Published online before print February 12, 2009, doi: 10.1161/CIRCRESAHA.108.191007
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© 2009 American Heart Association, Inc.
Cellular Biology |
Kv4 Potassium Channels Form a Tripartite Complex With the Anchoring Protein SAP97 and CaMKII in Cardiac Myocytes
From the Institut National de la Santé et de la Recherche Médicale and Université Pierre-Marie-Curie-Paris 6 (S.E.-H., E.B., N.N., G.D., A.C., S.N.H.), UMRS 956, Paris, France; Department of Pharmacology and Toxicology, and Service of Cardiology, University of Lausanne (B.G., H.A.), Lausanne, Switzerland; and The Allen Institute for Brain Science (A.J.), Seattle, Wash.
Correspondence to Dr Stéphane N. Hatem, UMRS-956, Faculté de Médecine Pierre-Marie Curie, 91 boulevard de l’Hôpital, 75013 Paris, France. E-mail stephane.hatem{at}upmc.fr
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Abstract |
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Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, Ito, in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited Ito in cardiac myocytes, whereas its overexpression by using an adenovirus increased Ito. Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.
Key Words: potassium channels • cardiac myocytes • MAGUK proteins • calcium/calmodulin-dependent protein kinase • dipeptidyl peptidase–like protein 6
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Introduction |
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In the heart, the transient outward potassium current, Ito, is among the main repolarizing currents that contribute to the early repolarization phase of the action potential and to its adaptation to changes in cardiac cycle length.1,2 During cardiac diseases, Ito is often altered, thus contributing to the risk of cardiac arrhythmias.3,4
There is a general consensus that voltage-dependent Kv4.2 and Kv4.3 channels are the main molecular determinants of cardiac Ito.5–8 These 
subunits tether with several partners to form functional channels. The best-known partner of Kv4 channels is the Kv channel-interacting protein KChIP.9 This protein assembles with the N terminus of the pore-forming Kv4 subunit and acts as a chaperone, regulating the channels surface expression and electrophysiological properties.9,10 Dipeptidyl peptidase–like protein 6 (DPPX) is another subunit that regulates the activation and inactivation properties of Kv4 channels.11,12
Membrane-associated guanylate kinase (MAGUK) proteins are important partners for the organization of several ion channels.13,14 The MAGUK protein SAP97 is abundantly expressed in myocardium and interacts with voltage-dependent Shaker channels Kv1.515,16 and Kir channels.17 As in other tissues, SAP97 may regulate the targeting of cardiac ion channels in the sarcolemma. Indeed, in neonatal rat myocytes, SAP97 overexpression causes the clustering and immobilization of Kv1.5 channels in the plasma membrane and increases the corresponding current.18
The cardiac Ito current is regulated by several protein kinases.19–21 For instance, inhibition of Ca2+/calmodulin kinase (CaMK)II accelerates Ito inactivation, resulting in an enhanced fast transient component of the outward current.20 An interaction between CaMKII and Kv4.2/3 subunits has been observed in heterologous expression systems.22,23 In HEK293 cells, CaMKII inhibition increases the rate of inactivation of the Kv4.3 current and slows its recovery from inactivation, whereas it has no effect on Kv4.2 current under basal conditions.23,24 However, the molecular determinants of the interaction between CaMKII and Kv4 channels are still largely unknown.
Recently, it has been reported that SAP97 regulates the localization of Kv4.2 channels in dendritic spines of hippocampal neurons.25 Here, we investigated whether in the heart too, SAP97 interacts with Kv4 channels. Using various biochemical and functional approaches, we obtained new evidence for the interaction between cardiac Kv4.2/3 channels and SAP97. SAP97 regulated Ito in both cardiac myocytes and heterologous expression systems. Moreover, we describe for the first time that the regulation of Kv4.2/3 channels by CaMKII depends on the expression of SAP97.
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Materials and Methods |
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An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org and provides a description of myocyte isolation and cell cultures, recombinant proteins, short hairpin (sh)RNA, adenovirus, immunohistochemistry, pull-down assays, electrophysiological recordings, and data analysis.
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Results |
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SAP97 Interacted With the C Terminus of Cardiac Kv4 Channels
Previous studies have shown that Kv4 channels interact with PDZ domains of MAGUK proteins via consensus amino acid sequences conforming to xS/TxV/L, located at their C terminus.14,25 Thus, to determine whether SAP97 interacts with Kv4 channels in the heart, we performed pull-down experiments using glutathione S-transferase (GST) fusion proteins comprising the last 50-aa residues of Kv4.2 and Kv4.3 channels, which contain the putative PDZ domain–binding motif Ser-Ala-Leu (SAL). Figure 1A shows that both GST-Kv4.2 and -4.3 fusion proteins interacted with SAP97 obtained from protein lysates of human atrial and rat ventricular myocardium. As shown in Figure 1A, SAP97 did not coprecipitate with GST fusion proteins of Kv4.2 and Kv4.3 C termini lacking the SAL motif (
-SAL). These results suggest that SAP97 and cardiac Kv4 channel subunits interact directly via the channel C terminus.
