Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels

Fei-Fei Yan, Chia-Wei Lin, Etienne A. Cartier, and Show-Ling Shyng

Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, Portland, Oregon

Submitted 18 May 2005 ; accepted in final form 24 June 2005

ABSTRACT

ATP-sensitive potassium (KATP) channels of pancreatic {beta}-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane KATP channels determines the sensitivity of {beta}-cells to glucose stimulation. The KATP channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of KATP channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of KATP channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic {beta}-cell line, INS-1, that express endogenous KATP channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of {beta}-cell KATP channels.

endoplasmic reticulum-associated degradation; sulfonylurea receptor-1; Kir6.2; proteasome inhibitors


THE PRIMARY FUNCTION of pancreatic {beta}-cells is to maintain glucose homeostasis by releasing insulin when the blood glucose level rises. Key to this glucose-insulin secretion coupling process is a membrane protein complex called the ATP-sensitive K+ (KATP) channel. KATP channels link plasma glucose levels to the insulin-secreting machinery by virtue of their sensitivities to intracellular nucleotides ATP and ADP, whose levels fluctuate as a result of glucose metabolism (1, 4). ATP promotes channel closure, whereas Mg2+-complexed ADP promotes channel opening. In this way, they translate metabolic signals to electrical signals, which in turn control insulin secretion. The extent to which KATP channels control {beta}-cell membrane potential is critically dependent on the level of channel expression at the cell surface. Reduced expression leads to inability of the {beta}-cell to shut down insulin secretion when plasma glucose falls (8, 23, 35, 42), whereas increased expression is expected to stabilize the cell near its resting membrane potential and raise the concentration of glucose required to elicit an insulin response.

Biogenesis of the {beta}-cell KATP channel, defined here as formation of functional channel complexes that properly traffic from the endoplasmic reticulum (ER) to the plasma membrane, requires initial coassembly of the inwardly rectifying potassium channel (Kir6.2) and the sulfonylurea receptor-1 (SUR1) into an octamer in a 4:4 stoichiometry (2, 3, 24). Upon successful assembly in the ER, the channel travels through the Golgi, where N-linked glycosylation of SUR1 is modified before reaching the plasma membrane (12, 43). Several molecular signals built into the channel proteins have been implicated in monitoring proper folding, assembly, and trafficking of the channel. These include two N-linked glycosylation sites in SUR1, a tripeptide-RKR-ER retention/retrieval motif present in both SUR1 and Kir6.2, as well as a putative forward trafficking signal present in the COOH terminus of SUR1 (14, 32, 43). Deletion of the N-linked glycosylation sites or the COOH terminal putative forward trafficking signal in SUR1 dramatically reduce channel expression at the cell surface (14, 32), whereas inactivation of the RKR retention/retrieval motif in SUR1 or Kir6.2 leads to surface expression of unassembled individual channel subunits or partially assembled channel complexes that are physiologically nonfunctional (37, 43). Aside from these molecular signals, the cellular events that might influence the efficiency of channel biogenesis and thereby the level of channel expression at the cell surface, remain largely unexplored.

Recent studies suggest that the biogenesis efficiency and/or surface expression of certain ion channels, including aquaporin, connexin, and ACh receptor (AChR), can be modulated via the ubiquitin-proteasome pathway (10, 26, 39), which plays a major role in ER-associated degradation (ERAD) of proteins that fail to fold or assemble properly (6, 11, 20). In this work, we sought to determine whether the ubiquitin-proteasome degradation pathway contributes to the biogenesis and surface expression efficiencies of KATP channels. We found in COS cells that both Kir6.2 and SUR1 are polyubiquitinated and that degradation of both channel subunits is slowed by proteasome inhibitors. Importantly, inhibition of proteasome function leads to an increase in the number of surface KATP channels, both in COS cells and in a rat pancreatic {beta}-cell line, INS-1, that expresses endogenous KATP channels. Our results suggest that degradation of nascent KATP channel subunits by the ubiquitin-proteasome pathway plays a role in setting the biogenesis efficiency, and thereby the level of surface expression, of KATP channels in pancreatic {beta}-cells.

MATERIALS AND METHODS

Expression constructs. FLAG epitope (DYKDDDDK)-tagged hamster SUR1 cDNA (referred to as fSUR1 hereinafter) in the pECE plasmid was constructed as described previously (8). Rat Kir6.2 cDNA (in pcDNA3) and hamster SUR1 cDNA with a V5 epitope (GKPIPNPLLGLDST) sequence added to the 3'-end (in pcDNA3.1, referred to as SUR1v5) were kindly provided by Dr. Carol A. Vandenberg.

