Department of Medicine, University College of London, London, United Kingdom
Submitted 20 January 2005 ; accepted in final form 12 April 2005
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ABSTRACT |
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phosphatidylinositol 4,5-bisphosphate; phorbol 12-myristate 13-acetate; receptor for activated C kinase; A kinase anchoring protein; carbachol; 5'-N-ethylcarboxyamidoadenosine
In our previous work (27), we have proposed that a Ca2+-independent PKC isotype inhibits Kir3.1/3.2A channels after stimulation of Gq/11-coupled, muscarinic M3 receptors, and that subsequent activation of the channels by co-expressed Gi/o-coupled adenosine A1 receptors is prevented. In this report, we have used a combination of electrophysiological, biochemical, and imaging techniques to investigate this phenomenon further. We show that purified PKC directly inhibits channel activity when applied to excised patches and we elucidate the specific PKC isotype responsible. Furthermore, we find that PIP2 also has a prominent role in mediating channel inhibition, which is dependent on PKC activity. Many have argued that either PKC or PIP2 regulate the channel: in this study, we provide evidence for a dual regulation of the channel by both PIP2 and PKC. Crucially, we hypothesize and provide evidence for a coordinated mechanism of channel inhibition, centered on the action of PKC, after Gq/11-coupled receptor activation. Furthermore, we identify the isoform of PKC involved in channel inhibition as PKC-.
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MATERIALS AND METHODS |
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Electrophysiological recordings. Whole cell currents were recorded as previously described (25) and we also performed cell-attached and inside-out patch recordings. Currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz, digitized at 5 kHz, and data acquired with the use of a Digidata 1322A and pCLAMP8 software (Axon Instruments). Single-channel data was analyzed using Fetchan 6.0 software (Axon Instruments) to generate events lists. The mean number of open probability channel (NPo) values was determined from 30-s sweeps at the appropriate test potential in each experimental condition.
For whole cell recordings, the bath solution contained (in mM) 140 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4) and the pipette solution contained (in mM) 107 KCl, 1.2 MgCl2, 1 CaCl2, 10 EGTA, 5 HEPES, 2 MgATP, and 0.3 Na2GTP (KOH to pH 7.2, 140 mM total K+). The pipette solution used in inside-out single channel experiments was identical to the whole cell bath solution as described above, whereas the inside-out single-channel bath solution contained (in mM) 140 KCl, 2.6 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.2). For cell-attached experiments, both pipette and bath solutions were identical to whole cell bath solution. For perforated-patch recordings the pipette solution contained (in mM) 140 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4), and was supplemented with 240 µg/ml amphotericin B. Drugs were bath applied using either a gravity-fed system or a localized application system (model MSC160; Bio-Logic). Data are presented as means ± SE (unless otherwise stated), where n indicates the number of cells/patches recorded from. Data were analyzed for statistical significance using one-way repeated-measures ANOVA tests with post Bonferroni or Dunnett's test as appropriate (P
0.05, P
0.01, and P
0.001).
Purification of PKC. PKC was purified by following a method modified from Allen et al. (1). Briefly, 250300 g wt adult Sprague-Dawley rats were euthanized by cervical dislocation in accordance with Home Office Guidelines for Animals (Scientific Procedures) Act 1986. The brains were removed and the hippocampi extricated into ice-cold PBS. The brain tissue was stored in cryovials at 80°C and used within 2 wk. All subsequent procedures were carried out at 4°C. Frozen tissue was thawed on ice and homogenized using a glass-on-glass Dounce homogenizer in solution A, which contained 20 mM Tris·HCl, 10 mM dithiothreitol (DTT), 1 mM CaCl2, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). This was then centrifuged at 35,000 g for 20 min and the supernatant separated and discarded. The pellet was then resuspended in solution B (identical to solution A, except that CaCl2 was reduced to 0.1 mM) and centrifuged again at 35,000 g for 20 min. This procedure was repeated twice. The pellet was resuspended and homogenized in solution C composed of 20 mM Tris·HCl, 10 mM DTT, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM PMSF, stirred at 4°C for 1 h, and then centrifuged at 100,000 g for 60 min. The resultant supernatant was then applied to a DEAE-Sephacel column preequilibrated with 20 mM Tris·HCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, and 10% glycerol (solution D). The column was washed with solution D and proteins bound to the column eluted with a linear 00.4 M NaCl gradient collecting 5-ml fractions at a flow rate of 10 ml/h. Fractions containing kinase activity were adjusted to 2.25 mM NaCl, 2 mM DTT, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM PMSF, and applied to a second column containing preequilibrated phenyl-Sepharose. Bound proteins were eluted using a linear 1.50 M NaCl gradient at a flow rate of 5 ml/h, and 2-ml fractions were collected. Fractions were assayed for PKC activity using the PepTag kit (Promega), which allows the detection of purified and active protein kinases on the basis of separation of phosphorylated and nonphosphorylated species on an agarose gel.
