© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current1
Department of Cardiovascular Pharmacology, SmithKline Beecham Laboratoires Pharmaceutiques, 4 Rue du Chesnay Beauregard, 35760 Saint-Grégoire, France
* Corresponding author. Tel.: +33-299-280-456; fax: +33-299-280-444; e-mail: antoine_bril@sbphrd.com
Received 18 March 1998; accepted 3 June 1998
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. Methods: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. Results: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to –40 mV and the steady-state inactivation curve had a midpoint of –47.4±0.3 mV and a slope of 5.9±0.2 mV. Rat ventricular Ito current was activated at potentials positive to –20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of –29.1±0.7 mV and 4.5±0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2±4.1 msec and 23.±1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54±0.13 mM and 0.14±0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogenous. Conclusion: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogenous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.
KEYWORDS K-channel; Gene expression; Transient outward potassium current; Kv4.3; Cadmium
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In most mammalian cardiac cells, including the human atrial and ventricular tissues [1–4]the transient outward current (Ito) plays an important role in the repolarisation process of the action potential and is responsible for the early repolarisation and the notch of the cardiac action potential. In rat myocytes, Ito is the principal repolarising current responsible for the overall time course of the action potential and its modulation is known to affect markedly the duration of the cardiac action potential [5]. Although the biophysical and pharmacological properties of the rat Ito current have been extensively investigated, its molecular structure still remains to be completely characterised. Amongst the genes coding for a transient outward potassium current, Kv1.4, Kv4.2 and Kv4.3 subunits have been found at the mRNA level to be expressed in adult rat atrial and ventricular tissues [6–10]. Recently, the Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of Ito [8, 11–15]. Among this family, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells [7, 8, 15–17]. Kv4.2 expression is highly abundant in rat myocytes and its distribution display a similar heterogeneity to that of the Ito current [6]. Furthermore, by expressing Kv4.2 in L-cells, Yeola and Snyders showed functional evidence that the Kv4.2 current is a major contributor to cardiac Ito [18]. In contrast, less is known regarding the other Shal isoform Kv4.3 recently cloned from rat brain, apart that this clone is highly expressed in rat atria and probably uniformly distributed in the ventricle [8, 19]. Expressed in oocytes, Kv4.3 encodes a current similar to that of rat and canine Ito current [8]. Recently, Fiset et al. [15]showed that antisense oligonucleotides directed against Kv4.2 or Kv4.3 mRNA similarly affected the transient outward potassium current in adult ventricular rat cells, demonstrating that both genes may be essential for the constitution of Ito channel in this species. The suppression of the rat myocardial Ito current by viral gene transfer of dominant negative Kv4 ion channel constructs further confirmed this hypothesis [17].
The aim of the present study was to express the rat Kv4.3 channel in a human cell line, the human embryonic kidney cell line (HEK-293) and to characterise its functional properties in comparison with the current recorded from isolated cardiac ventricular myocytes. In addition, because Ito and Kv4.2 have been shown to be more important in atrial and epicardial tissues, the localisation of the Kv4.3 mRNA was performed in the different parts of the rat heart using semi-quantitative polymerase chain reaction approaches.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Cell isolation
Male Wistar rats (250–350 g; Elevage St Antoine, Pleudaniel, France) were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23). The technique of cell dissociation used was derived from the method described by Mitra and Morad [20]. Briefly, rats were anaesthetised with sodium pentobarbital (60 mg/kg). Hearts were removed and perfused at 37°C through the aorta, according to the Langendorff method, at a constant pressure of 60 cm H2O. The composition of the standard perfusion solution was as follows (mM): NaCl 135, KCl 4, MgCl2 1, NaH2PO4 0.33, HEPES 10, glucose 10 (pH 7.4 with NaOH). Hearts were perfused for 5 min with this solution containing 1.8 mM calcium, for 5 min with standard solution alone (calcium-free solution), then for 35 min with perfusion solution containing collagenase (collagenase (Boehringer, type B) 1 mg/ml + protease (Sigma, type XIV) 0.16 mg/ml), and finally for 5 min with standard solution alone.