Immunoblot analysis. COSm6 cells (referred to as COS cells hereinafter) grown in 35-mm dishes were transfected with 0.6 µg of fSUR1 (or SUR1V5) and 0.4 µg of rat Kir6.2 using FuGene6 (Roche Applied Science, Indianapolis, IN). Cells were lysed 48–72 h later in 20 mM HEPES, pH 7.0, 5 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40 (IGAPEL) with protease inhibitors (Complete; Roche Applied Science, Indianapolis, IN). Proteins in the cell lysate were separated by SDS-PAGE (7.5% for SUR1 and 12% for Kir6.2), transferred to nitrocellulose membrane, and analyzed by incubation with appropriate primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ), and visualized using enhanced chemiluminescence (Super Signal West Femto; Pierce, Rockford, IL). The primary antibodies used were M2 mouse monoclonal anti-FLAG antibody for fSUR1 (Sigma, St. Louis, MO), mouse monoclonal anti-V5 for SUR1V5 (Invitrogen, Carlsbad, CA), rabbit polyclonal anti-Kir6.2 for Kir6.2, and mouse monoclonal anti-ubiquitin for ubiquitin (both from Santa Cruz Biotechnology, Santa Cruz, CA).

Metabolic labeling and immunoprecipitation. COS cells were transfected with SUR1 and Kir6.2 as described above. Forty-eight hours posttransfection, the cells were starved for 30 min in methionine/cysteine-free DMEM (Invitrogen) supplemented with 5% dialyzed FBS and 2 mM glutamine before being labeled for 30 min with 150–250 µCi/ml Trans35S-Label (MP Biomedicals, Irvine, CA) in the same medium. Labeled cultures were chased for various times in regular medium (DMEM plus 10% fetal bovine serum) supplemented with 10 mM L-methionine. At the end of the chase, cells were lysed in 500 µl of lysis buffer (50 mM Tris·HCl, pH 7.0, 150 mM NaCl, and 1% IGAPEL, with Complete protease inhibitors) on ice for 30 min. To ensure solubilization of channel proteins that might have aggregated as a result of proteasome inhibitor treatment, 1% SDS was included in the lysis buffer, which was later diluted (to 0.1% SDS) before immunoprecipitation. The cell lysate was centrifuged at 16,000 g for 5 min at 4°C, and the supernatant was immunoprecipitated by incubation with 10 µg anti-FLAG M2 antibodies (or 1 µl of anti-V5 antibody or 1 µl of anti-Kir6.2 antibody) per sample for 2 h at 4°C and then with protein A-Sepharose 4B (~2 mg/sample; Bio-Rad, Hercules, CA) for 2 h at 4°C. The precipitate was washed three times in lysis buffer, and proteins were eluted by incubation in SDS sample buffer at room temperature for 10 min. Eluted proteins were separated by SDS-PAGE, and the gel was subjected to fluorography. Protein bands were quantified using a Storm PhosphorImager (Amersham Biosciences).

Chemiluminescence assay. COS cells in 35-mm dishes were fixed with 2% paraformaldehyde for 20 min at room temperature 48–72 h posttransfection. Fixed cells were preblocked in PBS + 0.1% BSA for 1 h, incubated in M2 anti-FLAG antibody (10 µg/ml) for 1 h, washed four times for 30 min each time in phosphate-buffered saline (PBS) + 0.1% bovine serum albumin (BSA), incubated in horseradish peroxidase-conjugated anti-mouse secondary antibodies (Amersham, 1:1,000 dilution) for 20 min, washed again four times for 30 min each time in PBS +0.1% BSA, and twice for 5 min each time in PBS. Chemiluminescence signal was read in a TD-20/20 luminometer (Turner Designs) after 10-s incubation in Power Signal ELISA luminol solution (Pierce). For measuring channel internalization rate, cells were preincubated with anti-FLAG antibody at 4°C for 30 min to label surface fSUR1. After a brief washing, the cells were returned to 37°C to allow protein trafficking to resume for various times. Residual surface label at each time point was then determined by following the standard chemiluminescence assay protocol described above. The results of each experiment are the average of 2–3 dishes, and unless specified, each data point shown in figures is the average of 3–5 independent experiments.

Surface biotinylation and detection of biotinylated fSUR1. COS cells transiently expressing fSUR1 and Kir6.2 were washed twice with ice-cold HEPES buffer (20 mM HEPES, 150 mM NaCl, 1.8 mM CaCl2, pH 7.4). Biotinylation of surface proteins was carried out by incubating cells with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce) in HEPES buffer for 30 min on ice. The reaction was terminated by incubating cells for 5 min with HEPES buffer containing 20 mM glycine, followed by three washes in ice-cold PBS. Cells were either lysed immediately in Triton lysis buffer (20 mM HEPES, 125 mM NaCl, 4 mM EDTA, 1 mM EGTA, and 1% Triton X-100, pH 7.4, for 30 min at 4°C with rotation) or incubated further at 37°C in regular culture medium for another 30 or 60 min (to monitor surface channel stability) before being lysed. Cell lysate was cleared by centrifugation at 21,000 g for 20 min at 4°C, and biotinylated proteins were pulled down by incubation with Neutravidin-agarose beads (Pierce) for 3 h at 4°C with rotation. The beads were washed three times with lysis buffer, and proteins were eluted by incubation with SDS sample buffer containing 2.5% {beta}-mercaptoethanol for 30 min at room temperature. Eluted proteins were then separated by SDS-PAGE, and fSUR1 was detected using Western blot anti-FLAG antibody. In surface biotinylation experiments in INS-1 cells expressing endogenous KATP channels, we used similar procedures with the following modifications. INS-1 cells clone 832/13 [a gift from Dr. Chris Newgard (21)] were cultured in RPMI 1640 with 11.1 mM D-glucose (Invitrogen), supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM {beta}-mercaptoethanol. Surface protein biotinylation and pull down was carried out at 4°C as described above. Because of the absence of good antibodies for endogenous SUR1, we have used anti-Kir6.2 antibody to detect surface biotinylated KATP channel complex. Note that in this case, surface Kir6.2 itself is not biotinylated due to lack of extracellular lysine in the protein, but it is pulled down by Neutravidin beads via association with biotinylated surface SUR1. In control experiments where cells were not surface biotinylated, no Kir6.2 was detected, confirming that the Kir6.2 signal as shown in Fig. 5B was indeed surface Kir6.2.