Purification of G protein -subunits.
A His6 epitope was inserted at the NH2 terminus of G
1 to yield G
1-His6. This and G
2 were cloned into the pBUDCE4.1 vector and used to create stable HEK-293 cell lines. HEK-293 cells, containing G
1-His6
2 were harvested in 1x PBS, and the resulting pellet resuspended in lysis buffer A (10 mM HEPES, pH 7.4, 0.5 mM EDTA, and complete protease inhibitors) and left for 20 min on ice. One milliliter of resuspension buffer B (10 mM HEPES pH 7.4, 500 mM sucrose, 0.5 mM EDTA, and complete protease inhibitors) was added and mixed before the cells were fragmented with the use of a Dounce homogenizer. The debris was pelleted at 1,000 g for 15 min. The membranes were pelleted by ultracentrifugation (100,000 g for 60 min), followed by resuspension in buffer C (20 mM HEPES, pH 8.0, 100 mM KCl, complete protease inhibitors, and 1% CHAPS). The homogenized solution was left for 1 h on ice, followed by ultracentrifugation (100,000 g for 1 h). The resulting supernatant was mixed with preequilibrated Ni-NTA resin (Qiagen) on a rotary shaker for 2 h at 200 rpm. The material was loaded into a column and washed with buffer C + 10 mM imidazole for 8-column volumes. G
1-His6
2 was eluted with 100 mM imidazole (4 column volumes) and the resulting fractions pooled. Imidazole was removed by dialysis and the protein identified by Western blot analysis using the anti-G
antibody (Chemicon) or the anti-pentaHis antibody (Qiagen).
PKC translocation. For membrane localization experiments cells were washed in PBS and Dounce homogenized in ice-cold extraction buffer (20 mM Tris-Cl, pH 7.4, 2 µg/ml aprotinin, 100 µM tosyl-lysine-chloromethyl ketone, 1 µM pepstatin, 50 µg/ml PMSF, and 1 µg/ml diisopropylfluorophosphate). The extract was incubated at 4°C for 15 min and then centrifuged (14,000 rpm, 4°C). The supernatant was taken ("cytosol") and the pellet was reextracted as above. After clearance, the supernatant was discarded and the pellet was homogenized in extraction buffer containing 1% Triton X-100. The homogenate was incubated at 4°C for 30 min and centrifuged as above. The supernatant was taken ("membrane"). One-fifth volume of 5x Laemmli buffer was added to cytosol and membrane fractions and 10 µl of this buffer were analyzed by SDS-PAGE and Western blot analysis.
Western blot analysis. For Western blot analysis, proteins were analyzed by 9% SDS-PAGE and transferred to nitrocellulose as described (48). Nitrocellulose filters were probed and developed using the ECL Western detection system (Amersham) and quantified using ScionImage software (Scion).
Confocal fluorescence microscopy.
We used confocal microscopy to examine the translocation of green fluorescent protein (GFP)-tagged PKC- and PKC-
(30) in HEK-293 cells. Cells were imaged 24-h post-transfection at 37°C on a heated stage (Peltier MicroIncubator System) using a Radiance 2000 laser scanning confocal microscope (Bio-Rad). Excitation was measured with the use of a 488-nm laserline and emission was measured at >530 nm. Drugs were bath applied with the use of a gravity-driven perfusion system via a heated perfusion tube connected to a temperature controller (Scientifica). Images were acquired and analyzed using Bio-Rad software (Lasersharp and Laserpix, respectively).
Expression purification and phosphorylation of maltose-binding protein-fusion proteins. The cytoplasmic COOH termini of Kir3.1 (amino acids 180 to 501) and Kir3.2A (amino acids 194 to 425) were cloned into pMALc2 x (New England Biolabs) to create NH2 terminal fusion proteins with the maltose binding protein (MBP). Expression of the fusion proteins in Escherichia coli BL21(DE3) cells was induced using 0.3 mM isopropylthiogalactoside and incubated overnight at 25°C. Cells were harvested by centrifugation at 4,000 g for 30 min at 4°C. The resulting pellet was resuspended in column buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA) containing protease inhibitors (Roche protease inhibitor cocktail, EDTA free) and stored at 20°C until required. Cells were thawed and sonication (5 x 60 s) was used to lyse the cell membranes. After centrifugation (10,000 g for 30 min at 4°C), the supernatant was bound to amylose resin (New England Biolabs) preequilibrated with column buffer. The column was washed with 10 column volumes of column buffer before the protein was eluted with column buffer containing 10 mM maltose. Protein containing fractions were pooled and dialyzed against storage buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA). Protein concentration and purity were determined using the Bradford assay and SDS-PAGE, respectively. The phosphorylation assay was carried out as described previously (37).
