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Am J Physiol Heart Circ Physiol 291: H1978-H1987, 2006. First published April 28, 2006; doi:10.1152/ajpheart.01373.2005
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Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes

Olaia Colinas,1,* Mónica Gallego,2,* Raúl Setién,2 José Ramón López-López,1 M. Teresa Pérez-García,1 and Oscar Casis2

1Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, Departamento de Bioquímica y Biología Molecular y Fisiología e Instituto de Biología y Genética Molecular, Facultad de Medicina, Valladolid; and 2Universidad del País Vasco, Facultad de Farmacia, Departamento de Fisiología, Bilbao, Spain

Submitted 26 December 2005 ; accepted in final form 18 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K+ current (Ito) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of Ito, even at low [Ca2+]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of Ito. Because channels underlying Ito are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kv{alpha}-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca2+ was used in the pipette or when intracellular [Ca2+] ([Ca2+]i) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca2+-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca2+ increase, and the association was inhibited by KN93. These results suggest that both channels underlying Ito are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca2+]i increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca2+-dependent modulation by this enzyme of the associated Kv4.2 channels.

transient outward current; cardiac electrophysiology; immunoprecipitation

THE TRANSIENT OUTWARD K+ current (Ito) is the main repolarizing current in rat ventricular myocytes (27). The presence of large densities of this current in the rat minimizes the depolarizing effect of Ca2+ currents (ICa,L) and explains the lack of a clear plateau (phase 2) in the action potential (AP) of this species. Changes in Ito expression and/or kinetics significantly affect AP repolarization (15) and consequently the magnitude and time course of ICa,L, Ca2+ loading of the sarcoplasmic reticulum (SR), SR Ca2+ release, and contractility (33).

Ito currents are generally classified in two different types, "fast" (Ito,f) and "slow" (Ito,s), with different recovery from inactivation time constants (17, 29, 44). A general consensus exists that whereas Ito,f is mediated by Kv4.2 and/or Kv4.3 channels, Ito,s is generated primarily by Kv1.4 channels (26, 27, 29). Differences in the expression pattern of these channels between different species as well as within different regions of the heart produce marked differences in the densities of Ito,f and Ito,s that contribute to the regional variations in the length of AP (26, 27). In addition, regulation of channel expression or modulation of activation and/or inactivation parameters of Ito currents should also affect the shape of AP, modulating Ca2+ entry and contractility (33). Several signaling systems have been shown to modulate Ito (3, 5, 6, 14), including some using Ca2+/calmodulin-dependent protein kinase (CaMKII) (18). CaMKII is a particularly interesting enzyme in the heart, where Ca2+ is the key regulator of cardiac contraction. In fact, this signaling pathway regulates cardiac myocyte excitability and contractility in a very complex way affecting many different targets (24), including Ito. It has been reported that CaMKII modulates the rate of inactivation of Ito in human atrial myocytes (39), and CaMKII-induced phosphorylation has been described for the molecular correlates of Ito, Kv1.4, Kv4.2, and Kv4.3 expressed in heterologous system (32, 36, 40). Furthermore, analysis of Kv4.3 sequence reveals several putative sequence motifs likely phosphorylated by CaMKII, and, at least for one of them, direct phosphorylation by CaMKII has been demonstrated by site-directed mutagenesis studies (36).

The aim of this study was to determine the role of CaMKII in regulating physiological Ito activity in Sprague-Dawley rat ventricular myocytes and its molecular correlates expressed in human embryonic kidney (HEK)-293 cells. The rat right ventricle was chosen because in this preparation Ito is mainly carried by Kv4.2 and Kv4.3 and kinetically corresponds with Ito,f (42). Besides, right ventricular myocytes are an homogeneous population where no regional differences in the composition or the functional properties of Ito currents have been found (8, 16). Combining electrophysiological and biochemical approaches, we have obtained evidence that CaMKII modulates the rate of inactivation of Ito,f, although Kv4.3 and Kv4.2 are not equivalent targets for the enzyme. CaMKII seems to associate and phosphorylate Kv4.3 under low Ca2+ conditions, whereas phosphorylation of Kv4.2 requires an increase in cytoplasmic [Ca2+] ([Ca2+]i). An initial account of this work has appeared in abstract form (7).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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The present study was conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health and the protocols were approved by the Institutional Care and Use Committee of the University of the Basque Country.

Myocytes isolation. Young adult Sprague-Dawley rats (200–220 g wt) were anesthetized with an intraperitoneal injection of chloral hydrate (3 ml/kg) and killed by cervical dislocation. The hearts were removed and mounted onto a Langendorff perfusion apparatus. Single cells were obtained as previously described (8) and stored in suspension at 4°C for at least 2 h before the start of the experiments in a Kraftbrühe solution (20) containing (in mM) 10 taurine, 70 glutamic acid, 0.5 creatine, 5 succinic acid, 10 dextrose, 10 KH2PO4, 20 KCl, 10 HEPES-K, and 0.2 EGTA-K, adjusted to pH 7.4 with KOH.

