Cardiovascular Research 41 (1999) 188–199
Characterisation of Kv4.3 in HEK293 cells: comparison with the rat
ventricular transient outward potassium current 1
´
Jean-François Faivre, Thierry P.G. Calmels, Sabine Rouanet, Jean-Luc Javre,
*
Brigitte Cheval, Antoine Bril
Department of Cardiovascular Pharmacology, SmithKline Beecham Laboratoires Pharmaceutiques, 4 Rue du Chesnay Beauregard,
´
35760 Saint-Gregoire
, France
Received 18 March 1998; accepted 3 June 1998
Abstract
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 240 mV and the steady-state inactivation curve had a midpoint of
247.460.3 mV and a slope of 5.960.2 mV. Rat ventricular Ito current was activated at potentials positive to 220 mV and inactivated with
a half-inactivation potential and a Boltzmann factor of 229.160.7 mV and 4.560.5 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 213.264.1 msec and 23.61.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to
4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.5460.13 mM and 0.1460.02 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.  1999 Elsevier Science B.V. All rights reserved.
Keywords: K-channel; Gene expression; Transient outward potassium current; Kv4.3; Cadmium
1. Introduction
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
*Corresponding author. Tel.: 133-299-280-456; fax: 133-299-280444; e-mail: antoine [email protected]
]
1
See pages 16–18.
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)
Time for primary review 22 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 98 )00215-6
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
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 Lcells, 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.
2. Methods
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 378C through the aorta, according to the
Langendorff method, at a constant pressure of 60 cm H 2 O.
The composition of the standard perfusion solution was as
follows (mM): NaCl 135, KCl 4, MgCl 2 1, NaH 2 PO 4 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 (calciumfree solution), then for 35 min with perfusion solution
containing collagenase (collagenase (Boehringer, type B) 1
189
mg / ml 1 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, KH 2 PO 4 20, MgCl 2 3, EGTA 0.5,
HEPES 10, Taurine 20 (pH 7.4 with KOH). After filtration
(200 mm 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. 5310 5 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 mg of
plasmid DNA at 2 mg / ml in TE buffer (Tris–HCl 10 mM,
EDTA 1 mM, pH 8.0) and lipofectAMINE reagent (7.5
ml) (Life Technologies, Paisley, Scotland) were diluted
separately with OptiMEM 1 reduced serum medium (Life
technologies Ltd, Paisley, Scotland) to 100 ml final volumes, then mixed and incubated for 30 min at room
temperature. OptiMEM (800 ml) 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
378C in 5% CO 2 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% CO 2 incubator. Control transfections were performed by substituting plasmid pRP 141 by
pHook2 alone or by TE buffer.
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J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
2.4. Selection of transfected cells
2.6. Cardiac localisation of Kv4.3
Next day, transfected cells were harvested by incubation
with PBS / 3 mM EDTA at 378C for 5 min, collected by
centrifugation at 6003g, 5 min at 258C, and resuspended
in 1 ml of complete DMEM-F12 medium to which 1310 6
(5 ml) 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,
`
Compiegne,
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 ml of complete medium and pasted in the
center of a 35 mm culture dish.
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: 59-CAC CCC
AGA AGA GGA GCA CAT-39, antisense primer: 59-GGT
GGC CGG CAG GTT GGA GTT-39. 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.
2.5. 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, MgCl 2 1, CaCl 2 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 240 mV and the inward calcium current was inhibited
by the addition of 0.2 mM CdCl 2 to the external bath. The
internal medium contained (mM): K-Aspartate 80, KCl 60,
HEPES 10, Glucose 10, MgATP 2, EGTA 5, MgCl 2 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 MV when filled with the internal solution.
Experiments were performed at 20628C. 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 270 mV. The currents measured in rat
ventricular myocytes were divided by the cell capacitance
value to express ionic current amplitudes as densities
(pA / pF).
2.7. Data analysis
Results are expressed as MEAN6SEM. 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.
3. Results
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
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
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 haptencoated 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 240
mV (Fig. 1A, n524) and the steady-state inactivation curve
had a midpoint of 247.460.3 mV and a slope of 5.960.2
mV (Fig. 1B, n55).
Similar experiments were performed in rat ventricular
cells on the transient outward potassium current (Fig. 2).
Ito was activated at potentials positive to 220 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
250 mV were considered to fit the steady-state inactivation
curve of pure Ito . A half-inactivation potential of
229.160.7 mV and a Boltzmann factor of 4.560.5 mV
were obtained.
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 280 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
213.264.1 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
191
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
23.661.5 and 182.4647.7 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].
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 0.1160.01 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 247.460.3
mV in control conditions to 231.360.4 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,
110.162.8 ms) than in absence of cadmium (time constant
of recovery, 213.264.1 ms).
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 280 mV to 110 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. Halfmaximal effects were obtained with 1.5460.13 mM and
0.1460.02 mM on Kv4.3 and Ito , respectively, suggesting
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J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
Fig. 1. Voltage-dependent activation and inactivation characteristics of Kv4.3 expressed in HEK293 cells. A: Current-voltage curve from a holding potential
of 280 mV. Values correspond to mean time-dependent current amplitudes recorded from 24 cells for the corresponding depolarising pulses. Inset shows
individual traces obtained with progressive depolarisations from a holding potential of 280 mV to 170 mV (10 mV increment) for 510 ms. In this and
subsequent figures illustrating original recordings, the arrow indicates the 0 current level. B: Steady-state inactivation curve. Values correspond to mean
normalised time-dependent current amplitudes recorded from 5 cells during a test pulse to 110 mV after the membrane was clamped to the corresponding
voltage. The fitted Boltzmann curve was characterised by a half-inactivation voltage of 247.460.3 mV and a slope of 5.960.2 mV. Inset shows individual
traces obtained during this double stimulation protocol. Holding potential: 280 mV.
that 4-aminopyridine is 10 times more potent on rat Ito
than it is on Kv4.3.
