR1 (Lin et al., 2008). Cells at ϳ70% confluency were washed once with phosphate-buffered saline and then incubated for 1.5 h at 37°C in Opti-MEM containing low bovine serum and a mixture of viruses. The multiplicity of infection for each virus was determined empirically. After 90 min, 2ϫ growth medium was added, and the cells were incubated at 37°C until reaching appropriate density for the various experiments.

Affinity Purification of K ATP Channel Complexes
INS-1 (832/13) cells were transduced with adenoviruses carrying fSUR1, HA-Kir6.2, or fSUR1 and Kir6.2 as described above. Twenty-four hours after infection, the cells were lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 1% Triton X-100, with Complete protease inhibitors [Roche Diagnostics, Indianapolis, IN]) on ice for 30 min. The cell lysate was centrifuged at 16,000 ϫ g for 5 min at 4°C, and the supernatant was used for affinity purification by addition of 100 l of FLAG-or HA-antibody conjugated agarose beads (Sigma-Aldrich, St. Louis, MO) overnight at 4°C. After washing three times with the lysis buffer, bound proteins were eluted by incubation with FLAG peptide (250 g/ml, for fSUR1 sample) or HA peptide (10 g/ml, for HA-Kir6.2 sample) at room temperature for 30 min.

Proteomics and Mass Spectrometry Analysis
Affinity-purified samples were concentrated to a final volume of 20 l, mixed with Laemmli sample buffer, and electrophoresed briefly into 10% Bis-Tris gels (Invitrogen) at 200 V in 3-(N-morpholino)propanesulfonic acid buffer for 5 min. The gel was stained with Imperial Coomassie (Pierce Chemical, Rockville, IL), placed on a clean glass plate, and each sample was excised by cutting just below the dye front and removing the entire top of the lane including the bottom of the well. The excised gel pieces were then cut into 1to 2-mm sections, washed twice by shaking in 0.5 ml of water for 15 min, washed twice again by shaking in 0.5 ml of 1:1 solution of acetonitrile:100 mM ammonium bicarbonate for 30 min, and then dried by vacuum centrifugation. After reduction and alkylation of proteins in the excised gel slices by incubation first in dithiothreitol (10 mM in 100 mM ammonium bicarbonate) and then in iodoacetamide (25 mM in 100 mM ammonium bicarbonate), proteins were digested by rehydrating the gel slice on ice in trypsin digestion solution containing 100 mM ammonium bicarbonate, and 10 g/ml proteomics grade trypsin (Sigma-Aldrich). The excess trypsin solution was then removed and sufficient 100 mM ammonium bicarbonate was added to cover the gel slices; samples were incubated overnight at 37°C. The solution was then removed from the gel slices, additional peptides were extracted, the volume of the collected digests was decreased by vacuum centrifugation, and samples were transferred directly to autosampler vials for analysis.
The peptide mixture was injected onto a 1 mm x 8 mm trap column (Michrom BioResources, Auburn, CA) at 20 l/min in a mobile phase containing 0.1% formic acid. The trap cartridge was then placed in-line with a 0.5-ϫ 250-mm column containing 5-m Zorbax SB-C18 stationary phase (Agilent Technologies, Santa Clara, CA), and peptides were separated by a 2-30% acetonitrile gradient over 95 min at 10 l/min by using a 1100 series capillary high-performance liquid chromotography (Agilent Technologies). Peptides were analyzed using a LTQ linear ion trap fitted with an Ion Max Source and 34-gauge metal needle kit (Thermo Fisher Scientific, Waltham, MA). Survey mass spectrometry (MS) scans were alternated with three data-dependant tandem MS (MS/MS) scans by using the dynamic exclusion feature of the software to increase the number of unique peptides analyzed. Peptide identification was performed by comparing observed MS/MS spectra to theoretical fragmentation spectra of peptides generated from a protein database using Sequest, version 27, revision 12 (Thermo Fisher Scientific). A Swiss-Prot database created in April 2008 containing hamster, mouse, and rat sequences was used (Swiss Institute of Bioinformatics, Geneva, Switzerland). Scaffold software (Proteome Software, Portland, OR) was used to validate MS/MSbased peptide and protein identifications. Peptide and protein identifications were accepted if they could be established at Ͼ95% and 95% probability, respectively (Keller et al., 2002;Nesvizhskii et al., 2003) and contained at least two identified peptides matched per protein entry.