HEK-293 cell maintenance and transfections. HEK-293 cells were maintained in DMEM supplemented with 10% fetal calf serum (GIBCO), 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. Cells were plated on squared coverslips (24 x 24 mm) placed in the bottom of 35-mm Petri dishes at a density of 2–4 x 105 cells/dish the day before transfection. Transient transfections were performed using Lipofectamine 2000 (Invitrogen), with 1 µg of plasmid DNA encoding either rat Kv4.3, rat Kv4.2, or rat Kv4.2 and the vanilloid receptor (VR1). In all cases, 0.2 µg of plasmid DNA encoding the green fluorescent protein was included to permit transfection efficiency estimates (20–60%) and to identify cells for voltage-clamp analysis. Kv4.2 and Kv4.3 plasmids were provided by Dr. E. Marban (John Hopkins University), and VR1 was a gift of Dr. M. Izquierdo (IBGM, Valladolid, Spain).

Electrophysiological and fluorescence recordings. Recording of ionic currents was performed using the whole cell variation of the patch-clamp technique with an Axopatch 200B whole cell, patch-clamp amplifier (Axon Instruments). All experiments were performed at room temperature (20–22°C). Recording pipettes were obtained from borosilicate tubes (Sutter Instrument) and had a tip resistance of 1–3 M{Omega} when filled with the internal solution (in mM) containing 80 L-aspartic acid (potassium salt), 10 KH2PO4, 1 MgSO4, 50 KCl, 5 HEPES-K, 3 ATP-Na2, and 10 EGTA-K, adjusted to pH 7.3 with KOH. To elicit the Ca2+-independent Ito, we used an external solution containing (in mM): 86 NaCl, 1 MgCl2, 10 HEPES-Na, 4 KCl, 0.5 CaCl2, 12 dextrose, 2 CoCl2, and 50 tetraethylammonium-Cl, adjusted to pH 7.4 with NaOH. Ito was recorded by applying depolarizing pulses to +50 mV, starting from a holding potential (HP) of –60 mV. In addition to Ito, rat ventricular myocytes exhibit another TEA-resistant current called sustained current or Iss. Because this current is time independent, it was digitally subtracted. Peak Ito were normalized to cell capacitance (expressed as pA/pF).

To record Kv4 currents in HEK-transfected cells, we used an external solution containing (in mM): 141 NaCl, 4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH) and an internal solution containing (in mM): 125 KCl, 4 MgCl2, 5 ATP-Na2, and 10 EGTA, adjusted to pH 7.3 with KOH. To obtain 0.5 µM free Ca2+, total Ca2+ was calculated with the program CHELATOR (35). Titrated stock solutions of CaCl2 and EGTA were used to minimize errors due to impurities of EGTA and hydration of CaCl2.

Ionic currents were recorded by 500-ms voltage steps to +50 mV from a HP of –60 (myocytes) or –80 mV (HEK). Recovery of inactivation was studied in cardiac myocytes by applying two depolarizing pulses separated by an increasing interpulse interval in 25-ms steps. For application of drugs in the pipette solution and to eliminate artifacts due to changes in the seal conditions, the test pulse was preceded by a –10-mV short (10 ms) pulse. The capacitative response elicited was used to obtain the values of access resistance (Ra), membrane resistance (Rm), and capacity of the cell at each pulse by using the membrane test algorithm of the program pCLAMP [Axon Instruments (34), see Fig. 2A in this paper]. Only cells in which these parameters did not change in a significant way over time were chosen for further analysis.


Figure 2
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  		Fig. 2. Effects of several CaMKII inhibitors on the inactivation kinetics of Ito currents. A: protocol used for ensuring stability of the recording conditions (see METHODS). Computed access and membrane resistance (Ra and Rm, respectively) and capacitance (Cap) from one selected cell are shown in the plots. Only cells in which these parameters did not change in a significant way over time were chosen for further analysis. B: effect of selective CaMKII inhibitor autocamtide-2-related inhibitory peptide (ARIP II, top traces) and its myristoylated form ARIP-M (bottom traces) on the inactivation time course of cardiac Ito. C: {tau}s and {tau}f obtained with the different CaMKII blockers and in control cells over time. D: relative amplitude of slow component of current (As). Values are means ± SE of 6–10 cells. *P < 0.05; **P < 0.01.

 
Data analysis was performed with ORIGIN 7.5 (Microcal). Pooled data are expressed as means ± SE. Statistical comparisons between groups of data were carried out with the two-tailed Student t-test for paired or unpaired data, and values of P < 0.05 were considered statistically different.

All solution reagents were from Sigma, except ionomycin, KN93, calmidazolium, autocamtide-2-related inhibitory peptide II (ARIP-II), and its myristoylated form (ARIP-M), which were purchased from Calbiochem.

In a set of experiments, where Kv4.2 channels were coexpressed with the vanilloid receptor (VR1), 100 µM fura-2-pentapotassium salt (Molecular Probes) was included in the pipette solution to simultaneously record K+ currents and Ca2+ levels. Currents were elicited by depolarizing pulses applied every 10 s and fura ratio was measured every 2 s. Dual-wavelength measurements of fura-2 fluorescence were performed with the illumination system DX-1000 (Solamere Technology Group). A 100-W mercury lamp was used as light source (Optiquip). Light was focused and collected through a Nikon Fluor 40/1.30 objective. The wavelength for dye excitation was alternated between 340 and 380 nm, and fluorescence emission at 540 nm was collected with a SensiCam digital Camera (PCO CCD imaging). A binning 4 x 4 was applied to get images of 320 x 256 pixels (12 bits/pixel) at 0.5 Hz for both wavelengths. The illumination system and the camera were driven by Axon Imaging Workbench 4.0 (Axon Instruments).