Ventricles were then minced and gently shaken for 5 min in a KB solution [21]containing (mM): KCl 40, K-Glutamate 50, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, Taurine 20 (pH 7.4 with KOH). After filtration (200 µm Nylon mesh), ventricular cells were allowed to settle in this solution for 1 h. The supernatant was then substituted by the experimental control solution and the cells were kept at room temperature until experimental use.
2.2 Plasmid construction
The 2.0 Kb XbaI–HindIII DNA fragment encoding the rat Kv4.3 potassium channel was isolated from plasmid pXRKv43FLEX(5) [8]kindly provided by Dr. David McKinnon (Stony Brook University) and cloned into the pHook2 (Invitrogen Co, La Jolla, CA) mammalian expression vector [22]digested by NheI-HindIII to give the plasmid pRP141 (9.5 Kb). The resulting plasmid contained the rat Kv 4.3 cDNA under the control of the CMV promoter and thymidine kinase poly-adenylation signal.
2.3 Transfection and cell culture
Cell culture was carried out using standard procedures. HEK 293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM/NUT.F-12) with Glutamax1 (Life technologies Ltd, Paisley, Scotland), supplemented with 100 Units/ml penicillin–streptomycin (Life technologies Ltd, Paisley, Scotland), 10% foetal bovine serum, North America, FDA approved (Life technologies Ltd, Paisley, Scotland), and HEPES (12 mM). The HEK 293 cells were transfected with plasmid pRP 141 containing the full length cDNA in 35 mm culture dishes. 5x105 cells were plated into 35 mm culture dishes with 2 ml of appropriate complete growth medium. Cells reaching approximately 50% confluence were transfected 24 h later following lipofection methodology mainly described by Felgner and collaborators [23]. Briefly, for each transfection, 2 µg of plasmid DNA at 2 µg/µl in TE buffer (Tris–HCl 10 mM, EDTA 1 mM, pH 8.0) and lipofectAMINE reagent (7.5 µl) (Life Technologies, Paisley, Scotland) were diluted separately with OptiMEM 1 reduced serum medium (Life technologies Ltd, Paisley, Scotland) to 100 µl final volumes, then mixed and incubated for 30 min at room temperature. OptiMEM (800 µl) was added to bring the total volume of the DNA–liposome complexes to 1 ml. This mixture (1 ml) was added to dish culture of HEK 293. The cells and the complexes were incubated 6 h at 37°C in 5% CO2 incubator. Then, the OptiMEM medium containing liposomes/DNA complexes was substituted by complete DMEM-F12 medium and the cells were incubated overnight in a 5% CO2 incubator. Control transfections were performed by substituting plasmid pRP 141 by pHook2 alone or by TE buffer.
2.4 Selection of transfected cells
Next day, transfected cells were harvested by incubation with PBS/3 mM EDTA at 37°C for 5 min, collected by centrifugation at 600xg, 5 min at 25°C, and resuspended in 1 ml of complete DMEM-F12 medium to which 1x106 (5 µl) Capture-Tec beads (Invitrogen Co, San Diego, CA) were added. The cell/beads complexes were rotated for 30 min at room temperature on a MX1 mixer (Dynal, Compiègne, France). The bound cells were separated from the total population by placing the tube in a Capture-Tec magnetic stand (Invitrogen Co, San Diego, CA, USA). The bead pellet containing selected cells was washed twice by resuspension in 1 ml complete DMEM-F12 medium followed by gentle vortexing. Selected cells were resuspended in 100 µl of complete medium and pasted in the center of a 35 mm culture dish.
2.5 Electrophysiological recordings
Whole-cell configuration of the patch-clamp technique [24]was applied either on native rat ventricular cells or on HEK293 cells transfected with rat Kv4.3 twenty four hours after magnetic beads based selection. The external solution contained (mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, Glucose 10. The pH was adjusted to 7.4 with NaOH. For experiments performed in native rat cells, the inward sodium current was inactivated by a 10 ms prepulse at –40 mV and the inward calcium current was inhibited by the addition of 0.2 mM CdCl2 to the external bath. The internal medium contained (mM): K-Aspartate 80, KCl 60, HEPES 10, Glucose 10, MgATP 2, EGTA 5, MgCl2 1 and the pH was adjusted to 7.3 with KOH. When studied, 4-aminopyridine (4-AP) (Aldrich, Steinheim) was prepared directly to provide the desired concentration. Pipettes were made from borosilicate capillary tubing and had resistances of 2 to 4 M
when filled with the internal solution.