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
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
193
Fig. 2. Voltage-dependent activation and inactivation characteristics of the transient outward potassium current Ito of rat ventricular cells. A: Current-voltage
curve from a holding potential of 280 mV. Values correspond to mean time-dependent current amplitudes recorded from 9 cells for the corresponding
depolarising pulses. Inset shows individual traces obtained with progressive depolarisations from a holding potential of 280 mV to 170 mV (10 mV
increment) for 510 ms. B: Steady-state inactivation curve. Values correspond to mean normalised time-dependent current amplitudes recorded from six cells
during a test pulse to 110 mV after the membrane was clamped to the corresponding voltage. Only data obtained for pre-pulse potentials more positive
than 250 mV were considered to fit the steady state inactivation of Ito . The component resulting from inactivation of the delayed rectifier potassium current
was not considered in the fitting process. The fitted Boltzmann curve was characterised by a half-inactivation voltage of 229.160.7 mV and a slope of
4.560.5 mV. Inset shows individual traces obtained during this double stimulation protocol. Holding potential: 280 mV.
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
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J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
Fig. 3. Recovery from inactivation of Kv4.3 expressed in HEK293 transfected cells (A) and of Ito in rat ventricular cells (B). Left panels show experimental
currents elicited by two depolarising steps to 110 mV from a holding potential of 280 mV with variable interpulse delay. Right panels show mean
normalised time-dependent current amplitudes versus interpulse delay (n511 and 12 for HEK293 transfected cells and rat ventricular cells, respectively).
The continuous lines represent best-fit single and double exponential functions for rat Kv4.3 and Ito , respectively. Corresponding time constants are
indicated within the figure.
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.
4. Discussion
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 225 mV
[15,18,28]. However, the mid-point for inactivation was
more negative for Kv4.2 expressed in L-cells (245 to 240
mV) than for rat ventricular myocytes (around 230 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
247.460.3 mV but with an activation potential around
240 mV. Interestingly, when expressed in Xenopus oocytes
Kv4.3 activated at a potential around 240 mV and had a
mid-point of inactivation around 260 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 hetero-
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
195
Fig. 4. Effect of cadmium on Kv4.3 current measured in HEK293 transfected cells. (A) Time and dose dependent effect of cadmium on Kv4.3. The current
amplitude was measured in the absence (j) or in the presence of increasing concentrations of cadmium (10mM; d, 30mM;m, 100mM;., 200mM;♦,
300mM;1, and 1000mM;3). The concentration inducing a 50% inhibition of the current was 0.1160.01mM (Hill coefficient 50.7060.02, n55–8). (B)
Voltage dependence of steady-state inactivation of Kv4.3 in HEK293 cells in the absence (s) or presence (h) of 0.2mM of CdCl 2 . Values represent the
mean6sem of 7 cells. The best fits to Bolzmann equation were characterised for HEK293 cells without Cd 21 by a half-activation voltage of 247.460.3
mV and a slope of 5.960.2 mV and for HEK293 cells in the presence of Cd 21 by a half-activation voltage of 231.360.4 mV and a slope of 7.860.3 mV.
(C) Recovery from inactivation of Kv4.3 in HEK293 cells in the absence (s) or presence (h) of 0.2mM of CdCl 2 . The time constants for recovery from
inactivation was shifted from 213.264.1 msec in the absence of CdCl 2 to 110.162.8 msec in presence of CdCl 2 .
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J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
Fig. 5. Effect of 4-aminopyridine on Kv4.3 and Ito recorded from HEK293 transfected cells (A) and adult rat ventricular cells (B), respectively. Left panels
show experimental traces recorded in the absence and in the presence of 2 mM 4-aminopyridine. Right panels show dose and time-dependent variation of
mean normalised currents (n54–8) recorded in the absence (s) and in the presence of 2 mM (D), 5 mM (h) or 10 mM (앳) 4-aminopyridine. The
concentrations inducing a 50% inhibition of the current were 1.5460.13 mM (n54–8) and 0.1460.02 mM (n54–8) in HEK293 cells and rat ventricular
myocytes, respectively. Holding potential: 280 mV; Test pulse potential: 110 mV.
polymeric 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 IC 50 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 231.25 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
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
197
Fig. 6. Expression pattern of Kv4.3 mRNA in rat heart. A: Results obtained from three experiments of RT-PCR performed with increasing quantities of
total RNA extracted from the different parts of the rat heart. For each tissue sample, the quantities of RNA used were 500, 400, 300 and 200 ng of RNA
(from left to right for each group of four lines) B: mean results of the three experiments. RA: right atrium; LA: left atrium, RV: right ventricle, Epi:
epicardium, Mid: midmyocardium, Endo: endocardium.
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 in-
activation of rat Kv4.3 that we observed in HEK293 cells
(t 5213 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 (t )
198
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
for Kv4.2 was between 258 and 378 ms at 280 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 IC 50 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.
Acknowledgements
¨ for
The authors would like to acknowledge Sonia Saıdi
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.
J.-F. Faivre et al. / Cardiovascular Research 41 (1999) 188 – 199
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