Materials. Cell culture materials were from Invitrogen. Molecular biology reagents were obtained from New England Biolabs or Roche Molecular Biochemicals. All chemicals were from Sigma, Tocris, or Calbiochem. Drugs were made up as concentrated stock solutions in ethanol, water, or DMSO, and kept at 4°C or 20°C.
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RESULTS |
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We wanted to examine whether channel activity can be restored or rescued by PIP2. We made whole cell recordings from HKIR3.1/3.2/M3 cells using either control or PIP2-containing pipette solution. Under control conditions, in the absence of added PIP2, the magnitude of M3-mediated inhibition was 77.9 ± 2.2% (n = 36). However, in the presence of PIP2, this effect was significantly reduced by about one-half (M3-mediated inhibition: 39.6 ± 7.2%, n = 12, P = 0.0002), suggesting that PIP2 does play a part in the regulation process. To further investigate this rescuing effect of PIP2 we also performed a series of experiments using the HKIR3.1/3.2/M3 cell line co-expressing Gi/o-coupled A1-adenosine receptors. We have previously reported that stimulation of the M3 receptor prevented subsequent channel activation by the A1 receptor (27). In the current study, with the inclusion of PIP2 in the pipette the A1 response was rescued (Fig. 2).
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We were interested in elucidating whether a specific isoform of PKC may mediate channel inhibition. Because there are very few (if any) pharmacological agents that are isoform-specific, we made several molecular tools to identify which isoform(s) may play a role. Although constitutively active and kinase-dead mutants have been previously employed by several investigators to assess PKC function, evidence exists that these mutants lose their isotype-specific features observed for wild-type PKC isotypes (9, 14). However, ectopic expression of wild-type PKC isotypes has been associated with a range of specific biological inputs, suggesting that this a more useful approach to monitor isotype-specific function (2). We generated a series of myc-tagged PKC isotypes and analyzed their effects on whole cell Kir3.1/3.2A currents measured in the HKIR3.1/3.2A cell line. These experiments were done in the presence of co-expressed G1
2 to elevate basal currents (basal: 49.7 ± 4.4 pA/pF, n = 97; +
1
2: 241.3 ± 33.9 pA/pF, n = 64, P < 0.001). Both PKC-
I and PKC-
significantly reduced current density (PKC-
I: 91.2 ± 19.8 pA/pF, n = 12, P < 0.05; PKC-
: 95.1 ± 25.2 pA/pF, n = 15, P < 0.05) compared with control cells, whereas PKC-
, -
, -
, -
, and -
had no significant effects (Fig. 3B). Western blot analysis using an anti-myc antibody was used to confirm expression of these constructs, but it also revealed differences in expression levels (Fig. 3A). It is apparent from the electrophysiological data that PKC-
I and PKC-
had similar magnitudes of effects on current density even though they had different expression levels, as revealed by the Western blot in Fig. 3A. We thus titrated PKC-
I against PKC-
by reducing the amounts of PKC-
I construct transfected into the cells (50, 100, and 200 ng) and measuring the expression levels using the myc tag. We found similar expression levels using 100 ng PKC-
I cDNA and 1,000 ng PKC-
cDNA for transfection (Fig. 4A). These amounts were then used to measure the effect on current density in the G
1
2-expressing HKIR3.1/3.2A cells as described above. PKC-
I had no significant effects on current density (130.1 ± 32.5 pA/pF, n = 19; P > 0.05) compared with control cells, whereas PKC-
significantly reduced it (81.3 ± 16.7 pA/pF, n = 22, P < 0.01; Fig. 4B). The capacity of PKC-
to reduce current density was confirmed using a different PKC-
construct, in which expression was driven by a cytomegalovirus promoter. Again, PKC-
expression significantly reduced current density (69.0 ± 15.4 pA/pF, n = 11, P < 0.05).
