14-3-3 Is a Regulator of the Cardiac Voltage-Gated Sodium Channel Nav1.5
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Marie Allouis, Franoise Le Bouffant, Ron
参见附件。
Inserm UMR 533 (M.A., F.L.B., D.P., J.-J.S., J.N., H.L.M., J.M., D.E., I.B.), Université de Nantes, Nantes Atlantique Universités, l’institut du thorax, Faculté de Médecine, France
the Department of Physiology (R.W.), Academic Medical Center, University of Amsterdam, the Netherlands.
Abstract
The voltage-sensitive Na+ channel Nav1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3, interacting with the Nav1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Nav1.5 both in COS-7 cells expressing recombinant Nav1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Nav1.5 colocalized at the intercalated discs. We tested the functional link between Nav1.5 and 14-3-3 using the whole-cell patch-clamp configuration. Coexpressing Nav1.5, the 1 subunit and 14-3-3 induced a negative shift in the inactivation curve of the Na+ current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Nav1.5 cytoplasmic I interdomain interacting with 14-3-3. Reversal was also obtained with the dominant negative R56,60A 14-3-3 mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na+ channel acting as a cofactor for the regulation of the cardiac Na+ current.
Key Words: Na+ channel auxiliary subunit congenital heart disease
Introduction
Voltage-gated Na+ channels are responsible for initiation and propagation of the action potential in most electrically excitable cells. They are transmembrane glycoprotein complexes consisting of a pore-forming subunit and accessory subunits.1 The subunit has 4 homologous domains, each containing 6 transmembrane segments and an outer pore-forming loop. The 3 interdomain linkers (ID I to III), and the N- and C-terminal ends of the protein are cytosolic. Ten -subunit isoforms have been cloned from different mammalian tissues.2 Among accessory subunits regulating voltage-gated Na+ channels, 4 -subunit isoforms have been identified3 and shown to modulate the channel-gating properties.4 In the human heart, SCN5A and SCN1B encode the major voltage-sensitive Na+ channel subunit Nav1.5 (also called hH1) and the 1 subunit (h1), respectively. Recent studies have shown that Nav1.5 associates with other proteins that regulate its biosynthesis, localization, activity, and/or degradation.1 Given the major role of Nav1.5 in the cardiac action potential, the expression and activity of these auxiliary proteins are likely important determinants for the electrical excitability of the cardiomyocytes and cardiac conduction.
In the present study, we report and characterize the association of Nav1.5 with 14-3-3 proteins. 14-3-3 family members form a group of highly conserved 30-kDa cytosolic acidic proteins expressed in a wide range of organisms and tissues.5 In mammals, this family consists of 7 members encoded by separate and differentially expressed genes.6 14-3-3 proteins can interact with numerous cellular proteins and are crucial for signaling, cell growth, division, adhesion, differentiation, apoptosis, and ion-channel regulation.7 We show that 14-3-3 and Nav1.5 colocalize at the intercalated discs in cardiomyocytes. We found that the interaction of the 2 proteins altered the cardiac Na+ channel function. Considering that 14-3-3 proteins are expressed in cardiac myocytes, we also show that the Na+ current alterations attributable to 14-3-3 absence are expected to result in the alteration of the restitution of action potential duration and conduction velocity facilitating arrhythmia occurrence.
Based on these findings, we propose that the 14-3-3 protein is a novel member of the cardiac Na+ channel acting as a cofactor for the regulation of the cardiac Na+ current.
Materials and Methods
An expanded Materials and Methods section containing details for plasmids, yeast 2-hybrid screening, coimmunoprecipitation, pulldown assays, Western blotting, immunocytochemistry, electrophysiology, and computer simulations is available in the online data supplement at http://circres.ahajournals.org.
Yeast Two-Hybrid Screening
The first interdomain fragment of human Nav1.5 (hNav1.5 ID I) was used as bait. The yeast reporter strain L40, which contains the reporter gene HIS3 downstream of the binding sequence for LexA, was sequentially transformed with the pVJL10-ID I plasmid and with a mouse cDNA library, using the lithium acetate method8 and subsequently treated as previously described.9
Coimmunoprecipitation, Pulldown, and Western Blotting
African green monkey kidney-derived COS-7 cells (ATCC), mouse heart, anti–14-3-3 mouse monoclonal antibody (Santa Cruz Biotechnology) and anti-Nav1.5 rabbit polyclonal antibody (ASC-005, Alomone Labs) were used. GST-F11 fusion protein (F11 corresponding to residue 417 to 467 of human Nav1.5) has been used during pulldown experiments on mouse heart lysate.
Immunocytochemistry
Immunostaining was performed as described by Mohler et al10 on COS-7 cells expressing the human tagged Nav1.5-GFP and HA-14-3-3 and on freshly isolated rabbit cardiomyocytes11 using also anti-HA rabbit polyclonal antibody (Clontech) and Alexa Fluor 488- or 568-conjugated secondary antibodies (Molecular Probes).
Electrophysiology
Patch-clamp studies were performed on COS-7 cells transiently expressing hNav1.5 and h1 subunits, with or without human wild-type and/or double mutant R56,60A 14-3-3, using the whole-cell configuration at room temperature.
Computer Simulations
The functional role of the 14-3-3 protein in shaping the cardiac action potential was assessed by computer simulations using the human ventricular cell model by ten Tusscher et al.12
Statistics
Data are presented as mean±SEM. Statistical significance of the observed effects was assessed by means of the t test or 2-way ANOVA followed by a Tukey test for multiple comparisons when needed. A value of P<0.05 was considered significant.
