Kv1.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor
In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein–dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57±0.07 nA/pF, n=12; to 1.18±0.2 nA/pF, n=12, P<0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K+ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K+ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated.
Voltage-gated K+ channels (Kv channels) are intimately involved in the cellular regulation of excitation in all cardiovascular cells, and their activity depends on the presence of active channel subunits at the plasmalemma. Surface expression is regulated by changes in gene expression,1–3 interactions with accessory subunits, by phosphorylation, and by cellular components that regulate their trafficking to the cell surface. Trafficking can also provide an explanation for the mechanisms by which drugs may act to achieve their therapeutic actions.4 Although several groups have investigated motifs within K+ channels that affect trafficking,5–8 little is known about the molecules and machinery involved in these processes in the heart.
Surface expression requires movement from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane, and several studies have investigated channel determinants that affect this trafficking process. Motifs in the C termini and pore domains,6,7 of Kv channels have been implicated in their differential surface expression presumably through effects on forward trafficking.5 Functional expression of channels can also be regulated by the removal of extant channels from the cell surface, as recently shown for Kv1.2. Surface expression is modulated by tyrosine phosphorylation which promotes the endocytosis of the channel.9 Dhani et al,10 have implicated retrograde transport along the microtubule cytoskeleton in the regulation of ClC-2 chloride channel expression. ClC-2 interacts directly with the dynein motor and inhibition of this motor significantly increased the surface expression of the channel.
Here we have used a multi-pronged approach to show that dynein is also closely involved in the regulation of Kv1.5 surface expression, probably through its role in transporting newly formed endosomes from the cell surface to the cell interior. Direct interference with dynein function caused a significant increase in Kv1.5 surface expression as did disruption of the microtubule cytoskeleton or direct interference with endocytosis itself. We have identified specific residues in Kv1.5 that are essential to this process and shown the process to be relevant both in heterologous cell expression systems and in rat cardiac myocytes.
Materials and Methods
Many methods used in this study have been previously described.11 In electrophysiological experiments where controls were compared with cells overexpressing p50, dynamin inhibitory peptide, or compared with nocodazole pretreatment, control and experimental groups were prepared in parallel and the experimenter was blinded to their identity. Further methodological details can be found in the online data supplement available at http://circres.ahajournals.org.
Disruption of Dynein Function Increases Kv1.5 Surface Expression
Overexpression of p50/dynamitin is established as an effective and specific method for disrupting the dynein motor system.12–14 p50, an important component of the dynactin complex, is thought to link the cargo-interacting subunits to the dynein motor itself. Overexpression of this protein causes the dynactin complex to dissociate, decoupling the motor from its cargoes.12 As shown in Figure 1, overexpression of p50 in HEK cells significantly increased Kv1.5 currents on the day after transfection. Peak current densities at +80 mV were approximately doubled from 0.57±0.07 nA/pF to 1.18±0.2 nA/pF, P<0.01 (Figure 1A). This increase in response to p50 overexpression was not limited to HEK293 cells, and experiments in CHO cells yielded similar results (Figure 1B). Activation and inactivation kinetics were unchanged with increases in current across the whole activation range (Figure 1C), suggesting an increase in the number of functional channels at the cell surface.