Western Blotting and Immunoprecipitation Assay
INS-1 (832/13) cells were infected with SUR1 and Kir6.2 as described above. Twenty-four hours after infection, the cells were lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 1% Triton X-100, with Complete protease inhibitors) on ice for 30 min. The cell lysate was centrifuged at 16,000 ϫ g for 5 min at 4°C, and the supernatant was used for Western blot or immunoprecipitation. Immunoprecipitation was performed as described under Affinity Purification. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. The membrane was probed with appropriate primary antibodies including anti-FLAG (Sigma-Aldrich), anti-Hsp90␣/␤ (Santa Cruz Biotechnology), anti-Hsp40 (Abcam, Cambridge, MA), and anti-Hsc70 (Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), and visualized by enhanced chemiluminescence (Super Signal West Femto; Pierce Chemical).

Chemiluminescence Assay for Surface Expression
COSm6 cells or INS-1 cells in 35-mm dishes were fixed with 2% paraformaldehyde for 20 min at room temperature 48 h after transfection or infection.

Metabolic Labeling and Immunoprecipitation
COSm6 cells grown on 35-mm dishes were transfected with fSUR1 and Kir6.2 for 24 h. The cells were incubated in methionine/cysteine-free DMEM supplemented with 5% dialyzed fetal bovine serum for 30 min before labeling with l-[ 35 S]methionine (Tran 35 S-Label, 150 -250 Ci/ml; MP Biomedicals, Solon, OH) for 60 min at 37°C. Labeled cultures were chased in regular medium supplemented with 10 mM methionine at 37°C. At the end of the chase, the cells were lysed in 500 l of the lysis buffer described above. For immunoprecipitation, 500 l of cell lysate was incubated with 100 l of FLAG-antibody-conjugated agarose beads overnight at 4°C. The precipitate was washed three times in the lysis buffer, and the proteins were eluted with FLAG-peptide. The eluted proteins were separated by 8% SDS-PAGE, and the dried gels were analyzed using a Storm PhosphorImager (GE Healthcare).

Rb ؉ Efflux Assay
INS-1 cells were plated onto 12-well plates and cultured for 2 d to confluence. Cells were incubated for 12 h in culture medium containing 86 RbCl (1 Ci/ ml) with or without 17-(allylamino)-17-demethoxygeldanamycin (17-AGG; 100 or 200 nM). Before measurement of 86 Rb ϩ efflux, cells were incubated for 30 min at room temperature in Krebs-Ringer solution with metabolic inhibitors (2.5 g/ml oligomycin and 1 mM 2-deoxy-d-glucose). At selected times, the solution was aspirated from the cells and replaced with fresh solution. At the end of a 40-min period, cells were lysed. The 86 Rb ϩ in the aspirated solution and the cell lysate was counted. The percentage of efflux at each time was calculated as the cumulative counts in the aspirated solution divided by the total counts from the solutions and the cell lysate.

Patch-Clamp Recording
INS-1 cells were plated onto coverslips. Endogenous K ATP currents were assessed using inside-out patch voltage-clamp recording after 12-h pretreatment with 17-AGG. Micropipettes were pulled from nonheparinized Kimble glass (Thermo Fisher Scientific) on a horizontal puller (Sutter Instrument, Novato, CA) with resistance typically ϳ1-2 megaohms. The bath (intracellular) and pipette (extracellular) solution (K-INT) had the following composition: 140 mM KCl, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.3. ATP and ADP were added as the potassium salt. Recording was performed at room temperature and currents were measured at a membrane potential of Ϫ50 mV. In Figure 3, inward currents were shown as upward deflections. K ATP current was calculated as the difference between current in ATP-free (channels open) and in 1 mM ATP (channels closed) solutions.

Statistics
Data are presented as mean Ϯ SEM. Differences were tested using analysis of variance (ANOVA) when comparing three or more groups, and Dunnett's post hoc test was used to compare treated versus control group. When only two groups were compared, unpaired Student's t tests were used. Differences were assumed to be significant if p Յ 0.05.