Experiments were performed at 20±2°C. Ionic currents were recorded with a Biologic RK 300 amplifier (Biologic, Claix, France) or Axopatch-200A amplifier (Axon Instruments Inc, Foster City, CA, USA). Series resistance was compensated and currents were low-pass filtered at a cut-off frequency of 1 kHz (5 pole Tchebyschev filter). Currents, stimulus protocols and data collection were controlled by a microcomputer using pClamp software (Axon Instruments Inc, Foster City, CA, USA).
The amplitude of the transient outward potassium current recorded either in rat native ventricular cells or in HEK293 cells transfected with Kv4.3 was measured as the time-dependent amplitude of the current evoked by depolarising pulses. Rat ventricular cell capacitance was assessed by applying a 5 mV hyperpolarising pulse from a holding potential of –70 mV. The currents measured in rat ventricular myocytes were divided by the cell capacitance value to express ionic current amplitudes as densities (pA/pF).
2.6 Cardiac localisation of Kv4.3
The expression level of Kv4.3 mRNA in the different parts of the heart was determined using a semi-quantitative approach. To this aim, rat hearts were dissected out in right and left atria, septum, right and left ventricle. Furthermore, the left ventricle was separated according to the thickness of the tissue into endocardium, epicardium and midmyocardium. Total RNA was extracted using the acid guanidinium thiocyanate–phenol–chloroform method [25]. Samples were treated with RNase-free Dnase I, and first strand cDNA synthesis was done using 100 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Paisley, Scotland) per 200 ng of total RNA. Increasing concentrations of total RNA (200, 300, 400 and 500 ng) were used to conduct the RT-PCR in a semi-quantitative manner. Specific primers were designed to identify rat Kv4.3 and the following oligonucleotides were used for amplification: sense primer: 5'-CAC CCC AGA AGA GGA GCA CAT-3', antisense primer: 5'-GGT GGC CGG CAG GTT GGA GTT-3'. The products of the PCR were then run on 1% agarose gel electrophoresis and the intensity of the DNA fragments was scanned and quantified on an imaging densitometer (GS-670, Biorad SA) according to the quantity of total RNA used for the experiments.
2.7 Data analysis
Results are expressed as MEAN±SEM. The analysis of the electrophysiological data was performed using pClamp software (Axon Instruments, Foster City, CA, USA). Curve fitting was done by a non linear regression analysis using the single and double exponential functions and the standard Logistic algorithm provided by Origin 4.1 (MicroCal Software, Northampton, MA, USA). Comparison of the normalised RT-PCR fragment intensity measured from the different ventricular and atrial tissues was performed using a two factor repeated measures analysis of variance (ANOVA) followed by simple main effects and by multiple comparisons using the Sidak procedure as previously described [26]. All statistical analyses were performed by means of the microcomputer statistical program CRUNCH 4.0 (Crunch Software Corporation, Oakland, CA). A p value of less than 0.05 was considered as statistically significant.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Kv4.3 induces in HEK293 cells a transient outward current similar to Ito
To investigate the function of Kv4.3 expressed in HEK293 cells, the pHook2 vector was used. This plasmid allows selective isolation of transiently transfected cells by expressing a membrane anchored single chain antibody [22]. When transfected by either pHook2 or pRP 141, approximately 25% of the total treated cells compared to less than 10 cells (average 1 cell) with TE buffer were recovered 24 h after transfection using specific hapten-coated magnetic beads. The presence of magnetic beads do not interfere with electrophysiological recording. Among the selected cells complexed with magnetic beads, more than 95% were efficiently transfected and expressed the rat Kv 4.3 cDNA.