To confirm that increased Kv1.5 currents were indeed caused by increased surface expression, Kv1.5 localization was examined by immunocytochemistry/confocal microscopy and channel Proteinase K sensitivity was assayed. In Figure 1D, p50 overexpression caused a substantial redistribution of Kv1.5 staining. When expressed alone in HEK cells, Kv1.5 was distributed throughout the cell. Membrane expression was significant, but large amounts of the channel were cytoplasmic (Figure 1D, upper panel, center). p50 overexpression dramatically reduced internal staining (lower panels), whereas staining at the cell surface remained intense. This is most clearly demonstrated in the lone cell in the lower middle panel (white arrow), which escaped transfection with p50-GFP, but was transfected with Kv1.5. Protease-sensitivity experiments yielded results consistent with this. When applied externally to living cells, Proteinase K cleaves exposed protein on the exterior of the cell although cytoplasmic proteins are unaffected. This protease is thus a sensitive test for cell surface expression and is useful also for determining the relative proportions of the channel at the cell surface and in the cytoplasm.6 Uncleaved T7-tagged Kv1.5 migrates at 83 kDa; the digested N-terminal fragment (roughly, the cytoplasmic N terminus plus S1), migrates at ≈47 kDa. Densitometric analysis of the results shown in Lanes 3 and 4 of Figure 1E confirmed that the intensity of the 47 kDa Kv1.5 band relative to the upper band intensity was significantly increased in cells overexpressing p50 (1.52±0.09, n=3, paired t test P<0.05) compared with control cells. That is, p50 overexpression increased Kv1.5 surface residency by ≈50%. This result is consistent with the 2-fold increase in Kv1.5 expression detected electrophysiologically and with the redistribution of the channel seen in the imaging experiments. Given that p50 is well established as a specific disruptor of dynein motor function,12–14 the dynein motor is strongly implicated in the regulation of Kv1.5 surface expression.
Kv1.5 Is Present in Early Endosomes
Dynein motor disruption by p50 overexpression might increase Kv1.5 surface expression by interfering with endocytosis, because dynein is required for transport of endosomes from the cell surface to the interior.14 To see whether Kv1.5 is trafficked via endosomes, we used confocal microscopy to look for colocalization of Kv1.5 with EEA1, an early endosomal marker. As shown in Figure 2A, Kv1.5 and EEA1 colocalized in what appear to be small vesicles, ie, early endosomes, near the plasma membrane. Line-scan analysis across 9 groups of endosomes in this cell, as well as endosome-rich regions of several additional cells confirmed this. In all line scans the pixel correlation between Kv1.5 and EEA1 fluorescence was extremely high (P<0.0001, online Figure IA and IB). In contrast, there was no correlation of Kv1.5 and EEA1 associated pixels in line scans across regions where Kv1.5 was found but endosomal localization was not obviously present (see below).
If dynein disruption interferes with the removal of Kv1.5 from the plasma membrane, then direct inhibition of endocytosis should mimic this effect. Dynamin is responsible for pinching off the neck of the budding endocytotic vesicle and dynamin inhibitory peptide (DIP, Tocris, Bristol, UK) has been previously established to be an effective blocker of endocytosis.15 HEK293 cells stably expressing Kv1.5 were incubated overnight with 50 μmol/L DIP or a control scrambled peptide. As shown in Figure 2C, DIP incubation doubled Kv1.5 currents, an effect essentially identical to that elicited by p50 overexpression. Peak current densities at +80 mV were 0.36±0.05 nA/pF (n=15) in control cells and 0.76±0.05 nA/pF (P<0.01, n=11) in cells incubated overnight with DIP. Imaging analysis showed that colocalization of Kv1.5 and EEA1 in residual early endosomes was dramatically reduced in DIP-treated cells relative to controls. Line-scan analyses across these endosomes showed no significant pixel correlation between EEA1 expression and Kv1.5 (online Figure IC) with probability values between 0.065 and 0.420. These data suggest that dynein inhibition may increase Kv1.5 surface expression by interfering with the net removal of channels from the plasma membrane.