Identification of Molecular Chaperones Associated with K ATP Channel Proteins
To identify proteins that interact with K ATP channels in the early secretory pathway in ␤-cells, we overexpressed fSUR1 or HA-Kir6.2 in INS-1 cells (clone 823/13, a model ␤-cell line from rat; Hohmeier et al., 1997) by using recombinant adenoviruses (Lin et al., , 2008. The rationale for the overexpression approach is that it will enhance the probability of capturing molecular chaperones that are associated with each subunit during biosynthesis and folding by increasing fraction of folding intermediates present. The epitope-tagged channel subunits were used to facilitate affinity purification, and we have shown previously that the FLAG-or HA-epitope on SUR1 or Kir6.2, respectively, do not affect channel expression or function (Cartier et al., 2001;Lin et al., 2005). Note that viral transduction was used because INS-1 cells exhibit low transfection efficiency using nonviral transfection methods (Lin et al., , 2006Yan et al., 2007). Proteins that interact with the channel subunits were captured using an affinity purification approach by incubation of the lysate with agarose beads conjugated with anti-FLAG or anti-HA antibody followed by elution using FLAG-or HA-peptide. The eluted proteins were identified by tandem mass spectrometry as described under Materials and Methods.
In total, 10 experiments were conducted: three in INS-1 cells transduced with viruses for fSUR1 overepxression only (fSUR1 virus plus tTA virus; see Materials and Methods), two in cells transduced with the virus for HA-Kir6.2 only, and five in cells transduced with viruses for overexpression of fSUR1 and untagged Kir6.2. In all experiments, uninfected INS-1 cells were subjected to the same experimental procedure to serve as a negative control. Results of three representing experiments from the three different conditions are shown in Table 1. The chaperone proteins consistently copurified with the channel subunits are Hsp40, Hsc70, and Hsp90␣/␤. Calnexin and Hsp organizing protein (HOP) were also present in a subset of experiments in which fSUR1 was used as a bait (Table 1). In yet another proteomics experiment using COS cells transfected with fSUR1 and Kir6.2 cDNAs, Hsp40, Hsc70, and Hsp90␣/␤ were also found in the fSUR1 immunoprecipitate (data not shown).

Differential Association of K ATP Channel Subunits with the Hsp90 Chaperone Complex
To confirm the interaction between channel subunits and chaperone proteins identified by mass spectrometry, Hsp40, Hsc70, and Hsp90 were coimmunoprecipitated with fSUR1 or HA-Kir6.2 from INS-1 cells transduced with the fSUR1 or the HA-Kir6.2 virus, respectively, as described under Materials and Methods. Results revealed that all three chaperones interact with fSUR1 ( Figure 1A, left). Similarly, all three chaperones were detected in the HA-Kir6.2 immunoprecipitate ( Figure 1A, right). Note that overexpression of individual channel subunits in INS-1 cells did not significantly alter the abundance of the various heat shock proteins examined (input).
Because INS-1 cells express endogenous K ATP channel proteins albeit at a lower level than the exogenously expressed fSUR1 or HA-Kir6.2 (Lin et al., 2006), it is possible that association with fSUR1 or HA-Kir6.2 was mediated by endogenous channel subunits interacting with the epitopetagged exogenous channel subunits. We therefore expressed fSUR1 or HA-Kir6.2 individually in COS cells that do not have endogenous SUR1 or Kir6.2 and performed similar coimmunoprecipitation experiments. As shown in Figure  1B, Hsp90, Hsc70, and Hsp40 still coimmunoprecipitated with fSUR1, but they were not detected in the Kir6.2 immunoprecipitate. Although the lack of signal for these chaperone proteins in the Kir6.2 immunoprecipitate could be caused by factors such as the cell type used or the expression level of Kir6.2, the simplest interpretation of the results is that these chaperone proteins might interact preferentially with SUR1 subunit in the K ATP channel complex.