Myocyte membrane isolation. All procedures were performed at 4°C in a homogenization buffer (HF) containing 20 mM Tris·HCl (pH 7.4), 1 mM EDTA , and 2.5 µl/ml of the Sigma protease inhibitor cocktail. Myocyte suspensions were pelleted at 1,000 g for 10 min, resuspended in 4 ml HF, and then homogenized 1 min on ice. Nuclei and debris were pelleted by centrifugation at 500 g for 10 min. This procedure was repeated, and the supernatants from both low-speed spins were pooled and centrifuged at 40,000 g for 30 min. The pellet was resuspended in 1 ml HF and centrifuged again at 40,000 g for 30 min. The final pellet was solubilized in 0.5 ml of HF and stored at –80°C until used.

Immunoprecipitation and Western blotting. Twenty-four hours after transfection, HEK-293 cells were lysed in lysis buffer [20 mM Tris·HCl (pH 7.5), 138 mM NaCl, 3 mM KCl, 1 mM EGTA, 2 mM EDTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A]. The supernatant of a 10-min centrifugation at 10,000 g was precleared with protein A-sepharose beads (Amersham Pharmacia) for 2 h at 4°C with a rocking method. The precleared supernatant was incubated overnight at 4°C with rocking method with 2 µg of antibodies for immunoprecipitation: anti-CaMKII (Santa Cruz) and anti-phospho Ser/Thr (Cell Signalling). Protein A-sepharose beads were then added to each sample and the incubation continued for 2 h at 4°C. The complexed beads were collected by centrifugation at 1,800 g for 3 min at 4°C and washed three times with lysis buffer without Triton X-100. The bound proteins were eluted by incubating in SDS sample buffer with beta-mercaptoethanol and then centrifuged at 1,800 g for 3 min to pellet beads. The supernatant was heated for 5 min at 70°C, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. After blockade of the membrane with 5% nonfat dry milk in 1x phosphate-buffered saline with 0.1% Tween 20 (PBST), primary antibodies [anti-Kv4.3 or anti-Kv channel-interacting protein 2 (KChIP2); Santa Cruz or anti-Kv4.2; Alomone] were diluted in blocking solution at a final concentration of 1:200 and incubated for 1 h. The membrane was then washed with PBS and incubated withhorseradish peroxidase-conjugated secondary antibodies (1:2,000, donkey anti-goat or goat anti-rabbit; Santa Cruz) for 1 h. The protein signals were detected in an image station 2000R (Kodak) with chemiluminescence reagents (ECL, Amershan Biosciences).

Membranes isolated from myocytes were solubilized in RIPA buffer [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Igepal, 1% sodium deoxycholate, 2.5 µl/ml of the Sigma Protease inhibitor cocktail] and centrifuged at 48,000 g for 15 min. The pellet was resuspended in 150 µl of RIPA buffer and incubated 1 h at 4°C. Samples were cleared by centrifugation at 15,000 g for 25 min, and supernatants were incubated with 2 µg of the anti-CaMKII antibody overnight at 4°C. One-hundred microliters of 50% protein G-agarose were added, and the mixture was incubated for 3 h at 4°C. The beads were pelleted and washed three times in RIPA buffer. The bound proteins were eluted using 50 µl of SDS sample buffer and separated on 9% SDS-polyacrylamide gels. Western blot was developed as described above.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of CaMKII accelerates Ito,f inactivation. Recovery from inactivation of Ito currents recorded in rat myocytes isolated from right ventricle (Fig. 1A, right) was well described by a single exponential ({tau} = 33.3 ± 1.8 ms, n = 8), supporting the idea that Ito currents in the right ventricle are mostly mediated by a rapidly recovering component (Ito,f) (42). Typical Ito currents exhibited a biexponential time course of inactivation (Fig. 1), with a slow component (84.7 ± 3.4 ms) that slightly predominates (54.5 ± 2.3%) over a fast component (23.5 ± 0.8 ms). Perfusing the cells with KN93, a selective inhibitor of CaMKII (37), typically decreased the peak current amplitude and increased the rate of inactivation as already described (39). These effects have been attributed in part to a direct action of KN93 on K+ channels, acting as an open channel blocker (23). To avoid the direct effect of the drug on the channels (at least on the extracellular side), we have explored the effect of KN93 added to the internal solution (31). As depicted in Fig. 1A, when KN93 is included in the intracellular solution, the time course of inactivation is faster. This effect can be attributed to a decrease in the contribution of the slow component (from 54.5 ± 2.3% to 37.6 ± 2.4%), without any significant change in the {tau} constants (Fig. 1B). On the contrary, recovery of inactivation was unaffected by KN93 (right panels, Fig. 1A). Peak current was also unaffected, being 21.1 ± 2.5 pA/pF in control myocytes and 21.8 ± 1.3 pA/pF when KN93 was included in the pipette.