Nocodazole Treatment Mimics Dynein Inhibition in HEK Cells and Cardiomyocytes
If p50 overexpression exerts its actions through interference with dynein transport along microtubules, then disruption of the microtubule cytoskeleton should have similar effects on Kv1.5 currents. To test this prediction we used the microtubule depolymerizing agent, nocodazole, which strongly inhibits trafficking by dynein.16 Incubation of Kv1.5-expressing HEK cells with nocodazole substantially increased Kv1.5 currents (Figure 3A and 3B). Kv1.5 currents increased in a dose-dependent manner with a maximum effect at 70 μmol/L. p50 overexpression was additive to the nocodazole current increase at lower doses (open triangles, Figure 3B) where the drug had a submaximal action. However, current levels saturated with p50-overexpression and ≥70 μmol/L nocodazole. These results suggest that p50 overexpression is acting on a microtubule-dependent pathway, ie, very probably on dynein. Although the additivity of p50 overexpression and low-dose nocodazole may indicate that p50 acts also on a secondary pathway, it is more likely that p50 overexpression is not completely effective in preventing dynein motor complex assembly. Thus, only when microtubules are fully depolymerized at the higher nocodazole doses is the residual trafficking by dynein eliminated. In parallel experiments, atrial myocytes were isolated from adult rats and treated for 6 hours with 35 μmol/L nocodazole. I-V relations in Figure 3C and 3D show that sustained currents were enhanced ≈2× in the nocodazole-treated myocytes but the A-type currents, which underlie Ito, in these cells were unaffected (Figure 3E). As shown in Figure 3D, the increase in sustained current was across the voltage range that Kv1.5 is expected to be open.
Imaging experiments confirmed an enhancement of Kv1.5 surface expression in these myocytes. Nocodazole-treated and control myocytes were stained with anti-Kv1.517 and anti-tubulin and visualized by confocal microscopy. This treatment resulted in a loss of microtubule integrity, as evidenced by a loss of reticulate structure and a general dispersal of tubulin staining (data not shown). Nocodazole treatment dramatically increased the intensity of Kv1.5 staining at the myocyte surface compared with controls (Figure 4A and 4B). Line scan analysis on 10 randomly chosen control and nocodazole-treated myocytes showed that 26.9±2.1% (mean±SEM) of Kv1.5-positive pixels were within the quartile of the cell nearest to (and including) the plasmalemma in control cells and 40.5±2.6% (Figure 4C and 4D; unpaired t test, P<0.001) of positive pixels were within these boundaries in nocodazole-treated myocytes. The increased Kv1.5 staining in the outermost quartile supports the idea that enhancement of sustained currents in these cells by nocodazole was caused by a redistribution of Kv1.5 to the plasma membrane.
To determine the contribution of Kv1.5 to the increased sustained currents in nocodazole-treated myocytes, Kv1.5 blockers were used. 4-Aminopyridine is commonly used18 to block K+ channels by binding to the inner pore mouth. Susceptibility of the current to 4-AP eliminated the possibility that anion or other currents underlie the increase in myocyte current. In nocodazole-treated myocytes, 1 mmol/L 4-AP reduced sustained and A-type current (Figure 5A), confirming that nocodazole was indeed influencing potassium channel expression. However, whereas Kv1.5 reportedly underlies the bulk of the sustained current in rat atrial myocytes, other channels, like Kv2.1, may also be contributors to this current at the 1 mmol/L dose used here.19,20 To confirm that Kv1.5 underlies the enhanced sustained K+ currents seen after nocodazole treatment, a Kv1.5-specific blocker, 2-(3,4-Dimethylphenyl)-3-(4-methoxyphenethyl) metathiazan-4-one (DMM), a gift from Cardiome Pharma (Vancouver, Canada), was used. The IC50 for Kv1.5 block (1.5 nM) with this drug was >2 orders of magnitude lower than for Kv2.1 or other K+ channels and Na+ channels present in heart (Figure 5B), and 100 nM DMM completely blocked Kv1.5 in HEK cells (Figure 5C). More importantly, DMM also blocked the nocodazole-increased sustained current in rat atrial myocytes (Figure 5D).
Kv1.5 Coimmunoprecipitates With the Dynein Motor Complex
To test for an association between Kv1.5 and dynein, coimmunoprecipitations of Kv1.5 and the dynein intermediate chain (DIC), or p50 (an integral part of the dynein–dynactin complex) were performed on extracts of HEK cells heterologously expressing a T7-tagged Kv1.5 construct (Figure 6A), and rat ventricular myocyte lysates expressing the native channel (Figure 6B). A significant portion of Kv1.5 coimmunoprecipitated with the dynein intermediate chain in these experiments. Anti-T7 antibody brought down the DIC along with the targeted T7-tagged Kv1.5 in HEK cells and anti-Kv1.5 coimmunoprecipitated DIC in myocytes (Figure 6A, lower panel, and 6B). T7-tagged Kv1.5 was also efficiently coimmunoprecipitated with p50 (online Figure II). Given that dynein interacts with many cellular proteins and that plasmalemma-integrated Kv1.5 is unlikely to interact with dynein, this level of coimmunoprecipitation is highly significant. Thus, like ClC-2,10 Kv1.5 directly interacts with the motor complex that is likely trafficking it from near the cell surface in native cells to the interior.
