Molecular Coupling of a Ca2+-Activated K+ Channel to L-Type Ca2+ Channels via α-Actinin2
Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca2+-activated K+ channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca2+ concentration [Ca2+i] with changes in K+ conductance and membrane potential, associate with L-type Ca2+ channels; Cav1.3 and Cav1.2 through a physical bridge, α-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca2+ channels, instead, the 2 channels colocalize via their interaction with α-actinin2 cytoskeletal protein. The association of SK2 channel with α-actinin2 localizes the channel to the entry of external Ca2+ source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Cav1.3 Ca2+ channels. Null deletion of Cav1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca2+ channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.
Ca2+-activated K+ channels (KCa) are present in a variety of cells, where they integrate changes in [Ca2+i] with changes in K+ conductance and membrane potential.1,2 KCa channels are present in most neurons and mediate the afterhyperpolarizations following action potentials (APs). KCa channels can be divided into 3 main subfamilies: 1) the large-conductance Ca2+- and voltage-activated K+ channels (BK); 2) the intermediate-conductance KCa channels (IK, KCa3); and 3) the small conductance KCa channels (SK, KCa2).1–5
SK channels are encoded by at least 3 genes, KCNN1 (SK1), KCNN2 (SK2), KCNN3 (SK3).1,2,5 Previous studies have documented that Ca2+ entry through voltage-gated Ca2+ channels (VGCC) can activate either BK or SK channels in neurons. In addition, there is a unique specificity of coupling between VGCCs and BK or SK channels.6 However, the molecular mechanisms underlying the coupling of the Ca2+ channels and SK channels are not known.
Cardiac myocyte contains an intracellular scaffold, the cytoskeleton, which provides structural support and compartmentalization of intracellular components, as well as playing a role in protein synthesis, intracellular trafficking and organelle transport within the cell.7,8 Regulatory proteins and ion channels are often embedded within and physically bound to the cytoskeleton so that alterations in the cytoskeleton can directly affect their function. Furthermore, cytoskeletal proteins can modulate second messenger signaling pathways, including the β-adrenergic pathway.8 Previous work has demonstrated that ion channels anchored by cytoskeleton undergo highly specialized subcellular localization at the plasma membrane which is crucial to cell function. We have previously demonstrated the important functional roles of SK channels not only in mouse atrial but also in human atrial myocytes.9,10 Here, we presented data to provide insights into the molecular coupling of SK2 channel with voltage-gated Ca2+channel and represent the first report linking the molecular coupling of 2 different types of ion channels via cytoskeletal proteins.
Materials and Methods
A detailed Materials and Methods is described in the online data supplement available at http://circres.ahajournals.org.
Yeast Two-Hybrid Screens
Yeast two-hybrid (YTH) screening was performed with the GAL4 system using protocol of MATCHMAKER GAL4 Two-hybrid System 3 (Clontech, Mountain View, Calif).
Immunofluorescence Confocal Microscopy
Immunofluorescence labeling was performed as described previously.9,10 Immunofluorescence-labeled samples were examined using a Pascal Zeiss confocal laser scanning microscope.