Results
The Intracellular Interdomain ID I of Nav1.5 Interacts With 14-3-3
The yeast 2-hybrid was used to screen a mouse cDNA library with hNav1.5 cytoplasmic I interdomain (ID I) as bait (amino acids 417 to 711; Figure 1A). Among the 22 clones able to grow in the absence of histidine, we identified full-length 14-3-3 cDNA by sequencing and database searching. The specificity of the interaction was confirmed by cotransformation of the bait and 14-3-3 or GAL4 AD into the yeast L40 (Figure 1B). To refine the site responsible for the interaction site, we created LexA-fusion protein baits containing various fragments of Nav1.5 ID I (Figure 2A) and tested their ability to interact with 14-3-3 (Figure 2B). 14-3-3 interacted with Nav1.5 ID I fragment 1 (F1) and more precisely with its N terminus, ie, the 417 to 467 amino acid sequence of Nav1.5 (F11). A 99% identity between mouse and human 14-3-3 protein sequences and a 100% identity between residues 417 to 467 of mouse and human Nav1.5 were observed. We challenged the specificity of the interaction with other 14-3-3 isoforms. Using mouse and isoforms of 14-3-3, we observed that these isoforms were able to induce yeast growth (Figure 3). Indeed there are 82% and 86% homology between mouse protein sequences of and isoforms and and isoforms, respectively.
14-3-3 Interacts With Nav1.5 in Transfected COS-7 Cells and in the Heart
To provide biochemical evidence for the interaction between full-length Nav1.5 and 14-3-3, we immunoprecipitated the complex from IGEPAL extracts of Nav1.5-GFP–transfected COS-7 cells or mouse heart (Figure 4A and 4B) using antibodies against GFP, 14-3-3, or Nav1.5 proteins. When 14-3-3 immunoprecipitates were blotted and probed with the anti-GFP (COS-7 cells) or anti-Nav1.5 antibody (mouse heart), coprecipitation of the full-length channel was revealed. Inversely, 14-3-3 was detected in the immunoprecipitated Nav1.5 complex from mouse heart extract. The interaction of 14-3-3 and F11 of Nav1.5 was confirmed by pulldown experiments on mouse heart lysate showing the presence of 14-3-3 among the proteins specifically bound to GST-F11 (Figure 4C and 4D).
We determined the subcellular distribution of Nav1.5 and 14-3-3 using confocal microscopy. Immunostaining revealed colocalization of Nav1.5 and 14-3-3 in the plasma membrane of transfected COS-7 cells (supplemental Figure I). In rabbit cardiomyocytes, 14-3-3 localization was assessed by using a monoclonal antibody that detects members of the 14-3-3 protein family. Fluorescent labeling of polymerized actin with phalloidin–Texas Red stained the actin thin filaments inserting at the intercalated discs (Figure 5, top). A diffuse immunolabeling of 14-3-3 protein was observed in the cytoplasm. Clusters were seen at the intercalated discs, where 14-3-3 colocalized with Nav1.5 (Figure 5, bottom). Colocalization of 14-3-3 protein and Nav1.5 at the intercalated discs is consistent with a physiological interaction between these proteins in situ.
14-3-3 Modulates the Na+ Current by Direct Protein–Protein Interaction
To investigate the functional consequence of 14-3-3 on the channel activity, we used the whole-cell configuration of the patch-clamp technique. The presence of overexpressed human 14-3-3 (h14-3-3) in COS-7 cells expressing hNav1.5 and h1 subunits, did not modify the Na+ current density (–96.1±6.6 pA/pF; n=29 versus –99.3±5.2 pA/pF; n=32; in the absence and in the presence of exogenous 14-3-3, respectively; current measured at –20 mV; Figure 6A). The voltage dependence of the Na+ current activation and inactivation were also investigated. No changes were observed in the activation parameters (V1/2act: –35.7±0.9 mV, n=10, versus –34.6±1.1 mV, n=16; slope: 5.1±0.2 mV versus 5.4±0.3 mV; Figure 6B). On the other hand, the presence of exogenous 14-3-3 shifted the inactivation curve toward more negative values (V1/2inact: –79.2±1.2 mV, n=13, to –84.5±0.9 mV, n=21; P<0.001; Figure 6B) without change in the slope (–6.0±0.2 mV versus –6.1±0.1 mV). Inactivation was not significantly accelerated nor decelerated at any potential in the presence of exogenous 14-3-3 (not shown). Finally, recovery from inactivation was decelerated in the presence of exogenous 14-3-3 (2-way ANOVA: P<0.001; Figure 6C). The time course of recovery from steady-state inactivation was quantified by the time to reach 50% and 75% recovery (t1/2 and t3/4, respectively). Recovery from inactivation in the presence of 14-3-3 was slower (t1/2: 18.0±2.0 ms; t3/4: 52.3±6.3 ms) than in its absence (t1/2: 9.5±1.2 ms; t3/4: 22.5±5.6 ms; P<0.01). Coexpression of 14-3-3 and F1 prevented this deceleration as demonstrated by t1/2 and t3/4 values of 9.3±1.4 and 20.1±2.9 ms, respectively (P<0.01 versus +14-3-3; non-significantly different (NS) versus absence of 14-3-3).
To further evaluate the implication of protein-protein interaction in these changes, we used the F1 peptide (ie, amino acids 417 to 507 of Nav1.5) to compete with full-length Nav1.5 for association with exogenous 14-3-3. We first tested the effects of coexpressing Nav1.5 and F1 (pIRES-F1-Nav1.5). We observed no change in the current density (–121.8±21.2 pA/pF, n=19, in the presence of F1) nor in the inactivation curve (V1/2inact: –80.2±2.6 mV; slope: –6.2±0.3 mV; n=6; in the presence of F1; not illustrated). This suggests that endogenous 14-3-3 does not regulate overexpressed Nav1.5.
In cells expressing 14-3-3 and Nav1.5 (pIRES-14-3-3-Nav1.5), F1 (pcDNA3-F1) reversed the inactivation shift (V1/2inact: –79.8±1.2 mV; slope: –6.2±0.5 mV; n=8; NS in comparison with cells expressing Nav1.5 alone; Figure 6D), produced no change in current density (–97.7±22.3 pA/pF; n=16; NS in comparison with cells expressing Nav1.5 alone), and prevented the recovery from inactivation deceleration (2-way ANOVA: NS in comparison with cells expressing Nav1.5 alone; Figure 6C).