A Proline Rich Region in Kv1.5 Is Essential to Regulation of Channel Expression by Dynein
Both the N and C termini of Kv channels have been implicated in regulating their surface expression.5,11 Mutants lacking the bulk of either the N terminus (ΔN209) or C terminus of Kv1.5 (ΔC51) were tested for responsiveness to p50-overexpression (Figure 7A). The ΔC51 mutant behaved like WT, as currents were roughly doubled, but Kv1.5ΔN209 was wholly unresponsive to disruption of dynein motor function. A more conservative deletion (ΔN91) also proved unresponsive to p50 overexpression, and included in the deletion is a proline rich region (aa 65 to 93) harboring two SH3 binding domains that are consensus src kinase binding sites.21 As tyrosine phosphorylation has been previously implicated in Kv1.5 surface expression22 and specifically in the regulation of Kv1.2 endocytosis,23 this region seemed a likely candidate for involvement in internalization of the channel. Deletion of aa65–93 completely eliminated the increased Kv1.5 currents seen with p50 overexpression (Figure 7B).
The 2 consensus SH3-binding domains in this region are separated by 4 residues and both have the sequence RPLPPLP. We mutagenized the consensus sequences individually to RPLAALP (PI and PII, Figure 7C). Mutation of the second consensus domain (Kv1.5 P79/80A; P-II) had no effect on Kv1.5 responsiveness to p50 overexpression (Figure 8B), but mutation of the first consensus SH3-binding domain (Kv1.5 P68/69A; P-I) eliminated the responsiveness of Kv1.5 to p50 overexpression (Figure 8A). Interestingly, the baseline current densities in PI mutant cells were unusually high, resembling levels seen with WT Kv1.5 cells overexpressing p50 or the dynamin inhibitory peptide (DIP, Figure 2). This suggested that Kv1.5 P68/69A had lost its ability to be upregulated by interference with the motor, or perhaps had been relieved of negative regulation by the dynein motor because the channel no longer interacted with it. However, given that block of endocytosis with a dynamin inhibitory peptide mimics the effect of p50 (Figure 2C) and that tyrosine phosphorylation has been implicated in the Kv1.5 downregulation22 and in endocytosis of the related Kv1.2 channel,9 we preferred the hypothesis that this loss of responsiveness might be attributable to a defect in endocytosis of the channel, which was thus no longer available for retrograde transport. We therefore repeated the PI mutant experiment with DIP to test whether that peptide could still affect surface expression of the PI-Kv1.5. As shown in Figure 8C, DIP had no significant effect on the expression of PI channel currents, although still affecting PII currents. Kv1.5 P68/69A expression was high whether or not the cells were pretreated with DIP. Currents were 2 to 3× those of Kv1.5 P79/80A which served as a control in these experiments. Neither DIP, nor p50 overexpression, nor a combination of the two (Figure 8D) had any effect on the expression of the PI mutant Kv1.5 channel. We conclude that the N-terminal-most SH3-binding domain is essential for normal endocytosis of Kv1.5. Surface expression is regulated by endocytosis, followed by retrograde transport of the internalized channels in association with the dynein motor complex.