Figure 1
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  		Fig. 1. Calmodulin kinase II (CaMKII) blocker KN93 accelerates fast transient outward current (Ito,fast) inactivation without affecting the recovery of inactivation. A: transient outward current (Ito) recordings obtained in isolated right ventricular myocytes. Time course of inactivation (left) was explored either in control cells or in cells dialyzed with internal solution containing 40 µM KN93. Recovery of inactivation (right) was studied by applying the protocol depicted in inset. Ratio between the current amplitude obtained with both pulses is plotted against the interpulse interval. B: inactivation fitting parameters obtained in control conditions and in the presence of 40 µM KN93. Left and middle, absolute and relative amplitudes of the two components of inactivation (Af and As respectively). Right, time constants of inactivation ({tau}s and {tau}f) and the time constant of the recovery from inactivation ({tau}rec). Bars are means ± SE; n = 7–8 myocytes. **P < 0.01.

 
To further explore the selectivity of KN93 in inhibiting CaMKII, we have studied the effects of several other antagonists, such as calmidazolium chloride, a calmodulin antagonist, and ARIP II and its membrane-permeable derivative ARIP-M, which are potent and highly specific blockers of CaMKII (21). ARIP II (100 nM) was included in the pipette solution, and the acceleration of the inactivation time course was apparent after 12–15 min of dialysis. ARIP-M was perfused (10 µM) in the bath solution, and the effect on the kinetics of the current was reversible after removal of the drug. All these drugs have effects on the speed of inactivation similar to those described with KN93 (Fig. 2), decreasing the contribution of the slow component without changing the time constants (Fig. 2, C and D).

CaMKII coimmunoprecipitates with Kv4 channels in cardiac myocytes. The effect of CaMKII or calmodulin antagonists on inactivation kinetics of Ito when [Ca2+]i is heavily buffered, as in the experiments described in Figs. 1 and 2, was surprising. These experiments suggested that K+ channels were phosphorylated by CaMKII at rest in absence of an increase in [Ca2+]i. To test the genuineness of the effect of KN93 on CaMKII, we explored the possible association of the enzyme with K+ channels that underlie Ito,f currents in the rat: Kv4.2 and Kv4.3 (42). We looked for the existence of such complexes analyzing whether Kv4.3 or Kv4.2 copurified with the enzyme when CaMKII is immunoprecipitated from rat heart membrane lysates. When CaMKII was pulled down from the membrane lysates with a polyclonal antibody that recognizes all isoforms of the enzyme, a band of ~79 kDa was detected by immunoblot with either anti-Kv4.3 or with anti-Kv4.2 antibodies (Fig. 3). Parallel Western blot analysis of membrane lysates showed the presence of the same band when probing anti-Kv4.3 antibody, whereas a second, lower size band (~74 kDa) was also present when using anti-Kv4.2 channel antibody. Previous reports have also described the presence of Kv4.2 as a doublet of immunoreactivity in Western blot, demonstrating that the upper band represents Kv4.2 protein specifically trafficked to the cell membrane, whereas the lower one is a pool of channels that can exist either intracellularly or at the surface (40). In this context, our coimmunoprecipitation results would suggest that CaMKII associates with the upper, membrane-located fraction of Kv4.2 channels. When membrane lysates where preincubated with KN93 before the immunoprecipitation, the protein bands corresponding to Kv4 channels were almost undetectable, suggesting that inhibition of CaMKII blocked its association with the channels (Fig. 3). If, after being blotted with Kv4 antibodies, membranes were stripped and reprobed with an antibody against phospho-Ser and phospho-Thr (PSer/PThr), the bands corresponding to the channels were labeled in the control experiments but remained undetectable when lysates were preincubated with KN93 (data not shown). These results strongly support the hypothesis that Kv4 channels of rat cardiac myocytes are phosphorylated at resting conditions. However, as functional Ito,f currents are heteromeric, comprising Kv4.2/Kv4.3 {alpha}-subunits and the ancillary subunit KChIP2 (16, 26, 29), our experimental setting does not allow to discern whether CaMKII associated with either or both Kv4 subunits, hampering the study of possible differential effects of CaMKII on both channels.


Figure 3
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  		Fig. 3. CaMKII coimmunoprecipitates with Kv4 channels in rat cardiac myocytes. Myocyte membranes were immunoprecipated with anti-CaMKII and immunoblotted with anti-Kv4.3 or anti-Kv4.2 as indicated. Parallel Western blot analysis was performed to confirm identity of labeled bands. Arrows in the left indicate expected band sizes for Kv4.3 (79 kDa) and Kv4.2 (74 and 79 kDa). In both cases, incubation of the myocytes with KN93 inhibits association of CaMKII with the channel. Each blot is representative of 6–8 experiments in the same conditions. Bottom: Western blot of Kv channel-interacting protein 2 (KChIP2) performed in lysates from heart and untransfected human embryonic kidney (HEK)-293 cells as indicated. Two bands with apparent molecular masses of {approx}29 and 26 kDa, corresponding to the long and short isoforms, are apparent in heart lysates.