Whole-cell IK,Ca was recorded from transfected Human Embryonic Kidney (HEK293) cells at room temperature using conventional patch-clamp techniques as previously described.9,11 Whole-cell IK,Ca was calculated as the apamin-sensitive component using 10 nmol/L of apamin. Generation of Cav1.3 null mutant mice was previously described.12,13 Single atrial and ventricular myocytes were isolated from Cav1.3−/−, Cav1.3+/− and wild-type (WT, Cav1.3+/+) littermates in C57Bl/6J background as previously described.10
Interaction of SK2 Channel With α-Actinin2 Revealed by YTH System
To seek molecules that bind the α-subunit of the SK2 channel, we screened a human heart complementary DNA (cDNA) library using 3 different fragments of SK2 cDNA as baits (Figure 1a). Using bait consisting of amino acid (AA) residues 380 to 580 from the cytoplasmic carboxyl terminus of the human heart SK2 channel (SK2-C3), we obtained 9 independent positive clones. Each of the clones encoded a carboxyl-terminal fragment of α-actinin2 (Figure 1b). The clone containing the longest sequence (C9) encoded AA residues 316 to 894, representing approximately half of the fourth spectrin repeat and the 3 entire spectrin repeats plus the EF hands. The shortest clone contained roughly the last 2 spectrin repeats plus the EF hands. At the same time, we also obtained one positive clone (C44) encoding calmodulin consistent with previously published data showing the association of calmodulin with the calmodulin binding domain located in the C-terminal region of the SK2 channel.14
α-Actinin2 is an actin binding protein that contains an actin-binding domain at the N-terminus, 4 spectrin-like repeats in the central-rod domain and 2 EF hands in the C- terminus. The rod domain of α-actinin2 has been shown to be important for dimerization.15,16 In cardiac sarcomere, α-actinin2 dimer simultaneously cross-links 2 actin filaments.15,16 To map the interaction sites between SK2 channel and α-actinin2, 2 SK2 fragments (C1 encoding AA residues 380 to 487 and C2 encoding AA residues 481 to 580, Figure 1, a) were additionally tested as baits to clarify the domains necessary for the interaction with the α-actinin2 protein. As shown in Figure 1, c and d, both of the baits interacted with α-actinin2-C9 clone, but C1 displayed much stronger interaction than C2 on YTH high stringency medium. In contrast, SK2-N from AA residues 1 to 145 and SK2-M from AA residues 140 to 390 baits failed to exhibit any interactions with α-actinin2-C9 clone. Figure 1d, further illustrates the strong interaction between SK2-C1 and SK2-C3 baits with C9 α-actinin2 prey showing the same white color phenotype (as the positive colonies of pGADT7-T with p53), while SK2-C2 bait interacted only weakly with C9 α-actinin2 prey with a resultant pink color phenotype. Finally, SK2-N and SK2-M baits failed to interact with α-actinin2-C9 resulting in inactive ADE2 reporter gene on SD/-Leu/-Trp medium (red colonies, Figure 1d). The results were consistent using high and low stringency media. Taken together, our results suggest 2 binding sites for α-actinin2 on SK2 cytoplasmic carboxyl terminus with stronger interaction between SK2-C1 and α-actinin2 compared with SK2-C2.
Identification of Interaction Between α-Actinin2 and SK2 Channel in Native Tissues and Human Embryonic Kidney Cells Using Coimmunoprecipitation
In vitro biochemical methods were applied to further confirm the interactions between α-actinin2 protein and SK2 channel. We performed coimmunoprecipitation experiments using native human heart tissue as well as HEK 293 cells transfected with pSK2-IRES-EGFP plus pcDNA3-α-actinin2 (Figure 1, e and f). We first performed Western blots to document the presence of α-actinin2 protein in the human heart lysate and transfected HEK 293 cells showing the presence of a protein band at ≈ 98 kDa as expected for α-actinin2 (Figure 1e, lanes 1, 2, and 8).
To further verify the specificity of the antibody used in our experiments, we first documented the presence of SK2 channel protein in transfected HEK 293 cell lysate and detected the SK2 proteins as 2 distinct protein bands at ≈60 and 120 kDa belonging to SK2 monomer and dimer, respectively (Figure 1f, lane 1). Experiments were performed using immunohistochemistry and confocal microscopy in HEK 293 cells to further assess the specificity of the antibody (supplemental Figure I in online data supplement).
Anti-SK2 antibody which was bound to protein A-agarose precipitated α-actinin2 protein from both human atrial and ventricular tissues as detected on immunoblots using anti-α-actinin2 antibody shown in Figure 1e (lanes 6 and 7) as protein bands at ≈98 kDa. Similar data were obtained using HEK 293 cells transfected with pSK2-IRES-EGFP plus pcDNA3-α-actinin2 (Figure 1e, lane 9). Negative controls were shown in lanes 4 and 5.
The reverse experiments are shown in Figure 1f. Using anti-α-actinin antibody bound to protein A-agarose to precipitate the detergent-solubilized protein from transfected HEK 293 cells, SK2 channel protein could be detected on immunoblots using anti-SK2 antibody as shown in Figure 1f, lane 2. Negative control is shown in lane 3.