Inhibition of Hsp90 Reduces K ATP Channel Surface Expression
To determine the role of Hsp90 complexes in channel biogenesis, we examined the effects of 17-AGG, a geldanamycin derivative that inhibits the ATPase activity and client binding properties of Hsp90 (Kamal et al., 2003), on K ATP channel biogenesis efficiency and surface expression in COS cells. SUR1 has two N-linked glycosylation sites. As the SUR1/ Kir6.2 complex traffics through the Golgi network, SUR1 becomes complex glycosylated to give rise to the mature form that migrates slower on SDS-PAGE (referred to as the upper band; Figure 2) than the immature ER core-glycosylated form (the lower band). Western blots of lysates from cells expressing both fSUR1 and Kir6.2 showed that 17-AGG (100 nM for 12 h) significantly decreased the abundance of the mature complex-glycosylated SUR1 band (upper band), suggesting reduced processing efficiency of the channel protein (Figure 2A, left). In agreement, chemiluminescence assays showed that surface expression of K ATP channels in COS cells treated with a range of 17-AGG concentrations (25 nM-2 M) was reduced in a dose-dependent manner, with the effect being apparent at 25 nM and saturating at ϳ200 nM ( Figure 2B). Similar results were obtained in INS-1 cells infected with K ATP channel subunits viruses, with the effect  on surface expression being apparent at 25 nM and saturating at ϳ500 nM (Figure 2, C and D). In addition to reducing the abundance of the upper band, 17-AGG also reduced the signal of the lower fSUR1 band (Figure 2, A and C), suggesting that inhibition of Hsp90 function might accelerate degradation of SUR1 in the ER as has been reported for CFTR (Loo et al., 1998). Because the studies described so far were conducted on K ATP channels heterologously expressed in COSm6 cells or INS-1 cells in which protein overexpression might be a concern, we examined the effect of 17-AGG on functional expression of endogenous K ATP channels in INS-1 cells. Channel function was first assessed by 86 Rb ϩ efflux assays that monitor activity in response to metabolic inhibition in intact cells (Shyng et al., 1998). Compared with control, cells treated with 100 nM 17-AGG for 12 h showed substantially reduced 86 Rb ϩ efflux over a 40-min period, indicating reduced channel activity ( Figure 3A). Next, inside-out patch voltage-clamp recordings were used to directly measure K ATP current density in nucleotide-free solutions. In INS-1 cells treated with 50 or 100 nM 17-AGG, the averaged K ATP current was 38.81 Ϯ 6.07 and 37.30 Ϯ 8.44 pA, respectively, both significantly lower than that observed in control cells (78.71 Ϯ 12.95 pA; Figure 3, B and C). Because 17-AGG did not affect current amplitude or Mg-ADP response of K ATP channels when applied acutely to isolated membrane patches at concentrations used in the above-mentioned experiments (data not shown; also see Discussion), we concluded that the reduced K ATP channel activities were due to effects of 17-AGG on the number of channels expressed at the surface rather than on channel gating directly. Thus, inhibition of Hsp90 function by 17-AGG caused a decrease in endogenous K ATP channels present at the INS-1 cells membrane. These results support a role of Hsp90 in the biogenesis and surface expression efficiency of native K ATP channels.