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Fig. 5. Endogenous KATP channels in INS-1 cells are also subject to regulation by the ubiquitin-proteasome system. A: endogenous Kir6.2 expressed in INS-1 cells is polyubiquitinated. INS-1 cells treated with or without 10 µM MG132 for 3 h were lysed, and Kir6.2 was immunoprecipitated. The immunoprecipitate was probed using Western blot analysis with anti-Kir6.2 antibody (left) or anti-ubiquitin antibody (right). B: proteasome inhibitors increase the level of surface Kir6.2. In these experiments, surface KATP channel complexes were biotinylated (via the SUR1 subunit, see MATERIALS AND METHODS) and pulled down by Neutravidin-agarose beads. Kir6.2 was then detected using Western blot analysis. Results from two independent experiments are shown. In both experiments, cells treated with MG132 exhibited more surface Kir6.2 (shown is the monomeric form; arrow) than control cells. In both A and B, molecular mass markers (in kDa) are indicated to the left of the blots.

 
Whole cell patch-clamp recording. INS-1 cells were plated onto coverslips. Micropipettes were pulled on a horizontal puller (Sutter Instrument, Novato, CA). The pipette solution consisted of 140 mM KCl, 10 mM K+-HEPES, 1 mM K+-EGTA, and 1 mM MgCl2, pH 7.3. Recordings were made at room temperature with cells perfused with Tyrode solution composed of (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5 HEPES, 3 NaHCO3, and 0.16 NaH2PO4. Giga-ohm seals were formed before breakin, after which the cell was held at –75 mV and stimulated every 2 s with alternate ±10-mV pulses, each for 100 ms, to monitor the current. KATP currents were derived by subtracting the steady-state K+ current observed in 500 µM tolbutamide from that observed in 250 µM diazoxide. Whole cell KATP current was then normalized to cell surface area (measured as cell capacitance) to obtain the KATP current density, represented as pS/pF. In pharmacological experiments, cells received drug treatment 4–6 h before being subjected to patch-clamp recording.

RESULTS

KATP channel subunits are polyubiquitinated. Most membrane and secretory proteins that fail to mature or assemble properly in the ER are degraded through an ER-associated degradation process known as ERAD (6). Typical ERAD substrates are tagged by polyubiquitination, retrotranslocated into the cytosol, and targeted to the proteasome for degradation (11, 20, 28, 41), although exceptions that appear to follow a proteasome-independent pathway have been reported (7, 17, 29). To test whether the ubiquitin-proteasome pathway is involved in degradation of nascent KATP channel subunits, we first examined whether SUR1 and Kir6.2 transiently expressed in COS cells are polyubiquitinated. We have chosen to use a SUR1 tagged with a FLAG epitope at its extracellular NH2 terminus (referred to as fSUR1) for these experiments to facilitate detection; the construct has been previously shown to give rise to channels with properties indistinguishable from wild-type (WT) channels (8). The fSUR1 or Kir6.2 expressed in COS cells were subject to immunoprecipitation using anti-FLAG or anti-Kir6.2 antibodies (see MATERIALS AND METHODS), the precipitated proteins were then analyzed using Western blot analysis with antibodies against the channel proteins or ubiquitin. In COS cells expressing fSUR1 alone, ubiquitin labeling that appeared as a high molecular weight "smear" was already apparent (Fig. 1A, right). If the ubiquitinated SUR1 is targeted to degradation in the proteasome, then treating cells with proteasome inhibitors, such as lactacystin or MG132, may lead to accumulation of polyubiquitinated SUR1. Indeed, a more intense signal in the anti-ubiquitin blot was observed in cells treated with MG132 (10 µM, 4 h). Similar observations were made in cells coexpressing fSUR1 and Kir6.2 (Fig. 1A). Likewise, Kir6.2 was also found polyubiquitinated, either when expressed alone or together with fSUR1. In the anti-ubiquitin blot of Kir6.2 immunoprecipitates, discrete bands, which likely represent Kir6.2 with different numbers of ubiquitin moieties attached, can be discerned (Fig. 1B). These results demonstrate that both SUR1 and Kir6.2 are substrates for polyubiquitination, like most proteins that are targeted for proteasome-mediated ERAD. Note that in this and subsequent experiments, the results concerning fSUR1 have been confirmed using a SUR1 with a V5 tag placed at the cytoplasmic COOH terminus (SUR1V5). This is to ensure that any conclusion we draw is not dependent on the position and/or the nature of the epitope tag, although for space considerations only one construct is described or shown in the figures.