14-3-3 Forms Dimers for Functional Regulation of Nav1.5
14-3-3 proteins have a dimeric structure and monomeric proteins may not regulate their target. Double mutant R56,60A 14-3-3 has been shown to associate with wild-type 14-3-3 but to impair binding with ligands,13 resulting in a dominant negative activity (DN-14-3-3). Here, DN-14-3-3 prevented the inactivation shift induced by wild-type 14-3-3 (V1/2inact: –79.5±1.5 mV; slope: –6.2±0.3 mV; n=8; both NS in comparison with cells expressing Nav1.5 alone; Figure 6D). This suggests the requirement of 2 intact binding sites on 14-3-3 dimer to regulate the Na+ current.
14-3-3 Affects the Cardiac Action Potential
We performed computer simulations to assess the functional role of the 14-3-3 protein in shaping the cardiac action potential. The effects of the absence of 14-3-3 on INa were implemented into a human ventricular cell model12 by a +5.3-mV shift of the INa steady-state inactivation curve and a 70% increase in the rate constants governing fast and slow recovery from INa inactivation (see online data supplement). To validate this approach, we performed in silico voltage clamp experiments using the same protocols as in our experiments (Figure 7A through 7C). As illustrated in Figure 7A, these alterations did not result in significant changes in current density (for example, Figure 6A). When fitting Boltzmann curves to the in silico data (Figure 7B), no differences were observed in activation parameters (V1/2act: –42.1 mV; slope: 5.0 mV). In contrast, the inactivation curve was shifted toward more positive values in the absence of 14-3-3 (V1/2inact: –75.4 versus –79.9 mV; +4.5-mV shift) without change in slope (–7.1 mV). Figure 7B (inset) shows that this shift resulted in increased window current in the absence of 14-3-3, as also seen experimentally (Figure 6B). Finally, the rate of inactivation was not changed, whereas recovery from inactivation was accelerated in the absence of 14-3-3 (Figure 7A and 7C). As in the experiments shown in Figure 6, the time course of recovery from steady-state inactivation was quantified by the time to reach 50% and 75% recovery: t1/2 was 19.9 ms in the presence of 14-3-3 and 11.7 ms in its absence. Similarly, t3/4 was 39.8 ms in the presence of 14-3-3 and 23.4 ms in its absence. Thus, recovery in the model, which was designed for physiological temperature, was not significantly faster than in the experiments performed at room temperature. This model feature is based on the experimental observation that the relative amplitude of the slow components of recovery increases at close to physiological temperature.14
Figure 7D shows the effects of the absence of 14-3-3 through its action on INa at a pacing interval (basic cycle length [BCL]) of 800 ms (75 bpm). Changes to the shape of the action potential were mild. The 4- to 5-mV increase in action potential overshoot in the absence of 14-3-3 reflected the increase in peak Na+ current (Figure 7D, bottom) attributable to the higher availability of Na+ channels. This also resulted in a steeper upstroke (higher maximum upstroke velocity) in the absence of 14-3-3 (402 versus 334 V/sec in its presence; 21% increase; Figure 7F, top). At BCL=800 ms, the higher availability of Na+ channels was caused by the positive shift in steady-state inactivation rather than the accelerated recovery from inactivation (Figure 7F, bottom).
Because we expected the effects of faster recovery from inactivation to be augmented at higher pacing rate, we repeated our simulations with a pacing interval of 400 ms (150 bpm; Figure 7E). At this pacing interval, the control action potential (+14-3-3, solid line) had shorter action potential duration (APD) (222 versus 255 ms at a pacing interval of 800 ms) and lower maximum upstroke velocity (271 versus 334 V/sec). The combined effects of increased Na+ channel availability and accelerated recovery from inactivation in the absence of 14-3-3 resulted in an enhanced Na+ current (Figure 7E, bottom) associated with a 6-mV increase in action potential overshoot and a 34% increase in maximum upstroke velocity (Figure 7F, top). At BCL=400 ms, the higher availability of Na+ channels in the absence of 14-3-3 was not only attributable to the positive shift in steady-state inactivation but also to the accelerated recovery from inactivation (Figure 7F, bottom).
14-3-3 Changes Cardiac Electrical Restitution Properties
Changes in APD and conduction velocity (CV) restitution curves, relating APD and CV to the preceding diastolic interval, have been implicated in the susceptibility to cardiac arrhythmias.15–19 The significant increase in Na+ current in the absence of 14-3-3, preferentially at high pacing rate, facilitates action potential formation and conduction, and may thus affect APD and CV restitution. Therefore, we determined APD and CV restitution curves in the absence and presence of 14-3-3 using standard protocols (see online data supplement). Figure 8A shows that the APD restitution curve in the absence of 14-3-3 was steeper than under control conditions (+14-3-3). Notably, its slope exceeded 1 at short diastolic interval, whereas the slope of the control curve was <0.7. The CV restitution curve is shown in Figure 8B. In the absence of 14-3-3, conduction velocity was higher, especially at short pacing intervals. As a result, the CV restitution curve was flattened. Also, the absence of 14-3-3 allowed successful conduction at higher pacing rates and shorter diastolic intervals. The shortest pacing interval (5-ms resolution steps) resulting in successful action potential conduction was 240 ms in the absence of 14-3-3 (diastolic interval of 40 ms) and 280 ms (diastolic interval of 72 ms) in its presence.
Kagan et al13 have previously reported that the 14-3-3 protein also affects the rapid delayed rectifier current (IKr), ie, the current carried by the HERG channel. We have performed additional computer simulations to assess the effects of the 14-3-3 protein through its action on IKr. These effects appeared to be relatively modest (supplemental Figures II and III).
Discussion
In the present study, a direct interaction between the cardiac voltage-dependent Na+ channel Nav1.5 and 14-3-3 protein was identified using a 2-hybrid screen. The 14-3-3–binding region was determined in the first interdomain of Nav1.5 between amino acids 417 and 467. We also showed colocalization of 14-3-3 and Nav1.5 at intercalated discs of cardiomyocytes and both coimmunoprecipitation and pulldown experiments confirmed their physical association. Functional studies using the whole-cell patch-clamp configuration brought further light to the regulation of Na+ channel activity by 14-3-3.