 
CaMKII has different effects on Kv4.2 and Kv4.3 expressed in HEK cells. To overcome this problem, we used HEK-293 cells to express heterologously Kv4.2 or Kv4.3 channels because these cells do not express these channels endogenously. Furthermore, the expression of KChIP2 protein is almost undetectable (see Fig. 3). We tested the effect of KN93 and calmidazolium applied in the intracellular solution (Fig. 4A). Kv4.3-transfected cells exhibit inactivating currents well fitted by a biexponential function, with time constants that averaged 89.3 ± 2.3 and 21.8 ± 1 ms, and a 49 ± 3.2% contribution of the slow component to the total current. Peak currents averaged 5.59 ± 1.9 nA. Kv4.2-transfected cells exhibit similar currents, although with a faster inactivation. Time constants were in these cells 86 ± 4.5 and 16.2 ± 1.1 ms, and the slow component only represented a 26.5 ± 1.5%. Peak currents averaged 2.8 ± 0.6 nA. When KN93 (40 µM) or calmidazolium (20 µM) were included in the pipette, similar results to those obtained in native myocytes were obtained in Kv4.3-transfected cells (Fig. 4A, top), but no effect was observed in Kv4.2-transfected cells. In both cases, the peak current amplitudes were unaffected. The differential effect of CaMKII inhibitors on Kv4.3 and Kv4.2 channels suggest that Kv4.3 channels are phosphorylated at rest (with low [Ca2+]i), whereas Kv4.2 are not. These results were confirmed by studying the effects of ARIP-II and ARIP-M on the currents expressed by Kv4.3 channels. As in the case of cardiac myocytes, both drugs reproduced the effects of KN93, accelerating the time course of inactivation of the currents through the reduction of the contribution of the slow component of the current (Fig. 4, B and C) with no significant changes in the inactivation time constants (not shown).


Figure 4
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  		Fig. 4. Effect of CaMKII blockers in inactivation kinetics of cloned Kv4.3 and Kv4.2 channels. A: normalized traces obtained with depolarizing pulses in HEK cells transiently transfected with rat Kv4.3 (top) or rat Kv4.2 (bottom), in the absence (black traces, control) or in the presence (grey traces) of KN93 in the internal solution. Relative proportion of the slow inactivating component of the current and magnitude of both time constants of inactivation is shown in bar plots (n = 6–10. *P < 0.05, **P < 0.01). B: effect of ARIP II on Kv4.3 inactivation. Representative traces obtained 4 min (black) and 25 min (grey) after establishment of whole cell configuration are shown from a control cell and from a cell dialyzed with ARIP-II 100 nM. Average proportion of the slow inactivating component obtained in each case is shown in bar plot. n = 9. **P < 0.01. C: effect of ARIP-M on Kv4.3 inactivation. Traces were obtained in a cell before (control), during 10 µM application of ARIP-M , and after washout of the drug. Average changes in the inactivation parameters are shown in bar plot. n = 5. *P < 0.05.

 
Next, we explored the hypothesis that Kv4.2 could also be a substrate for CaMKII in the presence of increasing [Ca2+]i that would lead to activation of the enzyme. To test that possibility, we studied the time course of inactivation of Kv4.3 and Kv4.2 currents by using a pipette solution containing 0.5 µM free Ca2+ (Fig. 5). Inactivation parameters of Kv4.3 were not affected by this increase in [Ca2+]i, suggesting that CaMKII-mediated phosphorylation of the channels is already maximal at resting [Ca2+]i. In contrast, high Ca2+ decelerated Kv4.2 inactivation, significantly increasing the contribution of the slow component from a 26.5 ± 1.5% to a 34.8 ± 1.7%, again without affecting the average peak current amplitudes or the magnitude of the time constants of inactivation.


Figure 5
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  		Fig. 5. Effect of increasing intracellular Ca2+ concentration ([Ca2+]i) on the time course of inactivation of Kv4.3 and Kv4.2 channels. Left, records obtained from cells dialyzed either with control intracellular solution (10 mM EGTA) or with a pipette solution containing 0.5 µM free Ca2+. Each bar represents mean + SE data of 7–9 cells. **P < 0.01.

 
Ca2+ dependence of Kv4.2 inactivation. To confirm the effect of increasing [Ca2+]i on Kv4.2 inactivation, we contransfected HEK cells with Kv4.2 and VR1. VR1 is a ligand-operated channel that do not discriminate among monovalent cations but exhibits a notable preference for divalent cations (10), with a very high relative permeability for Ca2+ ions (PCa/PNa,K {approx} 10). The purpose of this set of experiments was to increase [Ca2+]i in a reversible manner by applying capsaicin, an agonist of VR1, and correlate the changes in [Ca2+]i with the changes in the time course of inactivation measuring simultaneously Kv4.2 currents and [Ca2+]i. A representative experiment is depicted in Fig. 6A. Activation of VR1 by the application of capsaicin was evidenced by an inward current at the HP (–80 mV, Ihold) and by an increase in the current recorded at the end of the depolarizing pulse to +50 mV (Iss). These currents disappeared after removal of the agonist. Also, application of capsaicin inhibited Kv4.2 peak current in a reversible manner. This effect can be explained by a direct interaction of capsaicin with the channels, because it has been reported as a reversible block of Ito by capsaicin (9). Finally, VR1 activation also elicited a [Ca2+]i transient. All of these changes were rapidly reverted upon agonist removal. By contrast, with a certain delay after VR1 activation (half-maximal time ~6 min), a maintained slowdown in the inactivation rate of Kv4.2 currents was apparent (in the example, the contribution of the slow component to the whole current increased from 20% to 30%). Figure 6B shows normalized sample currents obtained before and 15 min after application of capsaicin. Average results obtained in four different experiments are plotted in Fig. 6C. When KN93 was included in the recording pipette (Fig. 7), all the transient changes elicited by VR1 activation were also observed, but the increase in [Ca2+]i did not affect the time course of inactivation of Kv4.2 currents. Altogether, these results suggest that Ca2+-dependent changes in Kv4.2 current inactivation are mediated by CaMKII activation.