Dynein Motor Function Affects Kv1.5 Surface Expression
This is the first report of an association of a K+ channel with the dynein motor complex. In the only other report of an ion channel-dynein interaction, Dhani et al10 demonstrated that a chloride channel, ClC-2, bound the heavy and intermediate dynein chains in vitro and that the channel could be coimmunoprecipitated with the dynein motor complex from murine hippocampal lysates. As with Kv1.5, interference with dynein function increased surface expression of ClC-2. Here, our data unequivocally demonstrate that inhibition of dynein motor function in HEK cells with p50 overexpression, or nocodazole in myocytes increases the presence of Kv1.5 at the cell surface by about 2-fold, by electrophysiological, biochemical and imaging data (Figures 1, 3, and 4⇑⇑). p50 overexpression is well established as a powerful and specific means to inhibit dynein motor function. However, it remains possible that p50 overexpression has additional, as yet undescribed effects on the cell that are independent of its role in the dynein–dynactin complex. To exclude this possibility, nocodazole was used as a second method to inhibit dynein function, and caused significant increases in Kv1.5 currents in HEK cells and in rat cardiac myocytes (Figure 3), that could be abolished by the K+ current-selective agent 4-AP, and the more specific Kv1.5 antagonist, DMM (Figure 5). Although there is no requirement for a direct interaction between the complex and channel for trafficking by the motor (Kv1.5 could be dragged along in endosomes bound to the dynein motor complex independently of a specific Kv1.5-dynein association), we were also able to find a direct interaction between Kv1.5 and components of the dynein complex, p50, and DIC in HEK cells and native myocytes (Figure 6). Also, it is interesting that, at low doses of nocodazole, the effects of the drug and p50 overexpression on Kv1.5 expression in HEK cells are additive (Figure 3). Either p50 overexpression is insufficient to fully inhibit dynein function or there is at least one additional pathway by which Kv1.5 is trafficked.
Endocytosis and the Role of a Kv1.5 Consensus SH3 Binding Domain
The most likely mechanism by which dynein block increases Kv1.5 surface expression is indirect, by affecting the balance of channel delivery and removal from the plasma membrane. Valetti et al,14 have shown that endocytosis is normal in HeLa cells overexpressing p50, as is the recycling of endosomes to the cell surface. This does not mean, however, that Kv1.5 surface levels must remain in a steady state when retrograde trafficking is impaired. If even a fraction of the newly synthesized or internal pools of the channel continue to traffic to the cell surface, the failure of dynein to remove endocytosed channels would quickly result in an increased concentration of the channel in the endocytosing/recycling pool. Indeed, disruption of the dynein–dynactin complex by p50 overexpression has been shown to lead to a redistribution of endosomes toward the cell periphery in COS-7 cells,10,13 probably attributable to a failure of newly formed endosomes to be further internalized. Block of endocytosis using the dynamin-inhibitory peptide mimicked the effects of p50 overexpression and nocodazole treatment (Figure 2) and is consistent with this model. Mutation of 2 prolines to alanines in 1 Kv1.5 consensus SH3-binding domain also eliminated the Kv1.5 current response to p50 overexpression (Figures 7 and 8⇑). This is almost certainly because the mutant channel is not efficiently endocytosed. Current levels are high (similar to those of the wild-type channel when dynein function or endocytosis are inhibited) and, more tellingly, the mutant is also insensitive to dynamin inhibition (Figure 8).
SH3 domains are important for the recruitment of tyrosine kinases, like src, to a protein. Tyrosine phosphorylation is well known to downregulate Kv1.5 currents,22,24 src kinase has been shown to directly bind the proline-rich region in which this SH3-binding domain resides,22 and a direct role for tyrosine phosphorylation in the regulation of endocytosis has recently been demonstrated for Kv1.2, a related potassium channel.9 Thus, the simplest explanation for the role of this consensus SH3-binding domain in Kv1.5 is that it is required to promote tyrosine phosphorylation of the channel and that this in turn regulates channel internalization. Lacking this domain, the channel remains in the plasma membrane, and is inaccessible to dynein so manipulations of the motor have no effect on channel surface expression (Figure 8).
