Vol. 278, Issue 2, H484-H492, February 2000
Developmental differences in delayed rectifying outward
current in feline ventricular myocytes
Héctor
Barajas-Martínez,
Alejandro
Elizalde, and
José A.
Sánchez-Chapula
Unidad de Investigación Carlos Méndez, Centro
Universitario de Investigaciones Biomédicas, Universidad de
Colima, C.P. 28040, Colima, Mexico
 |
ABSTRACT |
In the present work,
we found that the delayed rectifying outward potassium current
(IK) in adult and neonatal cat ventricular myocytes
consists of both rapid and slow components, IKr and
IKs, respectively, which can be isolated
pharmacologically. Thus after complete blockade of
IKr with dofetilide, the remaining
IKs current is homogeneous, as shown by an envelope
of tails test. IKr maximum tail current density,
measured at 
40 mV, was similar in adult and neonatal myocytes.
IKs maximum tail current density in neonatal myocytes, measured at 40 mV, was significantly smaller than in adult myocytes. Activation kinetics of IKr and
IKs was similar in both age groups. However, the
IKr deactivation time course was significantly
faster in neonatal than in adult myocytes. Developmental differences in
the subunit composition of IKr that display
distinctly different deactivation kinetics are suggested.
cat; rapid delayed rectifying current; slow delayed rectifying
current; dofetilide
| |
INTRODUCTION |
THE DELAYED RECTIFYING OUTWARD potassium current
(IK) has been found to be important to the
repolarization of the cardiac action potential in different mammalian
species (11). In most mammalian species, IK has
been reported to consist of two different components that have distinct
time- and voltage-dependent and pharmacological properties (7, 14, 17,
20, 22). The rapidly activating, inwardly rectifying component,
IKr, is specifically blocked by
methanesulfonanilide class III antiarrhythmic agents such as E-4031,
sotalol, and dofetilide (20). The slowly activating component,
IKs, displays a linear current-voltage
(I-V) relationship and is insensitive to the
methanesulfonanilide agents (20).
In cat ventricular myocytes, it has been reported that
IK consists solely of the single rapidly activating
component, IKr (5). However, under experimental
conditions that minimize the contribution of IKr
(zero external K+), it was reported that
IK in cat ventricular myocytes consists of a single
component with time- and voltage-dependent properties similar to those
of the slowly activating, inwardly rectifying current
(IKs) (6). These inconclusive results led us to
reinvestigate the kinetic and pharmacological properties of
IK in cat ventricular myocytes. We found evidence
that IK in cat ventricular myocytes is composed of
two kinetically and pharmacologically distinct components,
IKr and IKs.
Age-dependent differences have been found in potassium current
densities and kinetics, which determine differences in action potential
duration (APD) and response to agents that prolong APD (4). In the
present work, we compared IK in adult and neonatal ventricular myocytes. The most striking difference we found between these age groups was that IKr deactivation was
faster in neonatal than in adult myocytes.
| |
METHODS |
Isolation of ventricular myocytes.
Experiments were performed on single ventricular myocytes obtained from
the right ventricle of adult (2.5-3.5 kg) and neonatal (0-4
days from birth) cats, using a method similar to that previously described (16). The hearts were mounted on a Langendorff apparatus, perfused for 8 min with normal Tyrode solution, and then switched to a
nominally calcium-free solution for 6 min. Afterwards, the hearts were
perfused for 12 (neonatal hearts) or 40 (adult hearts) min with a
calcium-free solution containing 1 mg/ml collagenase (Sigma type I) and
0.1 mg/ml protease (Sigma type XIV). The enzymes were washed out by
perfusion with a high-potassium, low-chloride saline solution for 8 min. Temperature of the solutions was maintained at 37°C. The free
wall of the right ventricle was dissected out and cut into small
pieces. Single cells were obtained by mechanical agitation with a
pipette. The cells were maintained in a high-potassium, low-chloride
solution at 4°C up to 10 h for later electrophysiological experiments.