Overexpression of Hsp90 Increases Surface Expression of K ATP Channels in COSm6 Cells
Given that the Hsp90 inhibitor reduces K ATP channel processing and surface expression, we next examined whether overexpression of Hsp90 might facilitate K ATP channel maturation and increase surface channel expression. There are two major cytosolic Hsp90 isoforms: the ␣ isoform that is highly inducible and the ␤ isoform that is thought to be more constitutive (Sreedhar et al., 2004). For our experiments, Hsp90␣ or ␤ cDNA or empty vector were transfected along with WT fSUR1 and Kir6.2 into COSm6 cells, and the resulting K ATP channel surface expression was analyzed by chemiluminescence assays. As shown in Figure 4A, overexpression of Hsp90␤ cDNA increased surface expression of K ATP channels by 29.64 Ϯ 6.29%. Overexpression of Hsp90␣ also improved surface expression slightly (by 9.46 Ϯ 4.82%), but this effect did not reach statistical significance. We therefore focused on Hsp90␤ in the experiments described below. To determine whether the increased surface expression of K ATP channels in cells overexpressing Hsp90␤ is a consequence of increased channel biogenesis, we performed metabolic pulse-chase labeling experiments to monitor the processing and maturation of newly synthesized SUR1 protein.
Overexpression of Hsp90␤ was first confirmed by Western blots ( Figure 4B). In metabolic pulse-chase experiments, the amount of SUR1 protein labeled at the end of the 1-h pulse period was higher in cells overexpressing Hsp90␤ than in control cells, indicating Hsp90␤ overexpression led to increased biogenesis of SUR1. Signals of both the lower and upper bands from seven experiments were quantified and are shown in Figure 4C. The signal of the mature complexglycosylated SUR1 upper band in Hsp90␤-cotransfected cells is consistently higher than that in control cells at all times during the chase period. These results provide evidence that Hsp90␤ function contributes to the biogenesis efficiency, thereby surface expression levels of K ATP channels.
To test whether overexpression of Hsp90␤ can alleviate the folding/surface expression defects of mutant K ATP channels, we examined the effect of Hsp90␤ on surface expression of several mutant channels characterized previously as being processing/trafficking-deficient (Cartier et al., 2001;Yan et al., 2004Yan et al., , 2007. The five mutants examined harbor mutation N24K, A116P, D310N, ⌬F1388, or D1472N in the SUR1 subunit. Of these, the N24K, D310N, and D1472N mutants exhibited improved surface expression levels in cells cotransfected with Hsp90␤ cDNA relative to cells cotransfected with a control empty vector. However, the A116P and ⌬F1388 mutants did not show improved surface expression ( Figure 5). Interestingly, the N24K, D310N and D1472N mutants have relatively milder processing/trafficking defects in that they do express at the cell surface to some extent even under control conditions, in contrast to A116P and ⌬F1388 that show virtually no surface expression. These results suggest that although up-regulation of Hsp90 can rescue the processing and surface expression of some mutants, effects are dependent on the nature of the mutation and severity of processing defect.

Hsp90 Exerts Its Effect on K ATP Channel Biogenesis via the SUR1 Subunit
Unlike CFTR or hERG channels, the K ATP channel complex is formed by two membrane proteins of very different size and topological complexity. In coimmunoprecipitation experiments conducted in COS cells expressing fSUR1 or HA-Kir6.2 alone, we detected association of the Hsp90 chaperone complex proteins with SUR1 but not Kir6.2 (Figure 1), suggesting the chaperone complex might target the SUR1 subunit to exert its effect on channel biogenesis. To address the question of whether Hsp90 promotes channel maturation and surface expression by facilitating the folding and processing of SUR1, Kir6.2 or both, we examined how manipulation of Hsp90 function affects surface expression of SUR1 and Kir6.2 individually. For these studies, we used an fSUR1 construct in which the RKR ER retention/retrieval motif has been mutated to AAA (fSUR1 RKR3 AAA ) and a HA-Kir6.2 construct in which the RKR motif at the C terminus has been deleted (HA-Kir6.2⌬C36). Previous studies have shown that mutation of the RKR motif to AAA or deletion of this signal allows individual subunits to express at the cell surface independently of the other subunit (Zerangue et al., 1999). Also, the FLAG-and HA-epitope tags are inserted in the extracellular domain of SUR1 and Kir6.2 respectively, to permit quantification of surface expression of the individual protein by using the chemiluminescence assay described under Materials and Methods (Cartier et al., 2001;Lin et al., 2005). In COSm6 cells expressing fSUR1 RKR3 AAA alone, inhibition of Hsp90 function by 17-AGG led to reduced steady state mature fSUR1 RKR3 AAA upper band level as well as the core-glycosylated immature lower band ( Figure 6A), similar to that observed for fSUR1 coexpressed with Kir6.2 (Figure 2A). Chemiluminescence assays also revealed reduced surface expression of fSUR1 RKR3 AAA in a 17-AGG concentration-dependent manner ( Figure 6B). In contrast, the steady state protein level of Kir6.2 or HA-Kir6.2⌬C36 expressed in COSm6 cells in the absence of SUR1 was not affected by the 17-AGG treatment based on Western blots ( Figure 6C), and surface expression of HA-Kir6.2 also unaffected as assessed by chemiluminescence assays ( Figure 6D).
Next, either fSUR1 RKR3 AAA or HA-Kir6.2⌬C36 were coexpressed with Hsp90␣ or ␤ in COSm6 cells and surface expression of each channel subunit measured by chemiluminescence assays by using anti-FLAG or anti-HA antibody respectively. As shown in Figure 7A, coexpression of Hsp90␤ resulted in 29.52 Ϯ 9.87% increase in surface expression of fSUR1 RKR3 AAA , similar to that observed for WT SUR1/Kir6.2 channel complex ( Figure 4A). By contrast, no significant difference in surface expression of HA-Kir6.2⌬36 was observed between control cells (vector) and cells overexpressing either Hsp90␣ or ␤ ( Figure 7B). These results suggest that Hsp90 improves the surface expression of K ATP channels by facilitating folding and processing efficiency of SUR1 rather than Kir6.2.