14-3-3-interacting consensus sequences have been extensively analyzed in mammalian systems (for review, see Bridges and Moorhead20). Two consensus phosphopeptide motifs (ie, RSPXpSXP and RXXXpSXP, where X is any amino acid and pS is phosphoserine) have been uncovered. 14-3-3 binding to its target has been shown to depend on phosphorylation of a serine or threonine. None of these motifs is present in the Nav1.5 417 to 467 sequence interacting with 14-3-3. However, many target proteins do not contain sequences conforming precisely to these motifs or do not need phosphorylation to bind.20 Noteworthy, the protein kinase A (PKA)-dependent mechanism of -adrenergic stimulation regulating functional expression of INa by phosphorylation of S525 and S528 is located in I to II linker, ie, close to the site interacting with 14-3-3.21 These residues could be protected by 14-3-3 from dephosphorylation, as proposed for HERG.13 In addition, INa of native cardiomyocytes22,23 or recombinant INa show a shift in channel inactivation and slower recovery from inactivation on adrenergic stimulation,21 as we observed with 14-3-3. However, unlike adrenergic stimulation, 14-3-3 induced neither an increase in current density nor a shift in voltage dependence of channel activation. Therefore, 14-3-3 effects on Nav1.5 differ from those expected of a protection of PKA sites from phosphatase, as shown for HERG.13
Our results suggest that 14-3-3 dimers regulate Nav1.5 channels. If so, 14-3-3 may contribute to the Na+ channel clustering at the membrane as for the H+–ATPase at the plant plasma membrane.24
The number of 14-3-3 targets and its effects are plethoric.20 Various interactions with ion transporters or channels have been reported in animals25–27 and plants.24,28 Among cardiac channels, the subunit of the ATP-dependent K+ channel, Kir6.2, and the voltage-dependent K+ channel HERG are regulated by 14-3-3.13,29 Dimeric 14-3-3 binding on the Kir6.2 C terminus containing the RKR motif, known as an endoplasmic reticulum localization signal, prevented channel retention. On the other hand, Kagan et al13 observed that 2 interaction sites exist on the HERG channel and that the association requires phosphorylation of the channel by PKA. In heterologous systems, PKA-dependent phosphorylation of HERG leads to a decrease in current amplitude,30 whereas coexpression and dimerization of 14-3-3 increases and accelerates the current activation.13 According to our results, no RKR retention signal is detected in the 14-3-3–binding site in Nav1.5 ID I. In addition, the Na+ channel trafficking to the cell membrane was not impacted by 14-3-3 expression because the current amplitude was not affected by the presence of 14-3-3.
Nav1.5 Na+ channel is crucial for coordinating cardiac muscle contraction and critical for the vulnerability of the heart to abnormal rhythm. Alterations in Na+ channel expression and function are known to have severe effects on cardiac excitability and conduction. The SCN5A gene encoding Nav1.5 is mutated in 4 different forms of congenital disorders: long QT3 syndrome, Brugada syndrome, progressive cardiac conduction disorder (Lenègre-Lev disease), and sick sinus syndrome.31–34 Our computer simulations demonstrate that APD restitution is steepened in the absence of 14-3-3, with a slope >1 at short diastolic interval. Such steeply sloped APD restitution curve may have strong proarrhythmic effects.15–19 We also observed that propagating action potentials could be elicited at shorter pacing intervals and diastolic intervals in the absence of 14-3-3, thus further enhancing the susceptibility to arrhythmias. The effects of increased conduction velocity and flattening of the CV restitution curve in the absence of 14-3-3 are less clear cut. Some studies suggest that these effects are proarrhythmic,15,18 whereas others suggest that they are antiarrhythmic.16,17 Cherry and Fenton have recently shown that CV restitution can have both proarrhythmic and antiarrhythmic effects.19 One can suspect that mutations of a Nav1.5 cofactor may induce cardiac disorders. In the same line, mutations in KCNE1, a regulator of KCNQ1, a voltage-dependent K+ channel, or mutations of ankyrin B, which interferes with Ca2+ homeostasis proteins, have been implicated in inherited cardiac arrhythmias.35,36 Using in silico models, we have shown that the absence of 14-3-3 could result in proarrhythmic changes in cardiac electrical restitution properties. Our computation data suggest that loss-of-function mutations in 14-3-3 could result in cardiac arrhythmias. Mice expressing 14-3-3 dominant negative double mutant under the control of the –myosin heavy chain promoter have been generated.37 As in NIH 3T3 cells transfected with DN-14-3-3, the activity of JNK1 and p38 mitogen-activated protein kinase is enhanced in cardiomyocytes of transgenic mice revealing the loss of activity of cardiac 14-3-3. Under basal conditions, transgenic mice had normal cardiac morphology, basal ventricular systolic function, and cardiomyocyte appearance.37 All 14-3-3 isoforms, except 14-3-3, are expressed in heart.5 Our results show that at least 3 highly homologous isoforms of 14-3-3 may interact with the cardiac Na+ channel. However, further studies in cardiac myocytes are needed to assess whether the different 14-3-3 isoforms could functionally replace each other to regulate Nav1.5.
Acknowledgments
We are grateful to Dr Andrey Shaw (Washington University, St Louis, Mo) for the generous gift of 14-3-3 cDNAs. We thank the expert technical assistance of Béatrice Leray, Marie-Jo Louérat, and Agnes Carcout.
Sources of Funding
Supported by grants from the Institut National de la Santé et de la Recherche Médicale (Inserm), Agence Nationale de Recherche (ANR COD/A05045GS to I.B.), and Vaincre la Mucoviscidose (to J.M.). M.A. is financially supported by the Association Thorax and the Centre Nantais de Recherche Cardio-Vasculaire; D.P., by Inserm. F.L.B., J.M., and I.B. are recipients of tenure positions supported by the Centre National de la Recherche Scientifique (CNRS).