Figure 6
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  		Fig. 6. Effect of controlled [Ca2+]i increase on the time course of inactivation of Kv4.2 currents. A: changes in [Ca2+]i and Kv4.2 currents measured in a HEK cell transfected with Kv4.2 and VR1. At indicated time, 100 nM capsaicin was added to the bath solution to stimulate Ca2+ entry trough VR1 channels. Figure shows the time course of changes induced by capsaicin in (from bottom to top) the peak current amplitude (Ipeak, open circles), the holding current at –80 mV (Ihold, open triangles), current at end of depolarizing pulse (Iss, open squares), [Ca2+]i (line plot), and proportion of current amplitude represented by slow component (open circles). Inset: two traces obtained at the indicated times (1 and 2). Symbols adjacent to traces reflect points where Ihold, Iss, and Ipeak were measured. B: representative normalized traces obtained before (1) and after (3) capsaicin application showing change in inactivation properties of the Kv4.2 currents. C: time course of inactivation of the currents recorded in four Kv4.2 + VR1-transfected cells. Bars represent the average (means + SE) proportion of slow current component amplitude before (1) and after (3) application of capsaicin. *P < 0.05.

 

Figure 7
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  		Fig. 7. Ca2+-induced changes in inactivation time course of Kv4.2 currents are prevented by KN93 treatment. A: representative experiment similar to that described in Fig. 6 but with 40 µM KN93 in the recording pipette. Capsaicin at two different concentrations was applied to bath solution, leading to a dose-dependent increase of [Ca2+]i (line plot) and to the appearance of an inward current at Ihold (open triangles). However, there were no changes in the proportion of the slow current component amplitude (open circles). B: representative traces obtained at times indicated in A as 1 and 2. C: average data from 3 similar experiments representing proportional amplitude of slow component of inactivation before (1) and after capsaicin application (2).

 
CaMKII coimnumoprecipitates in HEK cells with Kv4.3 but not with Kv4.2. The differential behavior of CaMKII in the modulation of Kv4.3 and Kv4.2 channels was further explored by testing the interaction between the enzyme and the channels with coimmunoprecipitation experiments. To look for the existence of a Kv4.3/CaMKII complex, control and Kv4.3-transfected HEK lysates were immunoprecipitated with anti-CaMKII and immunoblotted with anti-Kv4.3 (Fig. 8A, left). Kv4.3 channels are detected in CaMKII immunoprecipitates (middle) and absent in nontransfected HEK cells (left). The association is sensitive to KN93 because preincubation with this molecule makes the Kv4.3 band undetectable (right). These experiments were carried out in unstimulated cells, at resting [Ca2+]i, suggesting that association of CaMKII and Kv4.3 does not need a previous rise in [Ca2+]i. After establishing the existence of CaMKII/Kv4.3 complexes, we tested whether Kv4.3 channels were phoshorylated, either reprobing the blots with an anti-PSer/PThr (not shown) or immunoprecipitating HEK cells with anti-PSer/PThr (Fig. 8A, right). Kv4.3 channels were detected in these immunoprecipitates in transfected cells (Fig. 8A, right), suggesting that the channels are phosphorylated.


Figure 8
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  		Fig. 8. CaMKII coimmunoprecipitates in HEK cells with Kv4.3 but not with Kv4.2 channels. A: HEK- and Kv4.3-transfected HEK cell lysates were immunoprecipitated with anti-CaMKII (left) or anti-PSer/PThr (right) and immunoblotted with anti-Kv4.3. A band of ~82 kDa (arrows on left) could be detected in Kv4.3-expressing cells but was absent in nontransfected HEK cells. Pretreatment of cells with KN93 prevented the association between CaMKII and Kv4.3. B: immunoprecipitation with anti-CaMKII and immunoblot with Kv4.2 of HEK untransfected, transfected with Kv4.2 or transfected with Kv4.2+VR1 and stimulated with 100 nM capsaicin did not reveal association of CaMKII with Kv4.2 protein (expected size indicated by arrow). C: Western blot of HEK cells transfected with Kv4.2 after treatment with 5 µM ionomycin during 1 min (right lane) or 5 min (middle lane), or unstimulated (control, left lane) before cell lysis was blotted with anti-Kv4.2 (top) and reblotted with anti PSer/PThr (bottom). Arrow in left indicates expected band size for Kv4.2 protein. Untransfected HEK cells in which this Kv4.2 band was absent are also shown.