The Tyrode solution had the following composition (mM): 118 NaCl, 5.4 KCl, 1.05 MgCl2, 1.8 CaCl2, 24 NaHCO3, 0.42 NaH2PO4, 11 glucose,
and 20 taurine. The solution was equilibrated with 95%
O2-5% CO2 (pH 7.4). A nominally calcium-free
solution was prepared by omitting CaCl2 from the Tyrode
solution. The high-potassium, low-chloride solution had the following
composition (mM): 20 KCl, 10 taurine, 70 glutamic acid, 0.5 creatinine,
10 KH2PO4, 5 succinic acid, 10 glucose, 10 HEPES, and 0.2 EGTA; pH was adjusted to 7.4 with KOH. This solution was
equilibrated with 100% O2.
Data acquisition and analysis.
A few drops of the cell suspension were placed in a superfusion chamber
(0.5 ml) mounted on a stage of an inverted microscope (Nikon Diaphot,
Tokyo, Japan). The myocytes were allowed to settle to the bottom of the
chamber (5-10 min) and then were superfused with normal external
solution. Experiments were performed at 35°C, and the temperature
was controlled by an open bath microincubator (Medical Systems,
Greenvale, NY). Current recordings were obtained by using the whole
cell patch-clamp method (8) with an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA). Data acquisition and generation of
voltage-clamp pulse protocols were carried out with a LabMaster TL-1
interface controlled by pCLAMP 6.0 software (Axon Instruments).
Currents were low-pass filtered at 1 kHz and sampled at 2 kHz, and the
data were stored on the hard disk of a 486DX2 computer. Micropipettes
were pulled from borosilicate glass capillary tubes (WPI TW50, World
Precision Instruments, Sarasota, FL) on a programmable puller (Sutter
Instruments, Novato, CA). When micropipettes were filled with the
pipette solution, tip resistance ranged from 1 to 4 M
. Whole cell
capacitance and series resistance compensation (80%) were optimized to
minimize the duration of the capacitive currents and reduce voltage
errors. The capacitive transient response to a 10-mV hyperpolarizing
step was integrated and divided by the voltage step to estimate cell capacitance. Currents were normalized to capacitance of individual cells to represent I-V relationships. The calculated cell
capacitance was 19.2 ± 2.3 and 103.2 ± 6.7 pF for neonatal and
adult cells, respectively (mean ± SE, n = 30). After membrane
patch rupture, the cells were superfused with the
Ca2+-Co2+ solution for recording of membrane
currents. In the present work, Ca2+ current
(ICa) was blocked by using a low external calcium
chloride concentration and cobalt chloride. It has been recently
reported that divalent cations such as Ca2+,
Mg2+, and Co2+ produce a shift qualitatively
similar in the activation curve of human
ether-à-go-go-related gene (HERG) channels expressed in
oocytes to more depolarized potentials and a voltage-dependent blockade
(9). Therefore, qualitatively similar effects on
IKr are expected when Ca2+ and
Mg2+ are used or Co2+ is added to the external solution.
The normal external solution had the following composition (mM): 136 NaCl, 4 KCl, 1 MgCl2 1.8 CaCl2, 10 HEPES, and
11 glucose; pH was adjusted to 7.4 with NaOH. The
Ca2+-Co2+ solution had the following
composition (mM): 136 NaCl, 4 KCl, 2 CoCl2, 1 MgCl2, 0.5 CaCl2, 10 HEPES, and 11 glucose; pH
was adjusted to 7.4 with NaOH. The pipette solution had the following composition (mM): 80 K-aspartate, 10 KH2PO4, 50 KCl, 1 MgSO4, 5 HEPES, and 5 EGTA; pH was adjusted to 7.3 with KOH. Dofetilide was dissolved in DMSO as a 10 mM stock solution.
Results are presented as means ± SE. Statistical comparisons between
group means were evaluated by Student's t-test for independent
groups at a significance level of P < 0.05 (2-tailed test).
Curve fitting was performed with Fig.P software (Biosoft, Ferguson, MO).
| |
RESULTS |
Characterization of dofetilide-sensitive and dofetilide-insensitive
currents.
Figure 1 shows IK
obtained from an adult isolated ventricular myocyte. Figure 1A
shows total membrane currents induced by 3-s depolarizing pulses and
tail currents after repolarizing pulses to the holding potential of
40 mV. Data were recorded 3-5 min after patch membrane
rupture. At the holding potential of 40 mV, an outward holding
current caused mainly by the inward rectifying potassium current
(IK1) was recorded. Test pulses to membrane potentials from 30 to +10 mV first induced an instantaneous
decrease in outward current due to the inward rectifying properties of IK1 and then induced a time-dependent current that
showed a rapid component followed by a slower one. A sigmoidal onset of
activation was observed only during test pulses positive to +30 mV.