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Kv4.3 Channels and SAP97 Colocalized in Cardiac Myocytes
The localization of SAP97 and Kv4.3 channels was examined in freshly isolated rat atrial myocytes stained with anti-Kv4.3 and anti-SAP97 and visualized by means of confocal microscopy. Figure 1B shows that both Kv4.3 and SAP97 staining predominated at the periphery of the myocytes, at the plasma membrane. Only faint diffuse fluorescence was seen in the rest of cell body (Figure 1B, a and b). Overlay of the red and green images revealed (Figure 1B, c) marked overlap of the fluorescent signals, with an excellent pixel correlation (Pearson correlation coefficient: 0.85).26 These observations indicate that SAP97 and Kv4.3 channel subunits colocalize at the plasma membrane of rat atrial myocytes.
SAP97 Enhanced the Membrane Clustering of Kv4.3 Channels in CHO Cells
We immunostained CHO cells stably expressing Kv4.3 channels and transfected with green fluorescent protein (GFP)-tagged SAP97. As shown in Figure 1C, Kv4.3 channels were diffusely distributed throughout the cell body of control CHO cells transfected with enhanced GFP, with mild staining at the plasma membrane. In Kv4.3-CHO cells transfected with GFP-SAP97, staining predominated at the membrane periphery and was organized in clusters where the 2 proteins colocalized (Pearson coefficient: 0.53; n=3 cultures). In KChIP2a-deficient CHO cells, SAP97 and Kv4.3 are also colocalized and organized in clusters at the level of the plasma membrane (Figure I in the online data supplement), indicating that SAP97 induces the clustering of Kv4.3 channels at the plasma membrane independently of KChIP2a.
SAP97 Increased Kv4.3 Currents in CHO Cells
To study the functional consequences of the interaction between the SAP97 and Kv4 channel subunits, SAP97 was transiently expressed in a CHO cell line stably expressing the Kv4.3 channel subunit and KChIP2a. At all potentials, the current peak density was increased in cells overexpressing SAP97 (Figure 2A and 2B). Time-dependent inactivation of the current, which was best fitted by a double-exponential function, was slowed in SAP97 conditions, as indicated by the increase in both the 
fast and the slow of inactivation (Figure 2C). SAP97 had no effect on the voltage-dependent activation, inactivation, and recovery from inactivation of the current (Figure 2D and 2E).
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) and overexpressing SAP97 (
) CHO cells. C, The time constant (
In the absence of KChIP2a, and in keeping with defective channel trafficking,12 smaller outward currents were recorded in CHO cells (at +60 mV: Kv4.3 current: 43.1±8.2 pA/pF n=9 versus 177.1±24 pA/pF n=11; Kv4.2 current: 12.4±1.9 pA/pF n=9; supplemental Figure II). However, in these conditions, SAP97 still enhanced Ito density to the same extent (
2-fold) as in the presence of KChIP2a (Figure 2F and supplemental Figure II). In presence of SAP97, we still observed the canonical effects of DPPX on Kv4.3 inactivation and activation (supplemental Figure III). Together, these results suggest that SAP97 regulates the functional expression of Kv4 channels independently of KChIP2a and DPPX.
SAP97 Modulated Ito in Atrial Myocytes
Next, we studied whether SAP97 can modulate endogenous Kv4 channels of cardiac myocytes, by studying the consequence of SAP97 suppression on Ito in adult rat atrial myocytes using a shRNA targeting rat SAP97 (shSAP97). We first checked that shSAP97 inhibited endogenous cardiac SAP97 protein using Western blot assay (49±3% decrease, n=3) performed with proteins from rat cardiac cell cultures (inset in Figure 3A). After 3 to 4 days in culture, 20% of the adult atrial myocytes were green fluorescent and were used for the patch-clamp recording. In myocytes transfected with a scrambled shRNA (shSCR) (n=20), an outward current was recorded with both a transient component (Ito) and a large maintained (IKur) component. In myocytes transfected with shSAP97, a much smaller outward current was recorded (Figure 3B and 3C).