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To ensure that our preparation of rat brain PKC was clean and functional, we performed several control experiments: there were no effects of vehicle on channel activity, PKC activity required the presence of Mg2+ and ATP, and boiling the preparation to inactivate it prevented its effects (data not shown). We also confirmed our results with a commercially available preparation (Calbiochem). Thus PKC (1.6 U/ml) rapidly (12 s) and significantly reduced channel opening (control, 0.554 ± 0.06; +PKC, 0.084 ± 0.02, n = 6, P < 0.001), which recovered on removal of PKC (wash, 0.518 ± 0.05; n = 6). In the presence of the phosphatase inhibitor okadaic acid (OA; 10 µM), the reversibility of the effects of PKC on channel activity was prevented (Control, 0.455 ± 0.08; +PKC, 0.008 ± 0.004; +OA, 0.06 ± 0.008; and wash 0.413 ± 0.04; n = 5, P < 0.001, Fig. 10A), suggesting a mechanism involving phosphorylation and subsequent dephosphorylation.
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It has recently been shown by Mao et al. (30) that single point mutations introduced into Kir3.1 and Kir3.4 subunits (S185A and S191A, respectively) prevented PMA and Gq/11-receptor-mediated inhibition of the Kir3.1/3.4 channel. We generated the equivalent mutations in Kir3.1 and Kir3.2C (S185A and S178A, respectively): the wild-type heteromeric channel we have found to behave similarly at the whole cell level to wild-type Kir3.1/3.2A (data not shown). The double-mutant channel Kir3.1 (S185A)/Kir3.2C (S178A) carried currents which were robust, showed strong inward rectification, and which were inhibited by 100 nM PMA (data not shown). We then examined the modulation of these channels in response to M3 receptor stimulation. We found that carbachol still significantly and irreversibly inhibited the currents (40 ± 2% inhibition) similar to that observed with wild-type channels (49 ± 2% inhibition; Fig. 11, A and B); furthermore, purified PKC still inhibited channel activity when applied to inside-out patches (control, 0.430 ± 0.10; +PKC, 0.086 ± 0.040; wash, 0.346 ± 0.092, P < 0.05, n = 4; Fig. 11C). Therefore, these mutations had no effect on the ability of PKC (activated indirectly via a Gq/11-coupled receptor, directly via a phorbol ester or by using purified PKC itself) to inhibit Kir3.1/3.2C channels. It would seem that the phosphorylation sites identified by Mao et al. (30) are important specifically for Kir3.1+3.4 channels because in our hands the equivalent mutations clearly had no effects on M3-mediated inhibition of Kir3.1/3.2 channel.
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DISCUSSION |
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The involvement of PKC in this pathway is supported by our observation that direct pharmacological activation of PKC by phorbol esters and diacylglycerol analogues and indirect activation by Gq/11-coupled receptors leads to irreversible channel inhibition. This is further supported by our current findings that purified PKC protein inhibits channel activity when applied to excised patches, similar to the findings of Mao et al. (30) and Nikolov and Ivanova-Nikolova (34) with recombinant and native KAch channels, respectively. We found that channel activity rapidly recovered on removal of PKC indicating a close association of protein phosphatases with the channel, as suggested by Nikolov and Ivanova-Nikolova (34). This was also alluded to by Takano et al. (47), who demonstrated that the PP1/2A inhibitor OA, prevented recovery of channel activity following activation of Gq/11-coupled receptors in neurones. In a similar fashion, we also found that OA prevented the recovery of channel activity after PKC activation.
It has been proposed that PKC phosphorylates a serine residue on both Kir3.1 and Kir3.4 subunits because mutation of these residues to alanine in both subunits prevented the PKC mediated inhibitory phenomenon on the heteromeric channel (30). We introduced the equivalent mutations into Kir3.1 and Kir3.2C subunits and examined the properties of this heteromeric double mutant channel in response to 1) PMA treatment, 2) Gq/11 receptor stimulation, and 3) application of PKC to inside-out patches. Interestingly, all three mechanisms for investigating the effects of PKC on channel activity resulted in a similarly significant decrease in Kir3.1(S185A)/Kir3.2C(S178A) channel currents compared with wild-type Kir3.1/3.2C channels. This would seem to imply that the profile of PKC-phosphorylation of Kir3.1/3.2 heteromers is far more complex than that of Kir3.1/3.4, supported by the fact that Kir3.2 contains 26 phosphorylation sites compared with 19 in Kir3.4 (NetPhos predicted). Our biochemical data clearly shows that Kir3.1 is heavily phosphorylated by PKC, lending support to the notion that PKC can modify the channel on multiple sites. Perhaps with Kir3.1/3.2 channels, multiple sites are involved in a stepwise fashion. In addition, the environment of a Xenopus oocyte as used by Mao et al. (30) may differ substantially than the mammalian cells used in the current study.