 
When similar experiments were carried out in HEK cells transfected with Kv4.2 (Fig. 8B), we were unable to detect CaMKII/Kv4.2 complexes, even after increasing [Ca2+]i with capsaicin (in VR1-cotransfected cells) or with ionomycin. Nevertheless, we investigated the phosphorylation status of Kv4.2 channels in HEK-transfected cells, before and after ionomycin application (Fig. 8C, right). With the use of specific anti-Kv4.2 antibodies, the channels could be detected on Western blots as an ~85-kDa band (second lane), which was absent in nontransfected cells (first lane). Treatment with ionomycin split the putative Kv4.2 band in two, suggesting that Kv4.2 channels were phosphorylated after increasing [Ca2+]i. In fact, reprobing the membrane with anti-PSer/PThr revealed the Kv4.2 bands in ionomycin-treated cells.


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Our findings support the idea that Kv4.3 and Kv4.2 channels, the molecular correlates of cardiac Ito,f, are targets of CaMKII. However, the interaction of the enzyme with both channels looks clearly different. CaMKII associates with Kv4.3 even at low [Ca2+], and such association leads to channel phosphorylation. Phosphorylation of Kv4.3 channels is functionally relevant, making the time course of inactivation significantly slower. On the contrary, although CaMKII does not associate with Kv4.2 channels in resting conditions, increasing [Ca2+] both phosphorylates the channels and slows down the inactivation of Kv4.2 currents. Therefore, Kv4.3 associates with CaMKII and is phosphorylated even at low [Ca2+], whereas Kv4.2 can only be phospohorylated by the enzyme when Ca2+ levels increase. We did not carry out a detailed characterization of the effect of CaMKII on inactivation kinetics, but the change in the relative amplitude of the fast and slow component of inactivation could reflect a change in the relative contribution of open and closed-state inactivation (22, 41).

CaMKII-mediated phosphorylation of both Kv4.2 and Kv4.3 channels has been reported in several heterologous expression systems (36, 40), although these previous works show some discrepancies with the present study. The only reported effect of CaMKII on Kv4.2 currents is an increase in the levels of expression of Kv4.2 without changes in the biophysical properties of the currents (40). However, whereas in this study CaMKII was cotransfected with Kv4.2, we found that endogenous CaMKII is sufficient to phosphorylate transfected Kv4.2 channels when [Ca2+]i increases, although we could not demonstrate the association of the kinase and the channels by coimmunoprecipitation. The lack of success in these experiments could be explained by a weak, transient association of CaMKII to the channels upon stimulation, but the fact that endogenous CaMKII, and possibly other kinases, are capable of modulating transfected proteins in an heterologous expression system is a very interesting observation that has to be taken into account when these systems are use to study phosphorylation patterns. Regarding Kv4.3 channels, the available data are more concordant with our results, because CaMKII phosphorylation of transfected Kv4.3 channels has been shown to modify the kinetics of the current without changing its amplitude (36). However, whereas in our experimental settings the time course of inactivation is not modified by increasing [Ca2+]i, suggesting that maximal CaMKII phosphorylation of Kv4.3 channels is obtained at resting Ca2+ levels (pipette solution contains 10 mM EGTA), these authors found that Kv4.3 inactivation could be slowed by decreasing the calcium-buffer capacity of the pipette solution (36). Again, we can speculate that these discrepancies could reflect slight differences in the expression levels of endogenous CaMKII and/or endogenous phosphatases. Notwithstanding, the present work represents an important contribution to our knowledge of the role of CaMKII modulation of Kv4 channels, because we demonstrate that in the same expression system and with the same experimental conditions there is a differential modulation of Kv4.2 and Kv4.3 channels by CaMKII-dependent phosphorylation.

In rat ventricular cells both Kv4.3 and Kv4.2 contribute to Ito,f (27), and their association to form heteromultimers has been demonstrated in ventricular cells isolated from mice (16). This heteromeric association also includes KChIP2 accessory subunits. KChIPs are members of the neuronal calcium sensors subfamily of Ca2+ binding proteins (1). Although we cannot rule out the contribution of KChIPs to the modulation of Kv4.2 and Kv4.3 channels by CaMKII, we did not find any evidence of the presence of endogenous KChIP proteins in HEK cells (Fig. 3). Besides, data in Figs. 5–7 clearly indicate that Ca2+-dependent modulation of Kv4.2 kinetics is mediated by CaMKII. The effect of KN93 on Ito,f resembles the effect obtained on Kv4.3 currents expressed in HEK cells, and CaMKII coimmunoprecipitates with Kv4.3 channels in rat ventricular cells. These observations indicate that Kv4.3 channels are phosphorylated by CaMKII at rest, so that they could function tethering the kinase to the membrane and then facilitating the phosphorylation of their partners in the heteromultimer, the Kv4.2 channels, or even the phosphorylation of other transmembrane proteins. We have not further explored the mechanism of binding between Kv4.3 channels and CaMKII, but its dependence of KN93 (and calmidazolium) suggest that the channels bind to the regulatory domain of CaMKII, as has been reported for Eag channels (38) or the N-methyl-D-aspartate receptor NR2B subunit (4). Additionally, it has been reported that KN93 competitively inhibits Ca2+/calmodulin binding to CaMKII (37), and our coimmunoprecipitation results demonstrate that KN93 inhibits binding of Kv4.3 to CaMKII. If channels bind to the autoregulatory domain, the kinase could be activated and phosphorylate the channels in a Ca2+-independent manner. Because functional CaMKII is an holoenzyme with several Ca2+/calmodulin binding sites (19), it would be possible to speculate that the Kv4.3-bound kinase could also bind Ca2+/calmodulin and phosphorylate other membrane proteins (like Kv4.2 or Ca2+ channels) in a Ca2+-dependent manner.