There are 2 identical consensus SH3-binding domains in close juxtaposition in Kv1.5. However, mutation of the second SH3-binding domain does not affect the sensitivity of the channel to p50 overexpression. Perhaps the precise context of the domain is important. Also, although deletion of the entire proline rich domain (residues 65 to 93) eliminates the responsiveness of the channel to p50 overexpression, cells expressing this mutant do not display current densities above those of cells expressing the wild-type channel in the absence of p50 overexpression. Perhaps there are additional determinants in this region that affect delivery of the channel to the plasma membrane or otherwise affect its stability at that locale.
Implications for Forward Trafficking
Although pore6,7 and C-terminal5 motifs have been implicated in the trafficking of Kv channels to the cell surface, the mechanism(s) by which these motifs affect this movement remain unclear. They may relate to interactions with trafficking molecules or to channel assembly instead. However those signals operate, the fact that inhibition of dynein function and depolymerization of the microtubule cytoskeleton increases Kv1.5 surface expression has important implications for forward trafficking. Somehow, Kv1.5 is continuing to traffic to the membrane. Kinesins are unlikely to be involved in this process. Although they are generally responsible for anterograde transport,25 dynein and kinesin activities are coordinated. When retrograde transport is blocked, anterograde movement also ceases14 and vice versa.26 The dynactin complex, which connects the dynein motor to its cargo, may play an essential role in this regard. p150glued, a component of the dynactin complex, is required also for the functioning of at least 1 kinesin isoform.27 Whatever the case, kinesins are certainly unlikely to be functional in the nocodazole-treated cells, where the microtubule cytoskeleton has been depolymerized. At least a fraction of Kv1.5 forward trafficking must therefore use a nonmicrotubule-dependent pathway. One possibility is that the channel is trafficked via the actin cytoskeleton instead. Perhaps a fraction of Kv1.5 is carried to the surface by myosin V which has been implicated in anterograde trafficking in neurons, and in complex with kinesin has been shown to be involved even in long-range anterograde vesicular trafficking.28 Alternatively, simple diffusion within the cell might be enough to traffic Kv1.5 to the surface in the absence of a microtubule cytoskeleton. A high-affinity Kv1.5 anchoring protein in the plasma membrane or a preference of the channel for a plasma membrane-like lipid environment might be sufficient to drive the concentration of the channel at the surface in the time frames examined here. Clearly future work needs to address these possibilities.
Relevance to the Heart
Nocodazole-treatment increased Kv1.5 surface expression as assayed by confocal imaging and measurement of sustained K+ currents in rat cardiac myocytes. The sensitivity of these increased currents to the specific Kv1.5 antagonist DMM demonstrated that they were specifically underlain by increased Kv1.5 expression in these cells. In addition, Kv1.5 was shown to directly interact with components of the dynein complex (Figure 6). It is highly likely, therefore, that the microtubule cytoskeleton is involved in the internal trafficking of Kv1.5 in both heterologous and physiological (cardiac) systems. It will be of great interest to determine how this system is used by the heart to modulate the expression of Kv1.5 and other ion channels as a mechanism to regulate cardiomyocyte excitability, and how other channels are affected by perturbations of retrograde trafficking. It is possible that in disease states associated with channel remodeling, like atrial fibrillation and congestive cardiac failure, the trafficking or endocytosis mechanisms described above may be of key importance in determining the final population of surface-expressed channels. For example, it has been reported that microtubule abundance is increased in the myocytes of patients with congestive heart failure.29
The dynein motor has been strongly implicated in the retrograde trafficking of Kv1.5 in endosomes from the cell surface in HEK cells and myocytes, probably via a direct interaction between the channel and the dynein motor complex. This trafficking depends on efficient endocytosis of the channel, which is, in turn, dependent on an intact SH3-binding domain in the Kv1.5 N terminus. This is a previously unexplored avenue of Kv channel trafficking and regulation.
These studies were supported by funding to D.F. from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of British Columbia and Yukon, and to WSC from a University Graduate Fellowship. We thank Richard Vallee for the gift of human p50-GFP, and Yan Liu for help with cell preparation.
Original received May 6, 2005; revision received July 6, 2005; accepted July 19, 2005.
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