DISCUSSION
Pancreatic ␤-cell K ATP channels have been extensively studied in the past two decades. However, as the key signal The distribution of K ATP currents are shown as circles for each condition, note the break in the y-axis. The total number of patches is given above each bar. The current density in cells treated with 50 or 100 nM 17-AGG was significantly lower than that of control cells (p ϭ 0.03 and 0.05, respectively). transduction protein complex linking glucose metabolism to insulin secretion remarkably little is known about the molecular mechanisms involved in the channel's biogenesis process. In this work, we have begun to address these issues by identifying molecular chaperones that associate with the channel subunits in ␤-cells, by using the insulinoma cell line INS-1 as an experimental platform. We provide evidence that the Hsp90 chaperone complex participates in the biogenesis of K ATP channels. First, K ATP channels are associated with Hsp90, Hsc70, and Hsp40, components of the Hsp90 chaperone complex. Second, inhibition of Hsp90 function by 17-AGG markedly reduced surface expression of K ATP channels, whereas overexpression of Hsp90␤ significantly increased surface expression. In addition, we show that overexpression of Hsp90␤ improved surface expression of select misprocessing/mistrafficking SUR1 mutants. Finally, we present evidence that Hsp90 facilitates channel biogenesis by increasing the folding/processing efficiency of SUR1.

Comparison with Other Hsp90 Client Proteins
Hsp90 is an abundant cytosolic chaperone (Pearl and Prodromou, 2006). Unlike Hsc/p70, which acts by holding newly synthesized proteins in a folding competent state, Hsp90 is required for the folding of a subset of client proteins that are thought to have difficulties in reaching a native conformation (Nathan et al., 1997;Young et al., 2001). Even with proteins that share structural similarity or complexity, there can be a high degree of specificity. We now identify the K ATP channel as another Hsp90 client. K ATP channels are unique in that they are heterooligomers of SUR1 and Kir6.2-two membrane proteins with very different topological complexity (Inagaki et al., 1995;Conti et al., 2001). In understanding the biogenesis of such heteromeric protein complex, one has to consider the folding and processing of both channel subunits. Our study using SUR1 and Kir6.2 mutants that are able to express at the cell surface in the absence of the other assembly partner subunit shows that the two subunits exhibit differential dependence on Hsp90 for surface expression. Although surface levels of SUR1 are reduced by 17-AGG and augmented by overexpression of COS cells were cotransfected with fSUR1 and Kir6.2 subunits and either 0.6 g Hsp90 (␣ or ␤) or empty vector as control. Surface expression of K ATP channels was analyzed by chemiluminescence assays 48 -72 h after transfection. In cells cotransfected with Hsp90␤, channel surface expression levels were significantly higher (29.65 Ϯ 6.29%) than cells cotransfected with either empty vector or Hsp90␣. *p Ͻ 0.0003; N.S., not significant. (B) Western blot of Hsp90 from COS cells transfected with 0, 0.3, or 0.6 g of Hsp90␤ cDNA (per 35-mm culture dish). (C) Metabolic pulse-chase experiments were performed in COS cells transfected with K ATP channel subunits and Hsp90␤ or empty vector to assess channel maturation. Cells were pulse-labeled with [ 35 S]methionine for 1 h and chased for the indicated times, and fSUR1 was immunoprecipitated using anti-FLAG antibodies. An autoradiograph shows the changes in the signal of fSUR1 mature upper band and the core-glycosylated immature lower band over the 7-h chase period. The signal of the upper and lower band was quantified using a PhosphorImager and is expressed as percentage of signal of total signal observed at time 0 for the empty vector-transfected control sample (n ϭ 7 for each data point). The Hsp90␤-transfected samples showed significantly higher upper band signal at 3 and 7 h (p ϭ 0.02 and 0.03, respectively); the signal was also higher for the Hsp90␤ sample at 5 h, although the difference did not reach statistical significance (p ϭ 0.07).

