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 -cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane KATP channels determines the sensitivity of
-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
-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
-cell KATP channels.
endoplasmic reticulum-associated degradation; sulfonylurea receptor-1; Kir6.2; proteasome inhibitors
Biogenesis of the -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 -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
-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 4872 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 150250 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 4872 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 23 dishes, and unless specified, each data point shown in figures is the average of 35 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% -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
-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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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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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 -cell line, INS-1E clone 832/13, which closely resembles native
-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 8090 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 1213 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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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 -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
-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, 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
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 -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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