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received January 5, 2006; revision received April 24, 2006; accepted May 17, 2006.
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Inserm UMR 533 (M.A., F.L.B., D.P., J.-J.S., J.N., H.L.M., J.M., D.E., I.B.), Université de Nantes, Nantes Atlantique Universités, l’institut du thorax, Faculté de Médecine, France
the Department of Physiology (R.W.), Academic Medical Center, University of Amsterdam, the Netherlands.
Abstract
The voltage-sensitive Na+ channel Nav1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3, interacting with the Nav1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Nav1.5 both in COS-7 cells expressing recombinant Nav1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Nav1.5 colocalized at the intercalated discs. We tested the functional link between Nav1.5 and 14-3-3 using the whole-cell patch-clamp configuration. Coexpressing Nav1.5, the 1 subunit and 14-3-3 induced a negative shift in the inactivation curve of the Na+ current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Nav1.5 cytoplasmic I interdomain interacting with 14-3-3. Reversal was also obtained with the dominant negative R56,60A 14-3-3 mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na+ channel acting as a cofactor for the regulation of the cardiac Na+ current.
Key Words: Na+ channel auxiliary subunit congenital heart disease
Introduction
Voltage-gated Na+ channels are responsible for initiation and propagation of the action potential in most electrically excitable cells. They are transmembrane glycoprotein complexes consisting of a pore-forming subunit and accessory subunits.1 The subunit has 4 homologous domains, each containing 6 transmembrane segments and an outer pore-forming loop. The 3 interdomain linkers (ID I to III), and the N- and C-terminal ends of the protein are cytosolic. Ten -subunit isoforms have been cloned from different mammalian tissues.2 Among accessory subunits regulating voltage-gated Na+ channels, 4 -subunit isoforms have been identified3 and shown to modulate the channel-gating properties.4 In the human heart, SCN5A and SCN1B encode the major voltage-sensitive Na+ channel subunit Nav1.5 (also called hH1) and the 1 subunit (h1), respectively. Recent studies have shown that Nav1.5 associates with other proteins that regulate its biosynthesis, localization, activity, and/or degradation.1 Given the major role of Nav1.5 in the cardiac action potential, the expression and activity of these auxiliary proteins are likely important determinants for the electrical excitability of the cardiomyocytes and cardiac conduction.
In the present study, we report and characterize the association of Nav1.5 with 14-3-3 proteins. 14-3-3 family members form a group of highly conserved 30-kDa cytosolic acidic proteins expressed in a wide range of organisms and tissues.5 In mammals, this family consists of 7 members encoded by separate and differentially expressed genes.6 14-3-3 proteins can interact with numerous cellular proteins and are crucial for signaling, cell growth, division, adhesion, differentiation, apoptosis, and ion-channel regulation.7 We show that 14-3-3 and Nav1.5 colocalize at the intercalated discs in cardiomyocytes. We found that the interaction of the 2 proteins altered the cardiac Na+ channel function. Considering that 14-3-3 proteins are expressed in cardiac myocytes, we also show that the Na+ current alterations attributable to 14-3-3 absence are expected to result in the alteration of the restitution of action potential duration and conduction velocity facilitating arrhythmia occurrence.
Based on these findings, we propose that the 14-3-3 protein is a novel member of the cardiac Na+ channel acting as a cofactor for the regulation of the cardiac Na+ current.
Materials and Methods
An expanded Materials and Methods section containing details for plasmids, yeast 2-hybrid screening, coimmunoprecipitation, pulldown assays, Western blotting, immunocytochemistry, electrophysiology, and computer simulations is available in the online data supplement at http://circres.ahajournals.org.
Yeast Two-Hybrid Screening
The first interdomain fragment of human Nav1.5 (hNav1.5 ID I) was used as bait. The yeast reporter strain L40, which contains the reporter gene HIS3 downstream of the binding sequence for LexA, was sequentially transformed with the pVJL10-ID I plasmid and with a mouse cDNA library, using the lithium acetate method8 and subsequently treated as previously described.9
Coimmunoprecipitation, Pulldown, and Western Blotting
African green monkey kidney-derived COS-7 cells (ATCC), mouse heart, anti–14-3-3 mouse monoclonal antibody (Santa Cruz Biotechnology) and anti-Nav1.5 rabbit polyclonal antibody (ASC-005, Alomone Labs) were used. GST-F11 fusion protein (F11 corresponding to residue 417 to 467 of human Nav1.5) has been used during pulldown experiments on mouse heart lysate.
Immunocytochemistry
Immunostaining was performed as described by Mohler et al10 on COS-7 cells expressing the human tagged Nav1.5-GFP and HA-14-3-3 and on freshly isolated rabbit cardiomyocytes11 using also anti-HA rabbit polyclonal antibody (Clontech) and Alexa Fluor 488- or 568-conjugated secondary antibodies (Molecular Probes).
Electrophysiology
Patch-clamp studies were performed on COS-7 cells transiently expressing hNav1.5 and h1 subunits, with or without human wild-type and/or double mutant R56,60A 14-3-3, using the whole-cell configuration at room temperature.
Computer Simulations
The functional role of the 14-3-3 protein in shaping the cardiac action potential was assessed by computer simulations using the human ventricular cell model by ten Tusscher et al.12
Statistics
Data are presented as mean±SEM. Statistical significance of the observed effects was assessed by means of the t test or 2-way ANOVA followed by a Tukey test for multiple comparisons when needed. A value of P<0.05 was considered significant.