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Fig. 1. ATP-sensitive K+ (KATP) channel subunits expressed in COS cells are substrates for polyubiquitination. Untransfected COS cells, cells expressing either sulfonylurea receptor-1 (fSUR1) or inwardly rectifying K+ channel 6.2 (Kir6.2), or coexpressing both subunits were lysed, and fSUR1 or Kir6.2 was immunoprecipitated (IP) using the M2 anti-FLAG antibody or anti-Kir6.2 antibody. The immunoprecipitate was then probed by immunoblotting (IB) using antibodies against the channel protein or against ubiquitin. A: cell lysates immunoprecipitated with anti-FLAG (for fSUR1) and immunoblotted with anti-FLAG (left) or anti-ubiquitin (Ub, right). Solid arrow indicates the core-glycosylated fSUR1, and the open arrow indicates the complex-glycosylated form. Some higher molecular weight forms of fSUR1 that may represent polyubiquitinated fSUR1 or fSUR1 aggregates are shown in the anti-FLAG blot. B: cell lysates immunoprecipitated with anti-Kir6.2 and immunoblotted with anti-Kir6.2 (left; Kir6.2 indicated by the arrow) or anti-ubiquitin (right). In the anti-Kir6.2 blot, high molecular mass bands near 80 and 160 kDa are observed. These likely represent Kir6.2 dimers and tetramers as reported previously by others (31, 43). Note discrete bands representing different copies of ubiquitin can be discerned more easily for Kir6.2 than for SUR1, likely because the 12% gel used for Kir6.2 gave better resolution in the given molecular mass range. Also, in both A and B, cells treated with the proteasome inhibitor MG132 (10 µM for 4 h) exhibited enhanced signals in the anti-ubiquitin blots, especially for Kir6.2.

 
Proteasome inhibitors slow degradation rate of KATP channel proteins. To further demonstrate that proteasome is involved in degradation of nascent KATP channel subunits, we examined the effects of proteasome inhibitors on degradation of SUR1 using pulse-chase analysis. In cells expressing SUR1V5 (or fSUR1) alone, treatment of cells with 10 µM MG132 throughout the pulse-chase period significantly slowed the degradation of the protein (Fig. 2A). The amount of labeled SUR1V5 increased by ~30% at the end of 1-h chase. This initial increase is expected because the partially translated, labeled SUR1V5, which would have been degraded during the first hour of chase, is now allowed to complete translation due to inhibition of degradation and join the pool of labeled full-length SUR1V5. Similarly, degradation of Kir6.2 expressed alone in COS cells was significantly slowed by proteasome inhibitors (Fig. 2B). Note that for Kir6.2, cells were pretreated for 2 h with 10 µM MG132 before the pulse-chase analysis so that any effects of MG132 would not be missed, because degradation of Kir6.2 is more rapid. We also performed experiments where SUR1 and Kir6.2 were coexpressed. MG132 again slowed the overall degradation rates of newly synthesized channel subunits (see below; the degradation profile of fSUR1 is shown in Fig. 4A). Together, these data demonstrate that nascent KATP channel subunits are subject to degradation by the ubiquitin-proteasome pathway.



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Fig. 2. Proteasome inhibitors slow the degradation of SUR1 and Kir6.2. A: degradation of SUR1 in the presence of MG132. COS cells transiently expressing SUR1V5 were subject to pulse-chase analysis. In the drug-treated group, 10 µM MG132 was added to the medium at the time of the pulse and was present throughout the chase. Top, fluorogram of a representative experiment. Bottom, residual label during chase quantified by phosphorimaging. Note the signal increased transiently at the beginning of the chase before decreasing with time. The residual label at each time point is higher in MG132-treated cells than in control cells. Similar results were obtained using fSUR1 (not shown). B: degradation of Kir6.2 in the presence or absence of 10 µM MG132. Top, fluorogram of a representative experiment. Bottom, residual label during chase quantified by phosphorimaging. Note for Kir6.2, cells were pretreated with 10 µM MG132 for 2 h before the pulse-chase analysis. For both SUR1 and Kir6.2, only bands corresponding to monomeric form of the proteins (as opposed to oligomeric forms; see Fig. 1B) were included for quantification. Each data point is the mean ± SE of 3–4 independent experiments. {blacksquare}, control group; {circ}, MG132-treated group.

 


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Fig. 4. Proteasome inhibitors promote biogenesis of KATP channels without affecting the stability of surface channels. A: COS cells transfected with fSUR1 and Kir6.2 were pretreated with or without 10 µM MG132 for 1 h, pulse labeled for 30 min with 35S-Cys/Met and chased for the times indicated in the presence or absence of 10 µM MG132. Labeled fSUR1 were immunoprecipitated and analyzed by SDS-PAGE, followed by fluorography (left, a representative experiment). The core- and complex-glycosylated fSUR1 bands are indicated by the solid and open arrows, respectively. The amount of total (both top and bottom bands; dotted line) fSUR1 as well as the upper band fSUR1 (solid line) present at each time point was quantified and expressed as percentage of total protein labeled at time 0 and plotted over time (right). In cells treated with MG132, a higher percentage of immature core-glycosylated fSUR1 was converted to the mature form, indicating increased channel biogenesis efficiency. B: stability of surface KATP channels. Left, Western blots of residual surface biotinylated fSUR1 detected at 0, 30, and 60 min of chase. Right, stability of surface KATP channels monitored by chemiluminescence assays. In these experiments, surface channel complexes in control cells or cells pretreated with 10 µM MG132 for 4 h were labeled at 4°C with anti-FLAG antibody and chased at 37°C for 10 or 20 min. Residual surface FLAG-epitope at each time point was quantified by the chemiluminescence assay and normalized to that observed in control cells at time 0. Note in cells treated with MG132, the initial label was higher than that in control cells, consistent with that shown in Fig. 3B. All data points are the means ± SE of 3–5 independent experiments.