This study has provided further evidence reinforcing our previous hypothesis that a Ca2+-independent PKC isotype is responsible for the inhibitory phenomenon (27). PKC- would appear to be the strongest candidate because, after controlling for levels of expression, PKC-
was the only isotype, which caused a significant reduction in both basal and agonist-induced Kir3.1/Kir3.2A currents. Conversely, disruption of endogenous PKC-
activity through expression of dominant negative PKC domains significantly reduced the Gq/11-mediated channel inhibition. Translocation of GFP-tagged PKC-
, but not PKC-
, in our HKIR3.1/3.2A/M3 stable cell line after receptor stimulation provides further evidence for the selective activation and recruitment of PKC-
to the membrane, which can then act to inhibit Kir3.1/3.2 channel activity. The inhibition of the M3 receptor response by the regulatory domain of PKC-
does not itself indicate that PKC-
is the only isotype involved in the response. The regulatory domain contains the C1 module, which binds endogenous diacylglycerol and on this basis would inhibit most PKC isotypes, including classic PKCs. However, the fact that the PKC-
C2 module on its own antagonizes the M3 receptor response suggests that PKC-
is the most likely isotype involved. This module represents a unique fold within the PKC family (displaying only significant similarity with PKC-
, which is not expressed in these cells; Ref. 27) and as such would have a specific inhibitory effect (35). The mechanism by which this inhibition occurs is not clear. PKC-
C2 has been identified as a protein interaction module (8) and has inhibitory effects on PKC-
activity in other cellular contexts (29). It may therefore reduce the interaction of PKC-
with essential binding partners, e.g., receptor for activated C kinase (34) or A-kinase anchoring protein (13) so that channel modulation does not occur. The identification of these binding partners, and the biochemical characterization of the interaction with respect to isotype preference, will provide further insight into the mechanism of regulation of this class of channel.
Clearly, the native cellular environment is organized and expression is controlled, and therefore PKC recruitment will depend on stimulus, tissue expression, and subcellular microdomains (11, 32). Indeed, evidence has been presented that Ca2+-dependent PKC isotypes are recruited to atrial membranes after -adrenoceptor stimulation and can inhibit KAch currents (34). Therefore, it is tempting to speculate that endogenous Kir3 currents may be inhibited through pathways that stimulate different isoforms of PKC. In our single channel studies, PKC-mediated inhibition reversed rapidly on removal of the enzyme. Consequently, it is unlikely that PKC is causing the prolonged inhibition of channel activity after M3 receptor activation in whole cell conditions. Therefore, it is probable that the loss of phospholipid from the membrane is the cause of this.
Du and co-workers (12) have proposed that Kir3 channels have a comparatively low affinity (vs. other potassium channel family members) for PIP2 and thus are susceptible to PKC-mediated inhibition (as seen through phorbol ester stimulation; 12). Our experiments examining M3-modulation of currents under whole cell and perforated-patch conditions may help begin to explain the conflicting arguments for the mediator responsible for Kir3 current inhibition. For example, when the cellular environment remained largely intact (perforated patch) PKC activity was critical for channel inhibition. However, under whole cell conditions, although to a lesser extent, channel inhibition still occurred after PKC inhibition both pharmacologically and biochemically. The prolonged inhibition was manifested probably through loss of phospholipid content as supplementation of PIP2 restored and rescued channel activity. Equally, if the native cellular environment has a low phospholipid content, then it is likely that channel inhibition could manifest as apparently only a phospholipid-dependent mechanism after Gq/11-receptor activation (13). Conversely, on the basis of our perforated-patch experiments and inhibition of PKC activity, it is tempting to speculate that despite the comparitively low PIP2 affinity, the affinity of the channel for PIP2 may be sufficient to withstand physiological changes (through Gq/11-receptor mediated activity) in membrane phospholipid content in the absence of PKC modulation under conditions of high local concentrations of phospholipid.
It is our hypothesis that heteromeric channels are modulated primarily by PKC, and secondarily by PIP2 after activation of Gq/11-coupled receptors in HEK-293 cells. Thus we present a unifying mechanism for inhibitory regulation of Kir3.1/3.2 channels involving PKC- and PIP2 after Gq/11-receptor stimulation.
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ACKNOWLEDGMENTS |
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Present address for L. V. Dekker: Ionix Pharmaceuticals, 418 Cambridge Science Park, Milton Rd., Cambridge CB4 0PA, UK.
Present address for J. L. Leaney: Ion Channel Pharmacology Group, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Rd., Sandwich, Kent, CT13 9NJ, UK.
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FOOTNOTES |
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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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