Results
The Intracellular Interdomain ID I of Nav1.5 Interacts With 14-3-3
The yeast 2-hybrid was used to screen a mouse cDNA library with hNav1.5 cytoplasmic I interdomain (ID I) as bait (amino acids 417 to 711; Figure 1A). Among the 22 clones able to grow in the absence of histidine, we identified full-length 14-3-3 cDNA by sequencing and database searching. The specificity of the interaction was confirmed by cotransformation of the bait and 14-3-3 or GAL4 AD into the yeast L40 (Figure 1B). To refine the site responsible for the interaction site, we created LexA-fusion protein baits containing various fragments of Nav1.5 ID I (Figure 2A) and tested their ability to interact with 14-3-3 (Figure 2B). 14-3-3 interacted with Nav1.5 ID I fragment 1 (F1) and more precisely with its N terminus, ie, the 417 to 467 amino acid sequence of Nav1.5 (F11). A 99% identity between mouse and human 14-3-3 protein sequences and a 100% identity between residues 417 to 467 of mouse and human Nav1.5 were observed. We challenged the specificity of the interaction with other 14-3-3 isoforms. Using mouse and isoforms of 14-3-3, we observed that these isoforms were able to induce yeast growth (Figure 3). Indeed there are 82% and 86% homology between mouse protein sequences of and isoforms and and isoforms, respectively.
14-3-3 Interacts With Nav1.5 in Transfected COS-7 Cells and in the Heart
To provide biochemical evidence for the interaction between full-length Nav1.5 and 14-3-3, we immunoprecipitated the complex from IGEPAL extracts of Nav1.5-GFP–transfected COS-7 cells or mouse heart (Figure 4A and 4B) using antibodies against GFP, 14-3-3, or Nav1.5 proteins. When 14-3-3 immunoprecipitates were blotted and probed with the anti-GFP (COS-7 cells) or anti-Nav1.5 antibody (mouse heart), coprecipitation of the full-length channel was revealed. Inversely, 14-3-3 was detected in the immunoprecipitated Nav1.5 complex from mouse heart extract. The interaction of 14-3-3 and F11 of Nav1.5 was confirmed by pulldown experiments on mouse heart lysate showing the presence of 14-3-3 among the proteins specifically bound to GST-F11 (Figure 4C and 4D).
We determined the subcellular distribution of Nav1.5 and 14-3-3 using confocal microscopy. Immunostaining revealed colocalization of Nav1.5 and 14-3-3 in the plasma membrane of transfected COS-7 cells (supplemental Figure I). In rabbit cardiomyocytes, 14-3-3 localization was assessed by using a monoclonal antibody that detects members of the 14-3-3 protein family. Fluorescent labeling of polymerized actin with phalloidin–Texas Red stained the actin thin filaments inserting at the intercalated discs (Figure 5, top). A diffuse immunolabeling of 14-3-3 protein was observed in the cytoplasm. Clusters were seen at the intercalated discs, where 14-3-3 colocalized with Nav1.5 (Figure 5, bottom). Colocalization of 14-3-3 protein and Nav1.5 at the intercalated discs is consistent with a physiological interaction between these proteins in situ.
14-3-3 Modulates the Na+ Current by Direct Protein–Protein Interaction
To investigate the functional consequence of 14-3-3 on the channel activity, we used the whole-cell configuration of the patch-clamp technique. The presence of overexpressed human 14-3-3 (h14-3-3) in COS-7 cells expressing hNav1.5 and h1 subunits, did not modify the Na+ current density (–96.1±6.6 pA/pF; n=29 versus –99.3±5.2 pA/pF; n=32; in the absence and in the presence of exogenous 14-3-3, respectively; current measured at –20 mV; Figure 6A). The voltage dependence of the Na+ current activation and inactivation were also investigated. No changes were observed in the activation parameters (V1/2act: –35.7±0.9 mV, n=10, versus –34.6±1.1 mV, n=16; slope: 5.1±0.2 mV versus 5.4±0.3 mV; Figure 6B). On the other hand, the presence of exogenous 14-3-3 shifted the inactivation curve toward more negative values (V1/2inact: –79.2±1.2 mV, n=13, to –84.5±0.9 mV, n=21; P<0.001; Figure 6B) without change in the slope (–6.0±0.2 mV versus –6.1±0.1 mV). Inactivation was not significantly accelerated nor decelerated at any potential in the presence of exogenous 14-3-3 (not shown). Finally, recovery from inactivation was decelerated in the presence of exogenous 14-3-3 (2-way ANOVA: P<0.001; Figure 6C). The time course of recovery from steady-state inactivation was quantified by the time to reach 50% and 75% recovery (t1/2 and t3/4, respectively). Recovery from inactivation in the presence of 14-3-3 was slower (t1/2: 18.0±2.0 ms; t3/4: 52.3±6.3 ms) than in its absence (t1/2: 9.5±1.2 ms; t3/4: 22.5±5.6 ms; P<0.01). Coexpression of 14-3-3 and F1 prevented this deceleration as demonstrated by t1/2 and t3/4 values of 9.3±1.4 and 20.1±2.9 ms, respectively (P<0.01 versus +14-3-3; non-significantly different (NS) versus absence of 14-3-3).
To further evaluate the implication of protein-protein interaction in these changes, we used the F1 peptide (ie, amino acids 417 to 507 of Nav1.5) to compete with full-length Nav1.5 for association with exogenous 14-3-3. We first tested the effects of coexpressing Nav1.5 and F1 (pIRES-F1-Nav1.5). We observed no change in the current density (–121.8±21.2 pA/pF, n=19, in the presence of F1) nor in the inactivation curve (V1/2inact: –80.2±2.6 mV; slope: –6.2±0.3 mV; n=6; in the presence of F1; not illustrated). This suggests that endogenous 14-3-3 does not regulate overexpressed Nav1.5.
In cells expressing 14-3-3 and Nav1.5 (pIRES-14-3-3-Nav1.5), F1 (pcDNA3-F1) reversed the inactivation shift (V1/2inact: –79.8±1.2 mV; slope: –6.2±0.5 mV; n=8; NS in comparison with cells expressing Nav1.5 alone; Figure 6D), produced no change in current density (–97.7±22.3 pA/pF; n=16; NS in comparison with cells expressing Nav1.5 alone), and prevented the recovery from inactivation deceleration (2-way ANOVA: NS in comparison with cells expressing Nav1.5 alone; Figure 6C).