Ito modulation by CaMKII has been previously reported in human atrial myocytes (39) based on results obtained with a set of CaMKII inhibitors similar to that used in this report. However, it has been also reported that KN93 produces nonspecific inhibitory effects on delayed rectifier K+ currents in vascular myocytes, mainly a big inhibition of peak K+ currents, at doses as low as 5 µM (23). To minimize possible direct effects of KN93 on Kv4 channels, we have performed our study by applying CaMKII inhibitors directly in the patch recording pipette (31). With this approach, the only effect of KN93 on Kv4 currents is the slow down in the time course of inactivation. In addition, the fact that the effect of KN93 is mimicked by other more specific CaMKII inhibitors such as ARIP II and ARIP-M, together with the different response of Kv4.2 and Kv4.3 currents assessed with both electrophysiological and biochemical approaches, strongly support a link between changes in inactivation kinetic and CaMKII-induced phosphorylation.

The multifunctional CaMKII is highly expressed in the heart (13), where it has been found to be present at the cell membrane, in the cytoplasm, and in the nucleus of cardiac myocytes. CaMKII plays important functional roles in all of these locations, because the modulation of channels and transporters (2, 12, 43, 45) has the capability of linking cellular responses to changes in [Ca2+]i levels throughout the cardiac myocyte (24). The different subcellular location and intracellular targeting of CaMKII and its different substrate specificity suggest possible differential regulation of the CaMKII via different stores of Ca2+ or by virtue of different Ca2+ and/or Ca2+/CaM sensitivity (2, 46). These properties have also prompted the interest toward the search for CaMKII-anchoring proteins [by analogy with the best studied example of the targeting of PKA by AKAPs (25)]. The available data have provided evidence indicating that 1) CaMKII is dynamically targeted, because autophosphorylation of Thr286 strongly potentiates CaMKII interaction with several target proteins; and 2) CaMKII-binding proteins do not function only as anchoring proteins, but they are the downstream effector substrates for CaMKII (or proteins associated with those substrates) thereby bringing kinase and substrate in close proximity (11). Our findings regarding CaMKII modulation of Ito currents fit nicely in this hypothesis, because they suggest that Kv4.3 channels could function as CaMKII-anchoring proteins, allowing Ca2+-dependent modulation by this enzyme of the associated Kv4.2 channels. Also consistent with this hypothesis is the fact that CaMKII association (and modulation) of Kv4.3 channels does not seem to require an increase in [Ca2+]i, suggesting that the enzyme must be autophosphorylated and thereby active in a Ca2+-independent way.

In species like rats, where action potential (AP) repolarization is mainly determined by Ito, changes in the rate of inactivation are expected to have profound effects in AP duration and SR Ca2+ release (33). In fact, we have modeled a rat AP by using a model of rabbit AP (30) modified to simulate a rat AP (28), and we found that when Ito inactivation parameters are modified to reproduce the experimental results obtained with KN93 in the pipette solution, AP duration is reduced by {approx}50% (data not shown), clearly evidencing that CaMKII could play a prominent role in controlling repolarization of ventricular cells. Recently, several studies have linked different heart diseases to an increase in Ca2+ current by increased CaMKII activity, suggesting that CaMKII inhibition may be an effective antiarrhythmic therapy (2). Although the role of Ito is not so prominent in the human heart, CaMKII phosphorylation of Kv4 channels could also contribute to the pathophysiological effects of this kinase.


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This work was supported by Instituto de Salud Carlos III Grants G03/011 (Red Respira), G03/045 (Red Heracles), and PI041044 to J. R. López-López, Ministerio de Educación (MEC) Grant BFU2004-05551 to M. T. Pérez-García and MEC Grant SAF2005-00906 to O. Casis. O. Colinas is a fellow of the Spanish Ministerio de Ciencia y Tecnología, and M. Gallego and R. Setién are fellows of the University of Basque Country.


   ACKNOWLEDGMENTS
 
We thank Esperanza Alonso for excellent technical assistance. We also thank Pepe "el mago" (Puglisi JL) for help in modeling the rat AP.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Pérez-García, Departamento de Bioquímica y Biología Molecular y Fisiología, Edificio IBGM, Universidad de Valladolid, C/ Sanz y Forés s/n, 47003 Valladolid, Spain (e-mail: tperez{at}ibgm.uva.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* O. Colinas and M. Gallego contributed equally to this study. Back


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