Colocalization of SK2 Channel and α-Actinin2 Proteins in Isolated Mouse Cardiomyocytes
We next assessed the regional distribution of SK2 channel and α-actinin2 in isolated mouse atrial and ventricular myocytes. The specificity of anti-SK2 and anti-α-actinin antibodies to mouse heart tissues was examined by Western blotting (supplemental Figure IIa). Figure 2, a and A show photomicrographs of immunostaining using single anti-SK2 antibody illustrating distinct staining patterns around and in the Z-line in both atrial (Figure 2a) and ventricular (Figure 2A) cells. As expected, anti-α-actinin2 antibody displayed α-actinin2 (ACT2) localization in the Z-line (Figure 2, b and B). Double labeling (Figure 2, c and C) illustrated the localization pattern of SK2 channel to be similar to that obtained using single staining suggesting no overlap in the detection of the 2 fluorochromes (FITC and Texas-Red). Merged images show colocalization (yellow color) of SK2 channel protein and α-actinin2 along the Z-line (Figure 2, f and F). Further quantification was performed using profile scans (supplemental Figure IIb). Control experiments are shown in panel d, e, D, and E.
Previous studies have shown that transverse-tubular systems (T-tubular systems) are not as well developed in atrial cells compared with ventricular myocytes.17 Most of these prior studies are in rabbits and rats. Our immunostaining shows that SK2 channels are observed in transverse bands separated by ≈2 μm, which would suggest T-tubular localization. A smaller number of atrial myocytes (<10%) shows SK2 staining without specific localization in transverse bands (supplemental Figure IIIa). In addition, we have performed direct T-tubular systems identification using Di-8-ANEPPS (supplemental Figure IIIb).
We directly performed coimmunoprecipitation using mouse atrial and ventricular tissues (supplemental Figure IIIc). Close examination of the SK2 staining in the cardiac myocytes reveals that the SK2 immunoreactivity is not all confined to the transverse bands (Figure 2). The interactions of the channels are dynamic and at times in transit, it is reasonable to expect that, at any moment in time, not all of the channels will be colocalized within the transverse bands. Alternatively, it is possible that a separate population of the channels exists outside of the T-tubular system.
Modulation of Ca2+-Activated K+ Current by α-Actinin2 in HEK 293 Cells
To determine the functional significance of the interaction between α-actinin2 and SK2 channel, we compared whole-cell IK,Ca (apamin-sensitive K+ current) in HEK 293 cells using different plasmid composition. As shown in Figure 3a through e, representative whole-cell IK,Ca traces (apamin-sensitive current) elicited using a test potential of −100 mV from a holding potential of −55 mV are shown. Panel a represents a negative control from a nontransfected HEK 293 cell with no apamin-sensitive currents. IK,Ca density from HEK293 cells transfected with both α-actinin2 and SK2 channel was significantly larger than current density from HEK 293 cells transfected with SK2 channel alone (panels b versus c). Currrent density-voltage relations are summarized in Figure 3f showing significant increases in both the inward and outward current density by coexpression of α-actinin2 (*P<0.05). We further investigated the basis for the observed differences using 2 different α-actinin2 dominant-negative constructs, pBK-CMV-Act2-N (AA residues 1 to 251) containing only actin binding domain, and pBK-CMV-Act2-C (AA residues 344 to 745) containing only rod domain, both of which had previously been shown to compete with full-length α-actinin218 and may interrupt the association of SK2 channel with α-actinin2. We examined the functional effects of coexpressing full-length α-actinin2 and SK2 channel in the presence of dominant-negative trancated α-actinin2 proteins. As shown in Figure 3, d and e, coexpression of the dominant-negative constructs resulted in a reduction of the current density in HEK 293 cells transfected with α-actinin2 and SK2 channel. Panel g showed pooled data of current density elicited at the test potential of −100 mV from 6 different groups. IK,Ca density increased ≈4-fold when SK2 channel was coexpressed with α-actinin2 compared with SK2 channel expressed alone (**P<0.001). Furthermore, dominant-negative truncated α-actinin2 (AA residues 344 to 745) abolished the effects of full length α-actinin2 on SK2 current, reducing the current density by ≈53% (*P=0.04) and dominant-negative truncated α-actinin2 (AA residues 1 to 251) reduced SK2 current density by ≈33% (*P=0.04).