14-3-3 Forms Dimers for Functional Regulation of Nav1.5
14-3-3 proteins have a dimeric structure and monomeric proteins may not regulate their target. Double mutant R56,60A 14-3-3 has been shown to associate with wild-type 14-3-3 but to impair binding with ligands,13 resulting in a dominant negative activity (DN-14-3-3). Here, DN-14-3-3 prevented the inactivation shift induced by wild-type 14-3-3 (V1/2inact: –79.5±1.5 mV; slope: –6.2±0.3 mV; n=8; both NS in comparison with cells expressing Nav1.5 alone; Figure 6D). This suggests the requirement of 2 intact binding sites on 14-3-3 dimer to regulate the Na+ current.
14-3-3 Affects the Cardiac Action Potential
We performed computer simulations to assess the functional role of the 14-3-3 protein in shaping the cardiac action potential. The effects of the absence of 14-3-3 on INa were implemented into a human ventricular cell model12 by a +5.3-mV shift of the INa steady-state inactivation curve and a 70% increase in the rate constants governing fast and slow recovery from INa inactivation (see online data supplement). To validate this approach, we performed in silico voltage clamp experiments using the same protocols as in our experiments (Figure 7A through 7C). As illustrated in Figure 7A, these alterations did not result in significant changes in current density (for example, Figure 6A). When fitting Boltzmann curves to the in silico data (Figure 7B), no differences were observed in activation parameters (V1/2act: –42.1 mV; slope: 5.0 mV). In contrast, the inactivation curve was shifted toward more positive values in the absence of 14-3-3 (V1/2inact: –75.4 versus –79.9 mV; +4.5-mV shift) without change in slope (–7.1 mV). Figure 7B (inset) shows that this shift resulted in increased window current in the absence of 14-3-3, as also seen experimentally (Figure 6B). Finally, the rate of inactivation was not changed, whereas recovery from inactivation was accelerated in the absence of 14-3-3 (Figure 7A and 7C). As in the experiments shown in Figure 6, the time course of recovery from steady-state inactivation was quantified by the time to reach 50% and 75% recovery: t1/2 was 19.9 ms in the presence of 14-3-3 and 11.7 ms in its absence. Similarly, t3/4 was 39.8 ms in the presence of 14-3-3 and 23.4 ms in its absence. Thus, recovery in the model, which was designed for physiological temperature, was not significantly faster than in the experiments performed at room temperature. This model feature is based on the experimental observation that the relative amplitude of the slow components of recovery increases at close to physiological temperature.14
Figure 7D shows the effects of the absence of 14-3-3 through its action on INa at a pacing interval (basic cycle length [BCL]) of 800 ms (75 bpm). Changes to the shape of the action potential were mild. The 4- to 5-mV increase in action potential overshoot in the absence of 14-3-3 reflected the increase in peak Na+ current (Figure 7D, bottom) attributable to the higher availability of Na+ channels. This also resulted in a steeper upstroke (higher maximum upstroke velocity) in the absence of 14-3-3 (402 versus 334 V/sec in its presence; 21% increase; Figure 7F, top). At BCL=800 ms, the higher availability of Na+ channels was caused by the positive shift in steady-state inactivation rather than the accelerated recovery from inactivation (Figure 7F, bottom).
Because we expected the effects of faster recovery from inactivation to be augmented at higher pacing rate, we repeated our simulations with a pacing interval of 400 ms (150 bpm; Figure 7E). At this pacing interval, the control action potential (+14-3-3, solid line) had shorter action potential duration (APD) (222 versus 255 ms at a pacing interval of 800 ms) and lower maximum upstroke velocity (271 versus 334 V/sec). The combined effects of increased Na+ channel availability and accelerated recovery from inactivation in the absence of 14-3-3 resulted in an enhanced Na+ current (Figure 7E, bottom) associated with a 6-mV increase in action potential overshoot and a 34% increase in maximum upstroke velocity (Figure 7F, top). At BCL=400 ms, the higher availability of Na+ channels in the absence of 14-3-3 was not only attributable to the positive shift in steady-state inactivation but also to the accelerated recovery from inactivation (Figure 7F, bottom).
14-3-3 Changes Cardiac Electrical Restitution Properties
Changes in APD and conduction velocity (CV) restitution curves, relating APD and CV to the preceding diastolic interval, have been implicated in the susceptibility to cardiac arrhythmias.15–19 The significant increase in Na+ current in the absence of 14-3-3, preferentially at high pacing rate, facilitates action potential formation and conduction, and may thus affect APD and CV restitution. Therefore, we determined APD and CV restitution curves in the absence and presence of 14-3-3 using standard protocols (see online data supplement). Figure 8A shows that the APD restitution curve in the absence of 14-3-3 was steeper than under control conditions (+14-3-3). Notably, its slope exceeded 1 at short diastolic interval, whereas the slope of the control curve was <0.7. The CV restitution curve is shown in Figure 8B. In the absence of 14-3-3, conduction velocity was higher, especially at short pacing intervals. As a result, the CV restitution curve was flattened. Also, the absence of 14-3-3 allowed successful conduction at higher pacing rates and shorter diastolic intervals. The shortest pacing interval (5-ms resolution steps) resulting in successful action potential conduction was 240 ms in the absence of 14-3-3 (diastolic interval of 40 ms) and 280 ms (diastolic interval of 72 ms) in its presence.
Kagan et al13 have previously reported that the 14-3-3 protein also affects the rapid delayed rectifier current (IKr), ie, the current carried by the HERG channel. We have performed additional computer simulations to assess the effects of the 14-3-3 protein through its action on IKr. These effects appeared to be relatively modest (supplemental Figures II and III).
Discussion
In the present study, a direct interaction between the cardiac voltage-dependent Na+ channel Nav1.5 and 14-3-3 protein was identified using a 2-hybrid screen. The 14-3-3–binding region was determined in the first interdomain of Nav1.5 between amino acids 417 and 467. We also showed colocalization of 14-3-3 and Nav1.5 at intercalated discs of cardiomyocytes and both coimmunoprecipitation and pulldown experiments confirmed their physical association. Functional studies using the whole-cell patch-clamp configuration brought further light to the regulation of Na+ channel activity by 14-3-3.