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The consequences of SAP97 overexpression were studied in atrial myocytes using a recombinant adenovirus (AdSAP97).18 Two days after the infection, Ito and IKur were markedly increased (Figure 3D and 3E and supplemental Figure IV), and the Ito inactivation was slowed down, as indicated by the increase in fast and slow (Figure 3F). These results suggest that endogenous SAP97 modulates cardiac Ito.
SAP97 Was Necessary for CaMKII Regulation of Kv4.3
We then examined whether the interaction between Kv4 C terminus and SAP97 was involved in the increase in the outward K+ current. The Kv4.3--SAL subunits and SAP97 were coexpressed in CHO cells. In these conditions, SAP97 failed to increase Kv4.3--SAL–encoded current (Kv4.3--SAL: at +60 mV, 74.5±14 pA/pF, n=9; versus SAP97/Kv4.3--SAL: 57.7±10.8 pA/pF, n=8; P=NS).
Surprisingly, Kv4.3--SAL–encoded current showed a drastically increased rate of inactivation compared to the wild-type (WT) current (Figure 4A). The characteristics of the Kv4.3--SAL–encoded current resembled those of the Kv4-encoded current following CaMKII inhibition in HEK293 cells.22,23 In our conditions too, intracellular application of the CaMKII inhibitor KN93 was associated with gradual increase of the rate of inactivation of the outward current (Figure 4B). The inactive analog of KN93, KN92, had no effect on Kv4.3 channel–encoded current (data not shown). In CHO cells expressing Kv4.3--SAL, KN93 failed to modify the current inactivation (Figure 4B and 4C). Similar results were obtained using the CaMKII inhibitory peptide, AIP (Figure 4C). Of note the effect of DPPX on Kv4.3--SAL inactivation kinetics did not compensated for the lack of SAP97 binding, indicating distinct effects of the 2 partners on Kv channel gating (supplemental Figure III). Thus, in the absence of the SAL motif, Kv4.3 channel is no longer regulated by CaMKII.
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The SAL Motif of Kv4 C Terminus Was Necessary for the Interaction Between CaMKII and the Channel
We then examined whether the SAL motif is involved in the interaction between Kv4 channel subunits and CaMKII. Pull-down assays were performed using the GST fusion C terminus of WT and mutated Kv4 channels. As shown in Figure 5A and 5B, both Kv4.2 and Kv4.3 C termini precipitated CaMKII from rat ventricular myocardium protein lysates but not the Kv4--SAL C terminus (n=3). GST fusion C terminus also precipitated endogenous CaMKII of CHO cells (n=3) (supplemental Figure V).
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To examine whether SAP97 and CaMKII interact in cardiac myocytes, coimmunoprecipitation assays were performed with total rat heart proteins. The anti-SAP97 antibody precipitated a large 140-kDa band, the molecular mass of SAP97 (Figure 5C). Probing the membrane with the anti-CaMKII antibody revealed a doublet at around 50 and 60 kDa, corresponding to the molecular mass of 
B and C isoforms of CaMKII.27 To confirm the interaction of the 2 proteins, we also immunoprecipitated CaMKII (Figure 5C) and hybridized the membrane with the anti-SAP97 antibody that labeled a weak but distinct 140-kDa band corresponding to SAP97.
Furthermore, we performed pull-down experiments using the 50 last amino acids of Kv4.3 in cardiac myocytes transfected with the SAP97 shRNA. In SAP97-silenced myocytes, much less CaMKII could be precipitated with the Kv4.3 C terminus (n=3) (Figure 5D). In contrast and using the same pull-down approach, much more CaMKII could be precipitated with the GST-K4.3 C terminus in myocytes infected with the AdSAP97 (Figure 5E). Moreover, in infected myocytes overexpressing SAP97, Ito was more sensitive to CaMKII inhibition (at +60mV, AIP inhibition on fast was: 25.3±4.7%, n=7; versus 52±4.9%, n=9; P<0.01; supplemental Figure IV). Taken together, these data indicate that CaMKII/Kv4.3 channel interaction requires the SAL motif and involves SAP97.