 
Inhibition of proteasome function increases surface expression of KATP channels by increasing channel biogenesis efficiency in COS cells. Although a general function of ERAD is to remove unwanted proteins in the ER, recent studies (20, 26, 39) show that it can play a regulatory role in protein expression by controlling the degradation of WT proteins. Having established that newly synthesized KATP channel proteins undergo ERAD via the ubiquitin-proteasome pathway, we sought to determine whether inhibition of proteasome function would increase surface expression of KATP channels. To monitor channel expression at the cell surface, we first performed surface biotinylation experiment in which cell surface proteins were biotinylated using a membrane-impermeable biotinylation reagent Sulfo-NHS-SS-biotin, biotinylated proteins were then pulled down using Neutravidin-agarose beads, and fSUR1 detected using Western blot analysis with anti-FLAG antibody. As shown in Fig. 3A, the signal of surface biotinylated fSUR1 is stronger in cells treated with MG132 (10 µM for 4 h) than that in control cells, indicating increased channel expression at the cell surface. To better quantify and characterize the effects of proteasome inhibitors, we used a chemiluminescence assay that is much more sensitive than biochemical assays (8, 27). Exposure of COS cells to the proteasome-specific inhibitor MG132 (150 nM or 10 µM) or lactacystin (6 µM) (19) for 6 h increased surface expression of the channel by ~30–40% (Fig. 3B). In contrast, incubation of cells with lysosomal inhibitors (chloroquine at 100 µM or NH4Cl at 20 mM) or the calpain inhibitor N-acetyl-Leu-Leu-Met (ALLM; 10 or 100 µM) had no or little effect on surface expression.



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Fig. 3. Proteasome inhibitors increase surface expression of KATP channels in COS cells. A: biotinylated surface fSUR1 in control cells and cells treated for 6 h with 10 µM MG132. Duplicates are shown for each group. In both cases, cells treated with MG132 exhibited more surface fSUR1 than control cells. Note only one form of fSUR1, the complex-glycosylated form (open arrow), was detected, because the biotinylation reagent was membrane impermeable and reacted only with surface proteins. B: COSm6 cells transiently expressing KATP channels were treated with the indicated reagents for 6 h and then were processed for chemiluminescence assays to quantify channel expression level at the cell surface. The proteasome inhibitors, lactacystin (6 µM) and MG132 (0.15 or 10 µM) both significantly increased surface channel expression (n = 4, *P < 0.01 by Student's t-test). By contrast, inhibition of lysosomal function by chloroquine (100 µM) or NH4Cl (20 mM) or inhibition of the protease calpain by ALLM (10 or 100 µM; only 100 µM is shown) had little effect.

 
Treatment with proteasome inhibitors could potentially increase surface expression of KATP channels by stabilizing channels in the plasma membrane, as has been observed for {beta}-adrenergic receptors, growth hormone receptors, and AMPA-type glutamate receptors (13, 33, 38), or by promoting channel biogenesis, as seen in AChRs (10). To distinguish between these possibilities, we performed metabolic pulse-chase experiments in cells coexpressing fSUR1 and Kir6.2 to follow the efficiency at which nascent fSUR1 is converted to the mature complex-glycosylated form (upper band), which reflects the efficiency of channel biogenesis. In these experiments, MG132 treatment began 1 h before the pulse chase to ensure that the inhibitor had sufficient time to enter the cell and exert its effect. As expected, the overall degradation rate of fSUR1 was slowed in MG132-treated cells (similar results were obtained for Kir6.2; not shown). Importantly, the percentage of initially labeled fSUR1 that was converted to the upper band was consistently higher (~10%) than in control cells (Fig. 4A). In calculating the efficiency of conversion, the upper-band fSUR1 signal in control or drug-treated cells was divided by the initial label of the respective groups, thus correcting for potential effects of MG132 at the pretranslational or translational level. We next compared the stability of surface channels in control cells and cells treated with proteasome inhibitors. In surface biotinylation pulse-chase experiments, the signal of surface fSUR1 in both control cells and cells pretreated with MG132 (10 µM for 4 h) decreased rapidly in a 60-min chase period with similar rates (Fig. 4B, left). To better quantify the rate of surface channel degradation, we performed chemiluminescence assays using a similar pulse-chase experimental paradigm. Consistent with the surface biotinylation results, the signal of FLAG-antibody labeled channels decreased to ~50% of the initial value in 20 min, and no significant difference was observed between control and MG132-treated cells (Fig. 4B, right). Taken together, the above results led us to conclude that proteasome inhibitors increase surface expression of KATP channels by increasing the biogenesis efficiency of the channel without affecting the stability of surface channels.