We next investigated whether the disruption of cytoskeletal protein using cytochalasin D may interfere with SK2 current coexpressed with α-actinin2. HEK 293 cells transfected with SK2 channel and α-actinin2 were pretreated with 2.5 μmol/L cytochalasin D for 4 hours (Figure 3g). Pretreatment with cytochalasin D significantly reduced the SK2 current density by ≈ 50% (*P=0.04, Figure 3g).
L-Type Ca2+ Channel Interacts With α-Actinin2 Revealed Using YTH System and Coimmunoprecipitation Using Human Heart Tissue
In the central nervous systems, SK channels has been shown to be activated by Ca2+ influx through VGCC, however, the targeting mechanisms and scaffolding molecules that are necessary to guarantee the functional coupling of Ca2+ sources to SK2 channels are still unknown. Here, we directly investigated the functional coupling to LTCCs, Cav1.3 and Cav1.2, to the multimolecular complex formed by α-actinin2 and SK2 channels.
In a YTH assay, the C-terminal region of Cav1.3 (Cav1.3-C1), encoding AA residues 1505 to 2203, indeed displayed a very strong interaction with C9-α-actinin2 prey construct (see Figure 4, a, b, d and e). In contrast, no interaction was documented between the N-terminal region of Cav1.3 (Cav1.3-N) encoding AA residues 1 to 157 and C9-α-actinin2 prey construct (see Figure 4d). Similar to SK2 channel, there were 2 binding sites on the Cav1.3 cytoplasmic carboxyl terminus for α-actinin2 protein. However, in contrast to SK2 channel shown in Figure l, the interactions at the 2 binding sites to α-actinin2 were equally strong on the high (panel d) or low (panel e) stringency media and appeared as white colonies. Additional four α-actinin2 fragments in prey clones were constructed containing each of the spectrin region (Figure 4a). As demonstrated in Panels d and e using a prey construct containing only the first spectrin repeat (Spectrin-1), using high or low stringency media, only 1 spectrin repeat was sufficient for the interaction. Data from Spectrin 2 to 4 were identical but not shown.
Figure 4c shows 2 different C-terminal constructs from Cav1.2 Ca2+ channel. Similar to Cav1.3 channel, both constructs interacted strongly with C9-α-actinin2 prey construct in the YTH assay resulting in white colonies using high and low stringency media.
The interactions between α-actinin2 and Cav1.3 or Cav1.2 channels in native cells were further examined using coimmunoprecipitation performed using human heart homogenates. As shown in Figure 4f, anti-α-actinin2 antibody coupled to agarose beads precipitated both Cav1.3 (lanes 4 to 5, MW ≈200 kDa) and Cav1.2 (lanes 2 to 3, MW ≈200 kDa) Ca2+ channel proteins from human ventricular (H-V) and atrial (H-A) tissues. The reverse experiments are performed in panel g. In addition, we further demonstrated that SK2 channel forms complexes with Cav1.3 Ca2+ channel in human ventricular (lane 7) and atrial (lane 8) tissues. Anti-SK2 antibody coupled to agarose beads precipitated (IP) Cav1.3 Ca2+ channel proteins as shown in the immunoblot (IB) lanes 7 to 8 from human ventricular and atrial tissues.