HEK293 cells transfected with Kv4.3 and ventricular adult rat cells were submitted to different electrophysiological protocols to evaluate and compare biophysical and pharmacological properties of the Kv4.3 gene product and of the transient outward potassium current Ito. Fig. 1 shows the activation and inactivation patterns of Kv4.3 current. Transfection of HEK293 cells with Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current (Fig. 1, insets) whereas cells transfected with pHook2 plasmid alone did not show any measurable current (not shown). In HEK293 cells transfected with Kv4.3, the current activated for potentials greater than –40 mV (Fig. 1A, n=24) and the steady-state inactivation curve had a midpoint of –47.4±0.3 mV and a slope of 5.9±0.2 mV (Fig. 1B, n=5).
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Similar experiments were performed in rat ventricular cells on the transient outward potassium current (Fig. 2). Ito was activated at potentials positive to –20 mV (Fig. 2A). As reported by Apkon and Nerbonne [27], the voltage dependence of steady-state inactivation of Ito current contained two components (Fig. 2B). The most negative one was suggested to be due to inactivation of IK whereas the other component reflects Ito inactivation [27]. The present study being focused on Ito, the holding potential used was not negative enough to characterise adequately IK inactivation. Only data obtained for potentials positive to –50 mV were considered to fit the steady-state inactivation curve of pure Ito. A half-inactivation potential of –29.1±0.7 mV and a Boltzmann factor of 4.5±0.5 mV were obtained.
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3.2 The recovery from inactivation differs between Kv4.3 and rat cardiac ventricular Ito
The kinetics of recovery from inactivation are determinant in the functional role of the transient outward potassium current in the action potential and in its frequency-dependent properties. Recovery from inactivation was thus evaluated from both cell types with a holding potential of –80 mV and the results are illustrated in Fig. 3. In HEK293 cells transfected with Kv4.3, the time course of recovery from inactivation was best fitted by a single exponential equation. The time constant obtained was 213.2±4.1 ms. Conversely, in rat cells, recovery from inactivation has been shown to be best fitted by a double exponential function [27]. The rapid component was reported to reflect recovery from inactivation of Ito whereas the slow one was due to reactivation of IK. The stimulation protocol used in the present study was designed essentially to resolve the recovery from inactivation of Ito and interpulse delay did not exceed 1 s. The time constants that were obtained using this protocol were 23.6±1.5 and 182.4±47.7 ms for the rapid and slow components, respectively., showing that the recovery from inactivation was slower when Kv4.3 current alone was measured than in the case of Ito determined in rat cells. Although the recovery from inactivation of Kv4.3 current is slower than the native current, it appears to be faster than the recovery from inactivation measured for Kv4.2 current [15, 18, 28].
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3.3 Cadmium inhibits Kv4.3 current in HEK293 cells
Cadmium has been shown to cause a shift in the voltage dependence of the native rat ventricular Ito current [29, 30]and of the Kv4.2 current measured in L-cells [15, 28, 31]. Therefore we investigated the effects of cadmium on the Kv4.3 current in HEK-293 cells. The results summarised in Fig. 4 shows that cadmium induced a rapid and concentration-dependent inhibition of the Kv4.3 current. The concentration inhibiting 50% of the current encoded by Kv4.3 was found 0.11±0.01 mM, a concentration in a range similar to that used in rat myocytes to inhibit the inward calcium current [29]. Furthermore, Fig. 4B shows that cadmium (0.2 mM) induced a large rightward shift in the steady state inactivation of Kv4.3. In these experiments the half inactivation potential was shifted from –47.4±0.3 mV in control conditions to –31.3±0.4 mV in the presence of 0.2 mM of cadmium. Cadmium induced a similar effect on Kv4.2 expressed in L-cells [15, 28, 31]. Finally, and in contrast to Kv4.2 expressed in L-cells [15], the results shown in Fig. 4C clearly demonstrate that Kv4.3 recovery of inactivation was sensitive to cadmium. In presence of 0.2 mM of cadmium, Kv4.3 channels recovered from inactivation more rapidly (time constant of recovery, 110.1±2.8 ms) than in absence of cadmium (time constant of recovery, 213.2±4.1 ms).