Figure 5.
Hsp90 improves surface expression of select misprocessed mutant K ATP channels. The effect of Hsp90␤ abundance on surface expression of K ATP channels harboring SUR1 mutations known to disrupt normal processing and trafficking of the channel was assessed by chemiluminescence assays in COS cells cotransfected with various K ATP channel subunits (0.3 g of WT or mutant fSUR1, 0.2 g of Kir6.2 per 35-mm dish of cells) and either 0.6 g of Hsp90␤ or empty vector as control. Although Hsp90␤ improved surface expression of the N24K, D310N, and D1472N mutant (p ϭ 0.01, 0.01, 0.05, and 0.03 for WT, N24K, D310N, and D1472N, respectively), it did not significantly increase surface expression of the A116P or ⌬F1388 mutants.
Hsp90␤, those of Kir6.2 are not affected. These findings are consistent with the coimmunoprecipitation results obtained in COS cells in which the Hsp90 complex proteins were only detectable in the SUR1 but not Kir6.2 immunoprecipitates. To our knowledge, this is the first example in which differential dependences of individual components of a membrane protein complex on Hsp90 has been documented.
Identification of SUR1 as an Hsp90 client protein also underlines the question of how substrate specificity for this molecular chaperone is determined (Pearl and Prodromou, 2000). Although Hsp90 is important for the folding and maturation of CFTR and SUR1, it does not seem critical for the processing and maturation of two other ABC transporters, P-glycoprotein and MRP (Loo et al., 1998). In terms of sequence homology, P-glycoprotein belongs to the ABCB subfamily, whereas CFTR, SUR1, and MRP are in the ABCC subfamily (Vasiliou et al., 2009). With respect to membrane topology, CFTR and P-glycoprotein both contain a tandem repeat of six transmembrane helices, each set followed by an ATP-binding domain. SUR1 and MRP, in contrast, have an additional N-terminal transmembrane domain of five transmembrane helices that precedes the ABC core structure seen in the CFTR (Tusnady et al., 2006). Thus, prediction of Hsp90 substrates cannot be made simply based on similarity in sequence or topology. Future structure-function analyses of these closely related proteins may reveal features recognized by the Hsp90 chaperone complex.
Many cochaperone proteins interact with Hsp90 to regulate client protein folding and maturation. For example, Aha-1 is a cochaperone that stimulates the ATPase activity of Hsp90 and has been shown to play a role in CFTR folding. Knocking down Aha-1 by RNA interference rescued the trafficking defect caused by ⌬F508 (Wang et al., 2006). Another cochaperone, FK506-binding protein of 38-kDa (FKBP8; also called FKBP38), has been reported as a cochaperone of both CFTR and hERG potassium channels and knockdown of FKBP8 caused a reduction of processing and trafficking for both hERG and CFTR (Wang et al., 2006;Walker et al., 2007). Curiously, we did not observe Aha-1 or FKBP8 associated with wild-type K ATP channels in our proteomics experiments, and further studies are needed to determine whether these cochaperones are involved in the folding/processing of wild-type and mutant K ATP channels in INS-1 cells. Western blots showed no difference in steady-state Kir6.2 protein expression level between control and 17-AGG-treated group. (D) Chemiluminescence assays performed using cells expressing the HA-Kir6.2⌬C36 mutant construct (as Kir6.2 does not traffic to the cell surface in the absence of SUR1) showed no significant difference between control and 17-AGG-treated group (n ϭ 3).

Figure 7.
Overexpression of Hsp90 increases surface expression of fSUR1 RKR3 AAA but not HA-Kir6.2⌬C36. (A) COS cells were transfected with fSUR1 RKR3 AAA and 0.6 g of either Hsp90␣, Hsp90␤, or empty vector (as control). Surface expression of fSUR1 RKR3 AAA was analyzed by chemiluminescence assays by using anti-FLAG antibody. In cells overexpressing Hsp90␤, surface expression levels of fSUR1 RKR3 AAA were significantly higher (29.52 Ϯ 9.87%) than cells cotransfected with either empty vector or Hsp90␣. *p ϭ 0.02; N.S., not significant. (B) Same as A except that surface expression of HA-Kir6.2⌬C36 was examined using anti-HA antibody. No difference was observed between the control and Hsp90␣-or Hsp90␤cotransfected cells.