14-3-3-interacting consensus sequences have been extensively analyzed in mammalian systems (for review, see Bridges and Moorhead20). Two consensus phosphopeptide motifs (ie, RSPXpSXP and RXXXpSXP, where X is any amino acid and pS is phosphoserine) have been uncovered. 14-3-3 binding to its target has been shown to depend on phosphorylation of a serine or threonine. None of these motifs is present in the Nav1.5 417 to 467 sequence interacting with 14-3-3. However, many target proteins do not contain sequences conforming precisely to these motifs or do not need phosphorylation to bind.20 Noteworthy, the protein kinase A (PKA)-dependent mechanism of -adrenergic stimulation regulating functional expression of INa by phosphorylation of S525 and S528 is located in I to II linker, ie, close to the site interacting with 14-3-3.21 These residues could be protected by 14-3-3 from dephosphorylation, as proposed for HERG.13 In addition, INa of native cardiomyocytes22,23 or recombinant INa show a shift in channel inactivation and slower recovery from inactivation on adrenergic stimulation,21 as we observed with 14-3-3. However, unlike adrenergic stimulation, 14-3-3 induced neither an increase in current density nor a shift in voltage dependence of channel activation. Therefore, 14-3-3 effects on Nav1.5 differ from those expected of a protection of PKA sites from phosphatase, as shown for HERG.13
Our results suggest that 14-3-3 dimers regulate Nav1.5 channels. If so, 14-3-3 may contribute to the Na+ channel clustering at the membrane as for the H+–ATPase at the plant plasma membrane.24
The number of 14-3-3 targets and its effects are plethoric.20 Various interactions with ion transporters or channels have been reported in animals25–27 and plants.24,28 Among cardiac channels, the subunit of the ATP-dependent K+ channel, Kir6.2, and the voltage-dependent K+ channel HERG are regulated by 14-3-3.13,29 Dimeric 14-3-3 binding on the Kir6.2 C terminus containing the RKR motif, known as an endoplasmic reticulum localization signal, prevented channel retention. On the other hand, Kagan et al13 observed that 2 interaction sites exist on the HERG channel and that the association requires phosphorylation of the channel by PKA. In heterologous systems, PKA-dependent phosphorylation of HERG leads to a decrease in current amplitude,30 whereas coexpression and dimerization of 14-3-3 increases and accelerates the current activation.13 According to our results, no RKR retention signal is detected in the 14-3-3–binding site in Nav1.5 ID I. In addition, the Na+ channel trafficking to the cell membrane was not impacted by 14-3-3 expression because the current amplitude was not affected by the presence of 14-3-3.
Nav1.5 Na+ channel is crucial for coordinating cardiac muscle contraction and critical for the vulnerability of the heart to abnormal rhythm. Alterations in Na+ channel expression and function are known to have severe effects on cardiac excitability and conduction. The SCN5A gene encoding Nav1.5 is mutated in 4 different forms of congenital disorders: long QT3 syndrome, Brugada syndrome, progressive cardiac conduction disorder (Lenègre-Lev disease), and sick sinus syndrome.31–34 Our computer simulations demonstrate that APD restitution is steepened in the absence of 14-3-3, with a slope >1 at short diastolic interval. Such steeply sloped APD restitution curve may have strong proarrhythmic effects.15–19 We also observed that propagating action potentials could be elicited at shorter pacing intervals and diastolic intervals in the absence of 14-3-3, thus further enhancing the susceptibility to arrhythmias. The effects of increased conduction velocity and flattening of the CV restitution curve in the absence of 14-3-3 are less clear cut. Some studies suggest that these effects are proarrhythmic,15,18 whereas others suggest that they are antiarrhythmic.16,17 Cherry and Fenton have recently shown that CV restitution can have both proarrhythmic and antiarrhythmic effects.19 One can suspect that mutations of a Nav1.5 cofactor may induce cardiac disorders. In the same line, mutations in KCNE1, a regulator of KCNQ1, a voltage-dependent K+ channel, or mutations of ankyrin B, which interferes with Ca2+ homeostasis proteins, have been implicated in inherited cardiac arrhythmias.35,36 Using in silico models, we have shown that the absence of 14-3-3 could result in proarrhythmic changes in cardiac electrical restitution properties. Our computation data suggest that loss-of-function mutations in 14-3-3 could result in cardiac arrhythmias. Mice expressing 14-3-3 dominant negative double mutant under the control of the –myosin heavy chain promoter have been generated.37 As in NIH 3T3 cells transfected with DN-14-3-3, the activity of JNK1 and p38 mitogen-activated protein kinase is enhanced in cardiomyocytes of transgenic mice revealing the loss of activity of cardiac 14-3-3. Under basal conditions, transgenic mice had normal cardiac morphology, basal ventricular systolic function, and cardiomyocyte appearance.37 All 14-3-3 isoforms, except 14-3-3, are expressed in heart.5 Our results show that at least 3 highly homologous isoforms of 14-3-3 may interact with the cardiac Na+ channel. However, further studies in cardiac myocytes are needed to assess whether the different 14-3-3 isoforms could functionally replace each other to regulate Nav1.5.
Acknowledgments
We are grateful to Dr Andrey Shaw (Washington University, St Louis, Mo) for the generous gift of 14-3-3 cDNAs. We thank the expert technical assistance of Béatrice Leray, Marie-Jo Louérat, and Agnes Carcout.
Sources of Funding
Supported by grants from the Institut National de la Santé et de la Recherche Médicale (Inserm), Agence Nationale de Recherche (ANR COD/A05045GS to I.B.), and Vaincre la Mucoviscidose (to J.M.). M.A. is financially supported by the Association Thorax and the Centre Nantais de Recherche Cardio-Vasculaire; D.P., by Inserm. F.L.B., J.M., and I.B. are recipients of tenure positions supported by the Centre National de la Recherche Scientifique (CNRS).
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received January 5, 2006; revision received April 24, 2006; accepted May 17, 2006.
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