Endogenous KATP channel subunits are also affected by the ubiquitin-proteasome pathway. The studies described thus far were conducted on KATP channels heterologously expressed in COS cells where protein overexpression might be a concern. To determine whether ubiquitin-proteasome pathway-mediated ERAD also plays a role in the biogenesis efficiency of native KATP channel subunits expressed in insulin-secreting cells, we performed experiments using a rat {beta}-cell line, INS-1E clone 832/13, which closely resembles native {beta}-cells in glucose-stimulated insulin secretion response (21). To first confirm that endogenous channel subunits are also substrates for polyubiquitination, Kir6.2 in INS-1 cells was immunoprecipitated and its ubiquitination was probed using immunoblotting with an anti-ubiquitin antibody. To enhance the signal of ubiquitinated Kir6.2, cells were treated with the proteasome inhibitor MG132 (10 µM for 3 h), which allows accumulation of polyubiquitinated proteins. As shown in Fig. 5A, polyubiquitinated Kir6.2 was already detectable in control cells, and proteasome inhibitors dramatically increased this signal, implicating involvement of the ubiquitin-proteasome pathway in degradation of endogenous KATP channels. We next examined whether proteasome inhibitors also affect surface expression of the channel in INS-1 cells by probing the amount of surface Kir6.2 that could be pulled down by Neutravidin-agarose beads via association with biotinylated surface SUR1 (see MATERIALS AND METHODS). With the use of this approach, we found the level of surface Kir6.2 to be consistently higher in cells treated with MG132 (10 µM for 3 h) than in control cells, demonstrating that inhibition of proteasome function also increases surface expression of endogenous KATP channels.

Inhibition of proteasome function increases KATP current density in INS-1 cells. To better quantify the effect of proteasome inhibitors on surface channel expression in INS-1 cells, we employed a functional assay that measures KATP current density by whole cell patch-clamp recordings because no antibodies that recognize the extracellular domain of native SUR1 or Kir6.2 are currently available to allow quantification by chemiluminescence assays. Increased surface channel expression is expected to result in increased KATP currents. To elicit maximal KATP currents, cells were dialyzed with zero ATP pipette solution and perfused with 250 µM of the KATP channel opener diazoxide. Under this condition, the current gradually increased to a maximal level (usually ~80–90 s after breakin; Fig. 6A). The maximal K+ current seen in zero ATP and the presence of diazoxide was taken as the KATP current after subtraction of background K+ currents obtained by perfusing cells with 500 µM of the KATP channel inhibitor tolbutamide. This whole cell KATP current was then divided by the whole cell capacitance to obtain current density, which reflects the density of KATP channels in the plasma membrane. The averaged data from a total of 12–13 recordings from three independent experiments show that treatment of cells for 4 h with 150 nM MG132 led to a significant increase (~40%) in KATP current density. By contrast, inhibition of lysosome function by NH4Cl (12.5 mM) or inhibition of calpain by ALLM (10 or 100 µM) had little effect on KATP current density in INS-1 cells, demonstrating the specificity of the proteasome inhibitor effect. Control experiments were performed using inside-out patch-clamp recording to ensure that MG132 did not have an acute effect on KATP channel open probability or other background K+ currents (data not shown). However, because of its documented effect on KATP channel gating, chloroquine was not used as a lysosome inhibitor in these whole cell recording experiments (18). The electrophysiological data presented above are consistent with biochemical data and support a role of the ubiquitin-proteasome degradation pathway in determining the biogenesis and surface expression efficiency of native KATP channels.



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Fig. 6. KATP current density in INS-1 cells is increased after treatment with proteasome inhibitors. A: representative whole cell recordings of KATP currents in INS-1 treated without (top) or with (bottom) MG132 (150 nM) for 4 h. The lower concentration of MG132 was used in these experiments to avoid cytotoxicity; at this concentration, cell surface channel expression was increased by ~30% in COS cells (Fig. 3B). KATP current amplitude was plotted against time of recording (in seconds). The cell was held at –75 mV, and a brief (100 ms) +10-mV pulse was applied every 2 s to monitor the current. A gradual increase in current was observed after dialysis of the cell with ATP-free solution and simultaneous perfusion of the cell with a bath solution containing 250 µM diazoxide. After the current amplitude peaked, 500 µM tolbutamide was applied in the bath solution to inhibit KATP currents and to obtain background K+ currents from non-KATP channels. B: averaged KATP current density obtained in cells treated with the various pharmacological agents. K+ currents attributed to opening of KATP channels were first derived by substracting background K+ current observed in the presence of tolbutamide from the maximal K+ current observed in the presence of diazoxide. This KATP current was then divided by the total cell capacitance to correct for variation in current amplitude caused by variation in cell surface area. The value of each bar is the mean ± SE of 12–13 cells from 3 independent experiments. Only the MG132-treated cells showed significant differences compared with control cells (*P < 0.01, Student's t-test). For ALLM treatment, both 10 and 100 µM were tested; only 100 µM is shown. All data points are expressed as means ± SE. To avoid potential variation in KATP current density due to difference in INS-1 cell passage number, the KATP current density of cells treated with pharmacological agents was normalized only to that of control cells from the same culture and the same day of recording.