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) or in the presence of increasing concentrations of cadmium (10µM; 
, 30µM;
, 100µM;
, 200µM;
, 300µM;+, and 1000µM;x). The concentration inducing a 50% inhibition of the current was 0.11±0.01mM (Hill coefficient =0.70±0.02, n=5–8). (B) Voltage dependence of steady-state inactivation of Kv4.3 in HEK293 cells in the absence (
) or presence (
) of 0.2mM of CdCl2. Values represent the mean±sem of 7 cells. The best fits to Bolzmann equation were characterised for HEK293 cells without Cd2+ by a half-activation voltage of –47.4±0.3 mV and a slope of 5.9±0.2 mV and for HEK293 cells in the presence of Cd2+ by a half-activation voltage of –31.3±0.4 mV and a slope of 7.8±0.3 mV. (C) Recovery from inactivation of Kv4.3 in HEK293 cells in the absence (3.4 Rat Ito current is more sensitive to 4-AP than Kv4.3
The pharmacological regulation of Kv4.3 and Ito were assessed by evaluating the effect of different concentrations of 4-aminopyridine in both cell types. Fig. 5 shows the current traces obtained when the cells were depolarised from –80 mV to +10 mV and time-dependent effects recorded with 2, 5 and 10 mM 4-aminopyridine in HEK293 cells transfected with Kv4.3 (Fig. 5A) and with 2 and 5 mM in rat ventricular cells (Fig. 5B). Ito shows a greater sensitivity to 4-aminopyridine than Kv4.3. Half-maximal effects were obtained with 1.54±0.13 mM and 0.14±0.02 mM on Kv4.3 and Ito, respectively, suggesting that 4-aminopyridine is 10 times more potent on rat Ito than it is on Kv4.3.
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), 5 mM (
) 4-aminopyridine. The concentrations inducing a 50% inhibition of the current were 1.54±0.13 mM (n=4–8) and 0.14±0.02 mM (n=4–8) in HEK293 cells and rat ventricular myocytes, respectively. Holding potential: –80 mV; Test pulse potential: +10 mV.In both rat isolated myocytes and HEK293 cells expressing Kv4.3, 4-AP induced an apparent slowing of both activation and inactivation resulting in a crossover phenomenon (see left panels of Fig. 5). This effect was characterised by a reduced outward current at the beginning of the depolarisation and a delay in the time to reach the peak current. The second characteristics of this crossover phenomenon was that the current became greater than control when the depolarisation continued because the rate of decay was slowed. These changes in current time course suggest that 4-AP binds to the closed state of the Kv4.3 channel and unbinds in its inactivated state as already described in rat and ferret ventricular myocytes [32, 33]and on Kv4.2 channel expressed in oocytes [34].
3.5 Expression pattern of Kv4.3 mRNA in rat cardiac tissue
The results summarised in Fig. 6 show that Kv4.3 mRNA was observed in all the different parts of the rat heart. In the three experiments illustrated here (Fig. 6A), the level of expression was markedly higher in both the left and the right atria than in the ventricular tissue. The data obtained from the three experiments shown in Fig. 6A were analysed relatively to the expression level in the right atria. Arbitrarily, the intensity of each PCR Kv4.3 fragment was normalised to the intensity measured in the right atrium using 500 ng total RNA and expressed as a percentage ratio compared to the 100% assigned to the right atrium Kv4.3 fragment intensity obtained with 500 ng total RNA (Fig. 6B). Statistical analysis of the results shows that Kv4.3 was significantly more expressed in atrium compared to ventricle. In contrast, no significant difference was found between the expression level of Kv4.3 in the different ventricular tissues. Although these studies did not investigate a potential difference between the base and the apex of the ventricle, the results obtained suggested a homogenous distribution of Kv4.3 message.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The transient outward potassium current represents the principal repolarising current in rat cardiac myocytes and plays a major role in the early repolarisation of action potentials of many species including human. The results of the present study, which describes the electrophysiological and pharmacological characteristics of Kv4.3 expressed in a human cell line, clearly show that the Kv4.3 channel is a candidate to explain the molecular nature of Ito. However, these results also suggest that Kv4.3 alone cannot completely explain Ito.
Based on functional parameters, several molecular candidates have been proposed in the formation of Ito. The first candidate proposed was Kv1.4, a member of the Shaker family, because this protein when expressed in oocytes exhibited kinetics properties comparable to those measured in native cells [35]. However the recovery from inactivation of Kv1.4 was markedly prolonged when compared to Ito. Recently, members of the Shal family, Kv4 channel have been suggested to represent Ito more accurately.