Colocalization of LTCC, α-Actinin2 and SK2 Complex in Mouse Atrial Cells
We used the same approach to detect the distribution of L-type Cav1.2 and Cav1.3 Ca2+ channel and colocalization with α-actinin2 protein. Single staining using anti-Cav1.3 antibody showed that the localization pattern of Cav1.3 Ca2+ channel (Figure 4, h and i) was consistent and similar to that of SK2 channel protein in mouse atrial cells using anti-Cav1.3 antibodies from 2 different sources (Sigma and Alomone). The signals could be completely eliminated when primary antibody was pre-incubated with antigenic peptide (Figure 4j). In contrast, the distribution of Cav1.2 Ca2+ channel in atrial myocytes was much different from that of Cav1.3 channel (panel k). Double staining of Cav1.3 and α-actinin2 or Cav1.3 and SK2 channels was further analyzed, as shown in Figure 4, l and m. Similar to α-actinin2 protein, Cav1.3 channel showed the highest staining pattern along the Z-line. Furthermore, Cav1.3 channel was dramatically colocalized with SK2 channel along the Z-line (Merged images in panel 4m, yellow, see also quantification of the colocalization in Figure IV in the online data supplement). In contrast, immunofluorescence labeling of Cav1.2 revealed that Cav1.2 Ca2+ channel only partly colocalized with α-actinin2 protein and SK2 channel in mouse atrial cells (panels n and o, see quantification in Figure V in the online data supplement). We performed additional control experiments using single isolated mouse atrial myocytes from homozygous Cav1.3 null mutant mice (Cav1.3−/−) with anti-Cav1.3 and anti-α-actinin2 antibodies compared with WT mice (Figure 4p) showing lack of staining by anti-Cav1.3 antibody in Cav1.3−/− atrial myocytes.
Functional Coupling of SK2 and Cav1.3 Ca2+ Channels
To further test the functional significance of the colocalization between SK2 channel and Cav1.3 Ca2+ channel, we use a Cav1.3 null mutant mouse model which had previously been generated.12 We have previously documented significant cardiac phenotypes in the null mutant mice with atrial arrhythmias and abnormalities in the pacemaking cells.13,19 Figure 5, panels a and b show a significant prolongation of the cardiac repolarization in isolated atrial myocytes in the null mutant mice compared with the wild-type animals at all stimulation frequency tested. Using two-pulse protocol as shown in Figure 5, panel c, we first depolarized the cells from a holding potential of –55 to 10 mV for 30 ms to activate Ca2+ current and to initiate the release of Ca2+ from the sarcoplasmic reticulum. The cells were then stepped to +60 mV to increase the driving force for K+ while decreasing the driving force for Ca2+ current. The protocol was repeated in the absence and in the presence of apamin (500 pmol/). Calculated EK in this recording condition was ∼–90 mV. We demonstrated that the prolongation of the cardiac repolarization in the null mutant mice could be accounted for, at least in part, by the decrease in the density of the apamin-sensitive IK,Ca current density in the Cav1.3 null mutant mice compared with the WT animals. Summary data are shown in Figure 5d. Careful assessment of the AP data shows significant AP prolongation in the range of −40 to −50 mV, whereas the HEK293 cell data suggest a rather small current in this range. Even though the current maybe small at this voltage range, the impedance of the cells is relatively large after the inactivation of the transient outward current and hence, the small current may play an important role in the repolarization of the AP.
To further define the role of Cav1.3 Ca2+ channel in SK2 channel expression, we performed immunohistochemistry experiments as shown in supplemental Figure VI in the online data Supplement. In Cav1.3 null mutant mice, there was a significant decrease in the SK2 staining in atrial myocytes compared with the WT animals consistent with the functional data presented in Figure 5, a through d. Taken together, our data suggest that α-actinin2 may act as the anchor for the SK2 channel at the membrane and that the decrease in IK,Ca is not simply because of the loss of Ca2+ influx in the membrane microdomain.
In the present study, we demonstrate that SK2 channel physically and functionally binds α-actinin2. Moreover, SK2 channels do not physically interact with LTCCs, instead, the 2 channels colocalize via their interaction with α-actinin2 cytoskeletal protein. To confirm these interactions, a variety of approaches were used. To address the functional significance of the interaction between α-actinin2 and SK2 channel, whole-cell apamin-sensitive IK,Ca was detected in HEK 293 cells using different plasmid composition. Coexpression of α-actinin2 and SK2 channel significantly increased IK,Ca density while addition of the dominant-negative constructs resulted in a reduction of the current density in HEK 293 cells. Furthermore, LTCCs were found to be intimately interacting with the same complex composing of α-actinin-2 and SK2 channel in the cardiac myocytes using similar approaches. Even though SK2 channel did not interact directly with LTCCs as suggested by the YTH screen, both channels were colocalized via interaction with α-actinin2 cytoskeletal protein. Cav1.3 Ca2+ channel was found to colocalized with SK2 channel along the Z-line, while Cav1.2 Ca2+ channel only partly colocalized with α-actinin2 protein and SK2 channel. We further sought the functional significance of the colocalization of Cav1.3 and SK2 channels using Cav1.3 null mutant mice.12,13 We have previously shown that the Cav1.3 Ca2+ channel is predominantly expressed in atrial myocytes and that the null mutant mice showed evidence of atrial arrhythmias.19 Here, we further demonstrate that functions of SK2 channels are dependent on normal expression of Cav1.3 Ca2+ channel in the mouse atrial myocytes.