 
Do proteasome inhibitors promote surface expression of mutant channels with biogenesis/trafficking defects? Several SUR1 mutations identified in the insulin secretion disease congenital hyperinsulinism have been shown to cause KATP channel biogenesis/surface expression defects (5, 8, 9, 30, 35, 36, 42). Having established that proteasome inhibitors promote the biogenesis efficiency and surface expression of WT KATP channels, we sought to determine whether inhibition of proteasome function could overcome the surface expression defects caused by disease mutations. Three SUR1 mutations, A116P, V187D, and {Delta}F1388, which we have previously shown to result in ER retention and surface expression defects of KATP channels, were tested. Treatment of cells with 10 µM MG132 for 6 h did not cause a statistically significant increase in surface expression of the mutants (P > 0.01), unlike that seen in WT channels (Fig. 7A). The result suggests that the effectiveness of proteasome inhibitors on promoting channel biogenesis/surface expression may depend on the ability of the channel proteins to fold and assemble correctly. To further test this hypothesis, we examined the combined effect of glibenclamide and MG132 on surface expression of the A116P and V187D mutants. Sulfonylureas such as glibenclamide have previously been shown to significantly increase surface expression of the A116P and V187D mutants, presumably by acting as pharmacological chaperones to help mutant SUR1 fold more efficiently (42). We found that pretreatment of COS cells expressing the A116P or the V187D mutant with 5 µM glibenclamide led to a significant increase in mutant channel surface expression (P < 0.01) on subsequent exposure to the proteasome inhibitor MG132 (Fig. 7B). These findings support the notion that the effectiveness of proteasome inhibitors on enhancing channel surface expression is correlated with the ability of the channel proteins to fold and assemble correctly.



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Fig. 7. Effects of proteasome inhibitors on surface expression of mutant KATP channels in COS cells. A: COSm6 cells transiently coexpressing Kir6.2 and wild-type (WT) fSUR1 or fSUR1 bearing the A116P, V187D, or {Delta}F1388 mutation were treated with or without 10 µM MG132 for 6 h and processed for chemiluminescence assays to quantify channel expression level at the cell surface. Each data point represents the average of 6–8 samples from 2–4 independent experiments. *P < 0.01 by Student's t-test. B: cells expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated for 24 h with (Glib) or without (Control) 5 µM glibenclamide. For cells treated with both glibenclamide and proteasome inhibitor (Glib + MG132), 10 µM MG132 was added 18 h after the initiation of glibenclamide treatment and incubated further for 6 h. All data points are the average of 6–10 samples from 3–5 independent experiments. *P < 0.01 by Student's t-test (comparison between Glib and Glib + MG132).

 
DISCUSSION

Proteins retained in the ER are degraded mostly through the ubiquitin-proteasome ERAD pathway. Whereas a generally recognized function of this pathway is to prevent mutant or unassembled proteins from accumulating, it has also been shown to play an active role in regulating expression of normal proteins. Examples include T cell-receptor complex, HMG-CoA reductase, connexin, aquaporin-1, and most recently, the nicotinic AChR (6, 10, 20, 26, 39). The data presented in this study demonstrate that nascent KATP channel subunits are subject to degradation by the ubiquitin-proteasome pathway and that inhibition of this pathway leads to significantly increased expression of KATP channels at the cell surface. Involvement of the ubiquitin-proteasome pathway in KATP channel degradation is not unique to the COS cell system, where protein overexpression might be a concern (40). Endogenous KATP channel subunits expressed in INS-1 cells are also substrates for polyubiquitination, and surface channel expression is increased by inhibition of proteasome function. On the basis of these findings, we propose that the ubiquitin-proteasome pathway-mediated ERAD of KATP channel subunits likely contributes to the biogenesis efficiency and thereby the functional expression of KATP channels in native {beta}-cells.

Multiple lines of evidence suggest that proteasome inhibitors promote KATP channel surface expression by increasing channel biogenesis efficiency rather than affecting other cellular events. First, metabolic pulse-chase experiments demonstrate directly that the efficiency of channel biogenesis, as reflected by the efficiency of which fSUR1 is converted from the core-glycosylated form to the complex-glycosylated form, is higher in cells treated with proteasome inhibitors than in control cells (Fig. 4A). Second, pharmacological agents targeting other major cellular degradation pathways, including the lysosome and the cytosolic proteases calpains, had little or no effect on surface KATP channel expression, excluding indirect effects of the proteasome inhibitors on channel expression via inhibition of other degradation pathways. Third, the stability of surface KATP channels was unaltered by proteasome inhibitors, as assessed by two independent assays (Fig. 4B). One model consistent with our data is that the biogenesis efficiency of KATP channels is a balance between the rate of channel subunit degradation by the proteasome and the rate of channel assembly. Inhibition of proteasome activity slows the degradation of nascent channel proteins, causing their accumulation in the ER, thereby shifting the equilibrium toward channel assembly. A similar mechanism has recently been proposed for the hetero-pentameric nicotinic AChRs (10). Interestingly, metabolic pulse-chase experiments suggest that the biogenesis efficiency of KATP channels in COSm6 cells is rather low under our experimental conditions (fSUR1: Kir6.2 cDNA ratio ~1). Although one might argue that the low efficiency could be due to protein overexpression, the fact that endogenous KATP channel subunits are polyubiquitinated and that surface expression of KATP channels is also increased by proteasome inhibitors in INS-1 cells lends support to the idea that there exists an excess pool of channel subunits in {beta}-cells that can be rendered available for channel assembly when their degradation via the proteasome is inhibited.