When expressed in oocytes or in L-cells, Kv4.2 showed electrophysiological properties similar to cardiac Ito that were characterised by more appropriate kinetics (fast activation) during single depolarising voltage stimuli. As shown for rat cardiac myocytes the threshold for activation of Kv4.2 expressed in L-cells was around –25 mV [15, 18, 28]. However, the mid-point for inactivation was more negative for Kv4.2 expressed in L-cells (–45 to –40 mV) than for rat ventricular myocytes (around –30 mV) [15, 18, 28]. The results of the present study show that Kv4.3 expressed in HEK293 induced a current similar to Kv4.2 current with mid-point for inactivation of –47.4±0.3 mV but with an activation potential around –40 mV. Interestingly, when expressed in Xenopus oocytes Kv4.3 activated at a potential around –40 mV and had a mid-point of inactivation around –60 mV [8]. Therefore, based on these electrophysiological characteristics, our results suggest that Kv4.3 may play a role in rat Ito, but the differences noted in the activation and inactivation process further reinforce the current hypothesis proposing heteropolymeric structures for this channel. However, these differences could also be due to the conditions in which the currents were recorded.
The measure of Ito in native cells, including human myocytes [3], is commonly performed in the presence of cadmium to inhibit the calcium current. However, a direct effect of cadmium on Ito may explain the discrepancies between expressed and native channels. Therefore, we investigated the effect of cadmium on Kv4.3 current. Cadmium induced a dose dependent inhibition of the Kv4.3 current with a IC50 of 0.11 mM, a value similar to the concentration used in cardiac cells to inhibit the calcium current and to investigate Ito. Furthermore, we found that the steady state inactivation curve of Kv4.3 current was shifted to the right by more than 15 mV in the presence of cadmium, leading to a mid-point of inactivation of –31.25 mV, closely similar to the value measured in rat ventricular myocytes. This result suggests that cadmium may affect the electrophysiological characteristics of Ito.
It is well established that the transient outward current recorded in cardiac myocytes exhibits a rapid recovery from inactivation, as evidenced by a time constant for recovery from inactivation between 20 and 70 ms depending on the species (see table 1 in Yeola and Snyders [18]). Apkon and Nerbonne [27]reported that two time constants could be obtained from reactivation of the transient outward potassium current in rat ventricular myocytes. Whereas the rapid time constant obtained in the present study is in close agreement with the value reported for Ito by Apkon and Nerbonne [27](respectively 23.6 and 20 ms), the slow component, that has been attributed to IK, differed between the two studies (182.4 ms in the present report versus 520 ms in [27]). The reason for this discrepancy probably lies in the difference of the stimulation protocols used. IK channels were allowed to recover for up to 5 s in the study performed by Apkon and Nerbonne. In contrast, the interpulse delay did not exceed 1 s in our study, because we focused on the rapid component corresponding to Ito. The recovery from inactivation of rat Kv4.3 that we observed in HEK293 cells (
=213 ms), whereas slower than in native cells, was faster than for the other molecular candidates for Ito. Indeed, the time constant for recovery from inactivation () for Kv4.2 was between 258 and 378 ms at –80 mV [15, 18, 28]. Similarly, Kv1.4 failed to reproduce the rapid recovery kinetics of native Ito [35, 36]. Interestingly our preliminary results with the human Kv4.3 ortholog expressed in HEK293 show an even faster recovery from inactivation with a value below 140 ms. Finally, the presence of cadmium, as suggested by the present results, may also be taken into consideration in determining recovery kinetics for Kv4.3 because the recovery of inactivation was markedly accelerated by the presence of cadmium. In contrast, the results previously reported with Kv4.2 expressed in L-cells showed that cadmium has no effect on the recovery kinetics of this component of Ito [15].
Thus the present study clearly demonstrates that external cadmium chloride can affect Kv4.3 current and that characterisation of Ito in native rat cells may be influenced by the presence of cadmium in the external solution. Nevertheless, these effects of cadmium cannot account for all the differences noted between Kv4.3 current and native Ito, and particularly for the marked difference in reactivation kinetics observed between the two currents. It could therefore be hypothesised that in native tissues, Kv4 channels coassemble with auxiliary subunits to form functional Ito channels and that these unidentified subunits can modify the properties of the channel. Further work is necessary to identify if other proteins can interact with Kv4 channels and provide a more compete explanation of the gating properties of native Ito.