SK channels are voltage-independent, and are gated solely by Ca2+i.1,2,20 These membrane channels are heteromeric complexes that contain pore-forming α-subunits and the Ca2+-binding protein calmodulin. SK channels are coupled to the activation of different subtypes of VGCCs in a cell-type-specific manner.6,21 LTCCs activate SK channels only, without activating BK channels. In contrast, N-type Ca2+ channels activate BK channels only. P/Q-type VGCCs do not couple to either SK or BK channels. These data suggest an absolute segregation of coupling between KCa channels and the different subtypes of VGCCs. However, little is known regarding the targeting mechanisms and scaffolding molecules that are necessary to guarantee functional coupling of Ca2+ sources to the SK channels.
We provided data to support the existence of the multiprotein complexes and the crosstalk between SK2 channels and Ca2+ channels via cytoskeletal proteins. Specifically, our data suggest that α-actinin2 may act as the anchor for the SK2 channel at the membrane and that the decrease in IK,Ca is not simply because of the loss of Ca2+ influx in the membrane microdomain.
Importance of Cytoskeleton in Ion Channel Functions
Previous work has demonstrated that muscle actinin isoform, α-actinin2, binds and modulates the function of a voltage-gated K+ channel, Kv1.5,22,23 NMDA-type glutamate receptor,18 Cav1.2 Ca2+channel24 and polycystin-2.25 Here, we used SK2 C-terminus as bait to screen human heart library and obtained a total of nine independent clones, all of which corresponded to the C-terminal fragment of α-actinin2. Our data further suggest that α-actinin2 interacts strongly with SK2 channels in cardiac myocytes and localizes the SK2 channel to the Z lines of the sarcomere where the sarcolemma form invagination called transverse tubules (T tubules)-specialized regions of surface membrane which extend into the cell interior and are oriented in register with Z bands of the nearest myofibrils and involved in Ca2+ release, and re-uptake (see model in supplemental Figure VII in the online data supplement). Furthermore, both SK2 and Ca2+ channels, mainly Cav1.3 subtype, colocalize in the T tubules via interactions with α-actinin 2 which represents an important component of the actin cytoskeleton. Indeed, one recent study has demonstrated a tight feedback loop between SK channels in the dendritic spines and NMDA receptors which has previously been shown to be anchored to the actin cytoskeleton by its direct interaction with α-actinin2.18,26
The exact mechanisms for the decreased expression of SK2 in Cav1.3−/− mice remain unclear. LTCCs may act via α-actinin2 as the anchor for the SK2 channel at the membrane. On the other hand, it is possible that a change in Ca2+ homeostasis in the Cav1.3−/− mice may affect the expression of various genes including SK2 through an excitation-transcription control as previously described in neurons.27 Additional experiments are also required to further define the roles of global versus local Ca2+ rise as well as the roles of sarcoplasmic reticulum Ca2+ in the SK2 channel activation.
The authors are in debt to the UC, Davis Health System Confocal Microscopy Facility. The Cav1.3 Ca2+ channel clone was a gift from Dr Susumu Seino (Department of Cellular and Molecular Medicine, Chiba University, Japan), dominant-negative α-actinin2 constructs are gifts from Dr Richard L. Huganir (Howard Hughes Medical Institute, The Johns Hopkins University), and α-actinin2 plasmid was a gift from Dr David Fedida (University of British Columbia, Canada).
Sources of Funding
Supported by NIH/NHLBI (HL68507, HL75274), the VA Merit Review Grant (to N.C.) NIH/NHIBI (HL77281), and the VA Review Grant (to A.A.K.).
Original received May 19, 2006; resubmission received September 13, 2006; revised resubmission received November 06, 2006; accepted November 8, 2006.
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