SAP97 Silencing Mimicked the Effect of CaMKII Inhibition on Kv4.3 Current
To determine whether SAP97 inhibition can suppress the regulation of Kv4.3 current by CaMKII, we studied the effects of SAP97 silencing using a shRNA on current recorded in HEK293 cells that express endogenous SAP97. Regulation of Kv4.3 channels by CaMKII has been already characterized in this cell line, showing that CaMKII inhibition accelerates the current inactivation and slows its recovery from inactivation.22,23 In HEK293 cells cotransfected with Kv4.3 subunits and shSAP97, the outward current was reduced (Figure 6A and 6B), and its time-dependent inactivation was markedly accelerated (Figure 6C) compared with cells transfected with shSCR. In HEK293 cells transfected with shSAP97, steady-state inactivation was shifted leftward (Figure 6D) and the recovery from inactivation was slowed (Figure 6E) as observed following CAMKII inhibition in control conditions (not shown).23 Moreover, in cells transfected with the SAP97-shRNA, the current was insensitive to intracellular application of KN93 (fast reduction of 2.4±0.7%; n=4; P=NS) and AIP (fast reduction of 2.1±0.9%; n=8; P=NS, Figure 7A and 7B). In HEK293 cells transfected with the shSAP97, GST-Kv4.3 C terminus failed to precipitate CaMKII (Figure 7C). In HEK293 cells, SAP97 silencing suppressed Kv4.2 current (Figure 8A and 8B; n=15) but had no effect on its biophysical properties (Figure 8C through 8E). A much smaller outward current was also recorded in mutated (Kv4.2--SAL) than in WT-Kv4.2 channels but without difference in the kinetics of current inactivation between the two isoforms (at +60mV, Kv4.2: 180±20 pA/pF, n=14; versus Kv4.2--SAL: 55.0±10.9 pA/pF, n=7; P<0.001). High [Ca2+]i (0.5 µmol/L), used to stimulate the CaMKII,22 decreased the rate of inactivation of WT but not mutated (-SAL) Kv4.2 channels, an effect suppressed by SAP97-shRNA (Figure 8F and 8G). The CaMKII inhibitor AIP had no effect on Kv4.2 isoforms (data not shown).
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Two main findings emerge from this study. First, we found that SAP97 regulates the functional expression of Kv4 channels in the heart. Second, we describe a new property of this anchoring protein, as a molecular adaptor between Kv4 channels and CaMKII. We also provide the first direct demonstration that endogenous SAP97 regulates the excitability of cardiac myocytes.
Previous studies have shown that Kv4 channels containing the C-terminal sequence xS/TxV/L can bind to the PDZ domains of MAGUK proteins.14 For instance, the SAL motif is involved in the direct interaction of Kv4.2 channels with PSD95.28 Here, we found that SAP97 can be precipitated by Kv4 C terminus only in the presence of the SAL motif and that it fails to increase the mutated Kv4.3--SAL–encoded current. These results strongly suggest that the channel binds to PDZ domains of SAP97 protein by its C terminus, in agreement with the binding selectivity of PDZ domains, as observed by using protein microarrays and quantitative fluorescence polarization.29 This interaction between SAP97 and Kv4.3 subunits has marked consequences for the outward potassium current, as clearly indicated by the 2-fold increase in its density in CHO cells overexpressing SAP97. In cardiac myocytes, downregulation of endogenous SAP97 with shRNA drastically reduced the outward K+ current, whereas SAP97 overexpression resulted in an increase in Ito. In atrial myocytes, the fast component of Ito is believed to be due largely to functional expression of Kv4 channels.5 Thus, these results obtained with native cells strengthen the conclusion that SAP97 regulates cardiac Kv4 channels. Moreover, the modulation of IKur by SAP97 is in line with the interaction between SAP97 and Kv1.5 channels.15,16,30–32 Finally, these data obtained in cardiac myocytes constitute the first direct evidence for a role of endogenous SAP97 in cardiac excitability. In both cell types, SAP97 did not change the conductance properties of the channel, suggesting that it increases in the density of functional channels. This is further supported by the accumulation and clustering of Kv4.3 channels at the plasma membrane in CHO cells overexpressing SAP97 and their colocalization in the sarcolemma of cardiac myocytes. Collectively, these results are in line with the current idea that MAGUK proteins are part of a scaffold complex that anchors ion channels in the plasma membrane. This has been clearly established in the case of PSD95, which prevents the internalization of Kv4.2 channels.28 The effects of SAP97 on current and membrane expression of Kv4 channels were observed independently of KChIP2a and DPPX. This is in good agreement with a crystallography study showing that KChIP laterally clamps 2 neighboring Kv4.3 subunits, linking the N terminus in a 4:4 manner, far from the binding site of SAP97 on Kv4.3 C terminus33 and near the likely binding site of DPPX on S1-S2 domain of the Kv channel.11