4-Aminopyridine has been widely used to characterise the pharmacology of the transient outward current. Castle and Slawsky [32]showed that in isolated rat myocytes, 4-AP caused a rapid reduction in the peak amplitude of the inactivating component of Ito without affecting the sustained component. A closely similar finding is noted in the present study in which 4-AP dramatically reduced the time dependent amplitude of the current induced by Kv4.3 in HEK293 cells. Our results clearly show that the concentration dependence of 4-AP is markedly different from that of endogenous rat Ito. Indeed even with 10 mM a sizeable amplitude of the current remained activated. An effect closely similar was already observed on Kv4.2 expressed in oocytes where the IC50 for 4-AP (1.5 mM) [34]was identical to the one measured on Kv4.3 (1.54 mM, present study). Our results show that the effect of 4-AP on Kv4.3 is characterised by a ‘crossover’ of the current trace with the control trace suggesting a similar blocking mechanism as described for Ito measured in rat cardiac myocytes [32]and for Kv4.2 [34]. The current trace shown in Fig. 5 clearly indicates a slowing in the activation and the inactivation of the current. Although a more extensive investigation of the effect of 4-AP on this cloned channel remains to be finalised, these data suggest that 4-AP binds in the closed state and unbinds in the inactivated state of the channel as previously described in cardiac myocytes and oocytes expressing Kv4.2 [32, 34]. There are some differences between Kv4.3 and rat Ito currents, however, the electrophysiological and pharmacological characteristics of Kv4.3 suggest that this protein could play a role similar to that of Kv4.2 in the native current.
Our results finally show that the expression of the Kv4.3 gene is higher in the atria and appears to be homogeneously distributed across the ventricular tissue. Although these data were obtained using a semi-quantitative approach, the three sets of experiments performed led to a similar conclusion. Recently, two isoforms of the rat Kv4.3 have been identified [16, 37]. However, the primers used in the present study were not designed to discriminate between the short and the long variants of Kv4.3. The results of the present study reinforce the previous findings showing that Kv4.3 is uniformly expressed between endocardium and epicardium [8]. Moreover, it has recently been shown, using an immunohistochemistry approach, that the distribution of the Kv4.3 protein was homogenous across the ventricle in ferret heart [19]. Interestingly it is well known that both endogenous Ito current and Kv4.2 channel are distributed in a comparable non-homogeneous manner in the ventricle of mammalian hearts [3, 7, 19, 38]. Similarly, Kv4.2 has been shown to be more expressed in ventricle than in atrium [7]. Based on the present results, it could be suggested that Kv4.3 has no role in the electrophysiological heterogeneity observed with Ito. Furthermore, because the distribution of Kv4.3 across the ventricle is homogenous, the blockade of this channel by specific drugs might not alter the normal heterogeneity of Ito current. Finally, this channel might play a major role in the repolarisation of the atrial cell since expression of Kv4.3 seems more pronounced in the atria than in the ventricle.
In conclusion, the results of this work provide a characterisation of Kv4.3 channel expressed in HEK293 cell and further suggest that this protein could be as important as Kv4.2 in generating Ito in rat myocytes. All the results reported in the present study were performed with the short splice variant of the Kv4.3 gene [8, 16]. However, our preliminary data obtained by PCR cloning using human cardiac libraries showed that at least two splice variants can be obtained (unpubl. data) and they could both participate to the formation of the native Ito current. Therefore, further studies remain to be performed to characterise the relative role of the different molecular components of Ito, Kv4.2 and the different isoforms of Kv4.3 in controlling the duration of cardiac action potentials in both normal and pathologic situations.
Time for primary review 22 days.
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Acknowledgements |
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The authors would like to acknowledge Sonia Saïdi for the preparation of the manuscript and Dr David McKinnon (State University of New York at Stony Brook, Stony Brook, NY) for the gift of Kv4.3 cDNA.
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Notes |
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1 See pages 16–18.
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References |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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