However, the sigmoidal onset of current activation was evident at all
potentials studied in the presence of dofetilide (Fig. 1B).
Data were recorded in the presence of dofetilide 7-9 min after the
beginning of the drug superfusion. Figure 1C shows the
dofetilide-sensitive current (IKr) obtained by
digital subtraction of currents recorded in the presence of dofetilide
from control records. This current component did not exhibit an obvious
delay, and it fully activated at +10 mV. Figure 1 also shows the
I-V relationships for the time-dependent currents measured
during the depolarizing pulses (Fig. 1D).
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1.
Dofetilide-sensitive (IKr) and dofetilide-resistant
(IKs) delayed rectifier potassium currents in adult
cat ventricular myocytes. Currents recorded during 3-s pulses to
30, 10, +10, +30, and +50 mV applied from a holding
potential of 40 mV. Tail currents were obtained on return to
40 mV from indicated test potentials (Vm),
under control conditions (A) and in the presence of 5 µM
dofetilide (B). C: IKr obtained by
digital subtraction of currents recorded in the presence of drug from
those recorded under control conditions. D: time-dependent
currents (IK,act) obtained during 3-s depolarizing
pulses. Currents were measured from time initial capacitance spike had
settled to end of Vm (n = 13 cells).
IK,total, total delayed rectifier currents.
E: tail currents (IK,tail) measured at
40 mV, on return from indicated Vm
(n = 13 cells). IK,act and
IK,tail amplitudes were measured as indicated in
inset.
|
|
IKr and dofetilide-insensitive currents
(IKs) were similar to those described in guinea pig
ventricular myocytes (20). The isochronal I-V relation of the
time-activated, dofetilide-sensitive current showed inward
rectification, and the isochronal I-V relation of the
dofetilide-resistant, time-activated current was almost linear. The
dofetilide-sensitive tail current-voltage relation showed saturation at
+20 mV, and the dofetilide-resistant tail current-voltage relation did
not reach saturation, even at +50 mV. Figure
2 shows IK obtained
from neonatal ventricular myocytes. The voltage dependence,
rectification, and densities of the dofetilide-sensitive current
components recorded in neonatal myocytes were similar to those recorded
in adult myocytes. The voltage dependence of the dofetilide-resistant
current was similar in both neonatal and adult myocytes. The
dofetilide-resistant maximum tail current density was significantly
smaller in neonatal than in adult myocytes. The most striking
difference between currents recorded from these age groups was that the
deactivation time course of the total IK and
dofetilide-sensitive delayed rectifier outward currents was faster in
neonatal than in adult myocytes.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
IKr and IKs in neonatal cat
ventricular myocytes. Currents were recorded during 3-s pulses to
30, 10, +10, +30, and +50 mV applied from a holding
potential of 40 mV. IK,tail were obtained on
return to 40 mV from indicated Vm, under
control conditions (A) and in presence of 5 µM dofetilide
(B). Inset, enlarged IK,tail traces
after depolarizing pulses to 30, +10, and +50 mV. C:
IKr obtained by digital subtraction of currents
recorded in presence of drug from those obtained under control
conditions. D: IK,act obtained during 3-s
depolarizing pulses. Currents were measured from time initial
capacitance spike had settled to end of Vm
(n = 11 cells). E: IK,tail measured
at 40 mV on return from indicated Vm
(n = 11 cells). IK,act and
IK,tail were measured as shown in inset of
Fig. 1.
|
|
Envelope of tails test.
The envelope of tails test of representative adult and neonatal cells
in the presence and absence of dofetilide is shown in Fig.