Role of Hsp90 in Modulating Surface Expression of Misprocessing/Mistrafficking K ATP Channel Mutants
Studies of mutations identified in patients with congenital insulin secretion disease have shown that many SUR1 and Kir6.2 mutations affect channel function by preventing normal processing and trafficking of the channel (Ashcroft, 2005;Lin et al., 2006;Yan et al., 2007). In the present study, we have examined five such SUR1 mutations identified in congenital hyperinsulinism. Interestingly, only mutants that are considered to have mild processing/trafficking defects showed improved surface expression upon overexpression of Hsp90. Because Hsp90 is thought to act on substrates at a late stage of folding, it is possible that the A116P and ⌬F1388-SUR1 mutations render folding difficulties at an early stage that cannot be overcome by upregulation of Hsp90 function. Examination of more disease mutations that disrupt channel processing and trafficking to different degrees will further test this idea. Given our results, we speculate that the Hsp90 chaperone complex may play a role in modulating disease phenotype by modulating the expression of mutant channels at the cell surface. We have shown previously that many disease mutations disrupt both channel gating and biogenesis/surface expression (Lin et al., 2006;Yan et al., 2007;Pratt et al., 2009). In this case, the expression level of a mutant will modulate the severity of ␤-cell dysfunction by modulating the total channel functional output (Lin et al., 2006). It is conceivable that changes in Hsp90 expression under different physiological or pathological conditions could affect manifestation of certain disease mutations. This could potentially help explain why disease symptoms can vary among patients carrying the same mutations (Ashcroft, 2005).

Effects of the Hsp90␤ Inhibitor 17-AGG on K ATP Channels
In recent years, Hsp90 inhibitors such as 17-AGG have shown great promise in treating cancer by targeting oncogenic kinases for degradation (Solit and Rosen, 2006). Our study suggests that the use of these drugs may reduce the number of K ATP channels to alter glucose-stimulated insulin secretion response in pancreatic ␤-cells. It is also worth noting that although 17-AGG has little effect on K ATP channel gating at concentrations below 100 nM (those used in Figure 3), it does inhibit channel response to the stimulatory effect of Mg-nucleotides when applied to the cytoplasmic face of inside-out patches at concentrations above 200 nM (Pratt and Shyng, unpublished). Because K ATP channel stimulation by Mg-nucleotides is mediated by the NBDs of SUR1, 17-AGG may disrupt Mg-nucleotide binding or hydrolysis at NBDs to alter channel gating. Thus, 17-AGG at high concentrations probably targets ATPases other than Hsp90 (Chene, 2002). In this respect, functional data obtained with the use of 17-AGG need to be viewed with biochemical data and interpreted with caution. Recently, it has been reported that 17-AGG partially rescues the processing defect of a disease-causing aquaporin-2 mutation, although the precise mechanism of rescue is unknown (Yang et al., 2009). We, too, tested whether 17-AGG has a salutary effect on the five SUR1 trafficking mutants but saw no effects.
In summary, we have demonstrated in this study that Hsp90 plays a role in the biogenesis of SUR1/Kir6.2 K ATP channels, in particular the maturation of the SUR1 subunit. Alterations in Hsp90 expression or function affect the number of K ATP channels in the plasma membrane. Up-regulation of Hsp90 alleviates the processing/trafficking defects in some but not all disease-associated SUR1 mutations. Future studies examining the effect of overexpression or knock-down of other chaperones and cochaperones on the biogenesis of wild-type and mutant channel proteins will probably shed additional light on the molecular and structural mechanisms of channel folding and processing. Besides the SUR1/Kir6.2 K ATP channel subtype, several other K ATP channel subtypes containing the SUR2 isoforms are expressed in cardiac, vascular, and skeletal muscle tissues where they play important roles in linking metabolic changes to muscle activities (Minami et al., 2004). It would be important to determine whether Hsp90 similarly affects biogenesis and expression of these channels by interacting with the SUR2 protein.