One striking observation in this study was that Kv4.3--SAL channels were not regulated by CaMKII, unlike their WT counterpart. In both cardiac myocytes and cell lines expressing Kv4 channels, CaMKII inhibition is associated with a marked acceleration of Ito inactivation.20,22,23 Moreover, the site of phosphorylation by CaMKII has been identified as Ser550, located at the C terminus of Kv4.3 channels.23 Here, we found that the Kv4.3--SAL–encoded current showed a fast inactivation similar to that of WT channels, following CaMKII inhibition, and that it was not sensitive to several CaMKII inhibitors. Previous studies have shown that it is possible to coprecipitate CaMKII and Kv4.3 channels, but the nature of the interaction was unknown.22 We provide a strong body of evidence pointing to a role of SAP97 in this interaction. First, CaMKII precipitation by the Kv4.3 C terminus required the presence of the SAL motif. Second, CaMKII and SAP97 can be coprecipitated from cardiac protein lysate as observed in hippocampal rat brain.34 Third, in HEK293 cells, SAP97 inhibition with shRNA not only reduces the current amplitude but also mimics the effect of CaMKII inhibition on biophysical properties.22,23 Fourth, in both cardiac myocytes and HEK293 cells silenced for the shSAP97, the interaction between Kv4.3 C terminus and CaMKII was suppressed. The modulation of SAP97 expression having no effect on Kv4.2 current inactivation was consistent with the lack of regulation of Kv4.2 channels by CaMKII under basal conditions (ie, low [Ca2+]i),22,24 whereas the suppression of SAP97/Kv4.2 channel interaction inhibited the effect of high [Ca2+]i on current kinetics. Finally, in myocytes overexpressing SAP97, the coupling between CaMKII and Kv channels was enhanced.
Protein complexes containing CaMKII, SAP97 and ion channels or membrane receptors are already known and have been described for Kv4.2 channels25 and the N-methyl-D-aspartate (NMDA) receptor.34–36 CaMKII and PSD95 bind the same restricted region of the C terminus of some NMDA receptors (eg, NR2A).37 It has also been shown that SAP97 is the CaMKII target at amino acid residues 39 and 232.34 In hippocampal neurons, CaMKII can phosphorylate SAP97, resulting in increased colocalization between NMDA receptors and SAP97. The SAP97-PDZ1 domain interacts with both NR2A and GluR1 in neuronal cells, and CaMKII-mediated phosphorylation of Ser232 is critical for the interaction with NR2A but not with GluR1.34 SAP97 phosphorylation by CaMKII is also crucial for the targeting of SAP9738 and its effects on the trafficking of Kv4.2 channels from the endoplasmic reticulum to dendrites and spines of hippocampal neurons.25 In contrast with other protein kinases, there is an apparent diversity in CaMKII targeting mechanisms.39 For instance, in neurone CaMKII can bind to NMDA receptor, the PSD-enriched 180-kDa densin-180 protein and -actinin. The ability of CaMKII to interact simultaneously with multiple proteins could confer specific physiological function to this kinase. In the heart too, other partners than SAP97 might be involved in CaMKII interaction with channel multiprotein complexes.
Previous reports have shown that SAP97 can act as a link between signaling proteins and their targets. For instance, SAP97 interacts with the inward rectifier K+ channel via its PDZ domain and makes the current G protein–sensitive.40 Phosphorylation of β-adrenergic receptors by cAMP-dependent protein kinases depends on SAP97/AKAP interaction.41 The role of SAP97 as an organizer of signaling domains of cardiac myocytes has been also highlighted by the observation that SAP97 clusters with β-adrenergic pathways at the site of contact between myocytes and sympathetic neurons.42 Our study brings another clue for the role of SAP97 in the formation of signalosome in the heart.
In the heart, CaMKII regulates several ionic currents,43 including Ito,20 and is crucial for the adaptation of cardiac electric activity to intracellular calcium signal. The enhancement in the rate of Ito inactivation caused by CaMKII should shorten the action potential and consequently reduce the L-type calcium current and the release of calcium from the sarcoplasmic reticulum. Moreover, CaMKII can also participate in the occurrence of arrhythmias by its effects on ion channels and cardiac hypertrophy44–46 Thus, SAP97 that regulates the Kv4 interaction with CaMKII appears as an important molecular determinant of cardiac function.
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Acknowledgments |
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We thank I. Cantaloube for technical support.
Sources of Funding
We thank the Association Française contre les Myopathies and Agence Nationale de la Recherche (grant ANR-05-PCOD-006-01) for financial support. S.E.-H. is the recipient of a fellowship from the French Ministère de l’Education Nationale et de la Recherche Scientifique. Work in the laboratory of H.A. has been supported by a grant from the Swiss National Science Foundation (310030_120707).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received May 26, 2008; resubmission received November 12, 2008; revised resubmission received January 14, 2009; accepted February 2, 2009.
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