3. From a holding potential of 40
mV, the membrane potential was depolarized to +40 mV for various
durations and then returned to 40 mV. If IK
results from a single time-dependent current, then the ratio of tail
current to time-dependent current
(IK,tail/IK,act) for a given
depolarizing pulse will be constant, regardless of the pulse duration,
whereas if IK contains multiple components, the
ratio will vary with pulse duration. In the absence of dofetilide, tail
currents in adult and neonatal myocytes were larger than time-dependent
currents for short pulses, but as the pulse duration was lengthened,
the time-dependent current slowly increased in magnitude. In the
presence of dofetilide, the current ratio was constant, regardless of
the duration of the depolarizing pulse. IK,tail/IK,act obtained under
control conditions is larger than that obtained in guinea pig
ventricular myocytes (20). An explanation for such a difference may be
that in guinea pig ventricular myocytes, current density of the rapidly
activating, inward rectifying current, IKr, is
smaller than current density of the slowly activating current,
IKs (20), whereas in cat ventricular myocytes,
current density of IKr is larger than
IKs density (present study). Both pharmacological
evidence and the envelope of tails test evidence strongly suggest that
IK in adult and neonatal cat ventricular myocytes
is composed of two delayed rectifying currents, IKr
and IKs.
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
Envelope of tails test in adult and neonatal cat ventricular myocytes.
A: currents recorded in an adult cat ventricular myocyte before
(a) and after (b) 5 µM dofetilide and currents
recorded from a neonatal cat ventricular myocyte before (c) and
after (d) 5 µM dofetilide. B: plots of ratio of
IK,tail to IK,act before and
after 5 µM dofetilide in adult (n = 6; a) and
neonatal (n = 5; b) cells.
|
|
Reversal potential of delayed rectifier currents in neonatal and
adult ventricular myocytes.
Figure 4 illustrates the results from
experiments designed to assess the reversal potential of
IKr and IKs in adult and
neonatal ventricular myocytes. IKr was defined as
the dofetilide-sensitive current component and IKs
as the drug-resistant component. Tail currents were recorded during
repolarization at different membrane potentials ranging from 90
to 0 mV after an activating pulse (3-s duration) to +50 mV. Reversal
potential (Erev) in adult cells was 82.2 ± 0.4 and 76 ± 0.3 mV for IKr and
IKs, respectively. In neonatal cells
Erev was 79.4 ± 0.6 and 74 ± 0.2 mV for IKr and IKs,
respectively. The I-V relationships show the inward
rectification of IKr and a closely linear
relationship for IKs.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Current-voltage relation for IK,total,
IKr, and IKs in adult
(n = 5 cells; A) and neonatal (n = 4 cells;
B) ventricular myocytes. From a holding potential of 40
mV, 2 test clamp pulses were applied to voltages ranging from 90
to 0 mV with and without a preceding depolarizing clamp to + 50 mV for
3 s. Difference between initial IK,tail amplitudes
was plotted against test voltage at which current was elicited (see
inset).
|
|
Activation and deactivation kinetics of IK,
IKr, and IKs.
Figure 5 plots the amplitude of the tail
currents measured at 40 mV after a pulse to +30 mV, as a
function of the activating pulse duration in adult (Fig. 5A)
and neonatal (Fig. 5B) myocytes. Least-squares regression was
used to fit the sum of two exponentials to the data points for
IK and single exponentials for
IKr and IKs. The time constants
(
) of activation at three different membrane potentials (+10, +30,
and +50 mV of IK, IKr, and
IKs of adult and neonatal myocytes) are shown in
Table 1. No significant differences in the
activation time course of IK,
IKr, and IKs were recorded in
adult versus neonatal myocytes.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of IK,total, IKr,
and IKs activation in adult (n = 5 cells;
A) and neonatal (n = 5 cells; B) myocytes.
Activation time course was estimated using IK,tail
measurement. IK,tail were elicited by applying test
depolarizations to +30 mV, using different test pulse durations ( T,
inset). Currents were measured as peak
IK,tail during repolarization to 40 mV. Data
beginning at 50 ms for IK,total and
IKr and at 200 ms for IKs were
fitted to monoexponential functions to emphasize late phase of
activation.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Time constants of activation at different test potentials of
IK, IKr, and IKs in adult
and neonatal myocytes
|
|
The voltage dependence of deactivation was assessed by the following
protocol. Tail currents were recorded on repolarization after a
depolarizing pulse (3-s duration) to +50 mV. Tail currents were fitted
with two exponentials for IK,
IKr, and IKs. Figure 6A shows IK
tail current traces at different membrane potentials in myocytes from
adult and newborn cats. IK deactivation was faster in neonatal than in adult myocytes. Both fast (f) and
slow (s) time constants of IK
deactivation were smaller in neonatal than in adult myocytes (Fig.