To assess whether proteasome inhibitors can overcome KATP channel surface expression defects caused by disease mutations, we examined the response of three SUR1 mutants known to abolish or reduce surface channel expression (8, 42). Among them, {Delta}F1388 has been proposed to cause severe folding defects, whereas A116P and V187D appear to have milder defects that can be partially overcome by sulfonylurea treatment. None of the mutant channels showed a statistically significant increase in surface expression upon proteasome inhibitor treatment. However, after pretreatment with glibenclamide, the A116P and V187D mutant channels responded to proteasome inhibitors with a statistically significant increase in surface expression (Fig. 7B). These results suggest that for proteasome inhibitors to be effective, the channel proteins have to be competent in adopting correct conformation. For mutant proteins that are unable to fold correctly, inhibition of proteasome function may instead render accumulation of the mutant protein either in the ER or in intracellular inclusion bodies resembling aggresomes (25), as we have observed for the {Delta}F1388 mutant (E. A. Cartier and S.-L. Shyng, unpublished observations). Interestingly, a recent study (29) found that while some mutations in the Shaker potassium channel protein led to rapid degradation via proteasome-dependent ERAD, others led to degradation that was insensitive to the proteasome inhibitor lactacystin. We cannot rule out that such a pathway may also contribute to degradation of the mutant channel proteins we have studied here, partially accounting for why proteasome inhibitors did not improve their surface expression.

While not the focus of this study, in examining the effect of proteasome inhibitors on the stability of nascent channel subunits and surface channel proteins, two results came to light that warrant further discussion because they differ from previous reports published by others. First, we found that degradation of SUR1 (both fSUR1 and SUR1V5) expressed alone in COSm6 cells is biphasic (Fig. 2A), with a fast component (~80%, half-life ~1 h) and a slow component (~20%, half-life ~18 h). This is in contrast to that reported by Crane and Aguilar-Bryan (15), who found unassembled SUR1 to be very stable, with a single half-life of ~25.5 h. Criteria used for degradation curve fitting, as well as several differences in experimental details, including epitope tag, detergent used in the lysis buffer, and the amount of cDNA used for transfection could potentially account for this apparent discrepancy. We are currently investigating these variables systematically to resolve this issue. Regardless, while the intrinsic metabolic stability of SUR1 in the absence of Kir6.2 may have implications for the channel assembly process, it does not affect the conclusion of the present study, especially considering that the effect of proteasome inhibitors is observed in INS-1 cells expressing endogenous channels. Another result worth noting is the rapid degradation of surface KATP channel proteins we observed in COSm6 cells. Both surface biotinylation experiments and chemiluminescence assays yielded similar results indicating that the half-life of surface channel proteins is ~20 min (Fig. 4B). This result is different from that reported by Hu et al. (22), who found little internalization of surface KATP channels expressed in COS-7 cells in a 30-min period using chemiluminescence assays. Difference in the cell used for transfection and the Kir6.2 construct (Hu et al. used a Kir6.2 tagged with a HA-epitope in the extracellular domain) could potentially contribute to the difference in results. Again, the absolute stability of surface channels does not affect our conclusion as long as MG132 has no effect on this stability.

Modulation of protein expression by the ubiquitin-proteasome system has been reported under certain physiological conditions. For example, aquaporin-1 water channel surface expression is increased by hypertonic stress via inhibition of protein degradation by the ubiquitin-proteasome pathway (26). Upregulation of gap junctions at the cell surface is observed when cells are challenged with mild oxidative or thermal stress, which reduces retrotranslocation of newly synthesized connexin back to the cytosol for proteasome degradation (39). Conversely, downregulation of multiple postsynaptic density proteins due to increased degradation via the ubiquitin-proteasome pathway has been reported during activity-dependent synapse remodeling (16). It will be interesting to determine in the future whether KATP channel degradation through the ubiquitin-proteasome pathway can also be modulated to alter surface channel expression in vivo during development or under stressful physiological conditions. Such modulation would be expected to alter the sensitivity of {beta}-cells to glucose stimulation and affect insulin secretion. It is worth noting that during submission of our paper, Tanaka et al. (34) reported similar upregulation of cardiac KATP channels (SUR2A/Kir6.2) in cells treated with proteasome inhibitors, suggesting that the mechanism likely applies to other KATP channel subtypes.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57699 and Juvenile Diabetes Research Foundation Grant 1-2001-707 (both to S.-L. Shyng).

ACKNOWLEDGMENTS

The INS-1E cell line clone 832/13 was kindly provided by Dr. Christopher B. Newgard. Rat Kir6.2 and V5-tagged hamster SUR1 cDNAs were gifts from Dr. Carol A. Vandenberg.

Present address for E. A. Cartier: Department of Pharmacology, MS 357280, University of Washington, Seattle, WA 98195-7280.

FOOTNOTES


Address for reprint requests and other correspondence: S.-L. Shyng, Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239 (e-mail: shyngs{at}ohsu.edu)

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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