6C). The of deactivation of IKs were smaller in neonatal than in adult myocytes, but the difference was only
significant at a few membrane potentials. The of deactivation of
IKr in neonatal cells were significantly different
from those in adult cells at all membrane potentials.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
Decay time course of tail current of IK,total,
IKr, and IKs in adult and
neonatal cat ventricular myocytes. A:
IK,total tail currents recorded at variable
Vm (indicated at right) after 3-s
depolarizing pulse to +50 mV in adult (a) and neonatal
(b) myocytes (note different time scales in a and
b). Best-fit curves with indicated time constants () are
drawn through current records. B: mean ± SE values of fast
(f) and slow (s) time constants of
IK,total deactivation at different
Vm in adult (n = 5 cells) and neonatal
(n = 4 cells) myocytes. C: mean ± SE values of
f and s of IKr
deactivation at different Vm in adult (n = 4 cells) and neonatal (n = 4 cells) myocytes. D: mean ± SE values of f and s of
IKs deactivation at different
Vm in adult (n = 4 cells) and neonatal
(n = 3 cells) myocytes. * Statistically significant
difference between means.
|
|
Accumulation of activation during trains of pulses.
The slow deactivation of IKr in cat adult myocytes
predicts accumulation of activation at high stimulation rates, but on
the other hand, in cat neonatal myocytes, deactivation is fast and small or no accumulation is expected. Trains of sixteen 200-ms-duration pulses were applied to +30 mV, at a frequency of 2 Hz, from two different holding potentials, 40 and 80 mV (Fig.
7). In adult myocytes, with a holding
potential of 40 mV at the end of a train of 16 pulses, tail
current amplitude induced by the last pulse was 22% greater than tail
current induced by the first pulse. When the holding potential used was
80 mV, no significant differences in tail current amplitude were
observed during the train. In neonatal myocytes, no significant
differences in tail current amplitude were observed during the train at
holding potentials of 40 and 80 mV (Fig. 7).
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Accumulation of activation during a train of pulses. From 2 different
holding potentials (HP), 40 mV (A) and 80 mV
(B), trains of 16 pulses of 200-ms duration to +30 mV, followed
by repolarization to 40 mV, were applied at a frequency of 2 Hz
in adult (n = 4 cells) and neonatal (n = 4 cells) cat
ventricular myocytes. Amplitude of IK,tail measured
at 40 mV, normalized to amplitude of 1st pulse of train was
plotted against pulse number (HP 40 mV, C; HP 80
mV, D).
|
|
| |
DISCUSSION |
IK in cat ventricular myocytes consists of both
IKr and IKs.
Because of the importance of IK in modulating
cardiac action potential repolarization, and its possible role as a
target for agents with antiarrhythmic as well as arrhythmogenic
potential, it is important to study its characteristics. There are two
main findings in the present work. First, we have shown that in adult and neonatal cat ventricle, IK consists of two
different current components, IKr and
IKs, similar to those previously recorded in other
mammalian species (20, 14). Second, we found that IKr and IKs and activation time
courses are similar in adult and neonatal ventricular myocytes.
However, current deactivation is faster in neonatal myocytes. The
presence of two kinetically distinct current components in
IK of adult and neonatal ventricular myocytes was
demonstrated on the basis of independent kinetics and pharmacological evidence.
The envelope of tails test for total IK was not
satisfied, suggesting that more than one current component was present.
However, the test was satisfied once the rapidly activating component, IKr, was blocked by the specific
IKr blocker dofetilide. IKr, defined as the dofetilide-sensitive component, activated by
depolarizing pulses in cat ventricular myocytes, shows a threshold
potential of 30 mV and a strong inward rectification. In canine
and guinea pig ventricular myocytes, IKs tail
current density is greater than that of IKr. In
contrast, in both adult and neonatal cat ventricular myocytes, we found
that IKr tail current density is greater than that
of IKs, as suggested by previous work (5). The
presence of both IKr and IKs
suggests that both participate in cat ventricular repolarization.
However, the greater density and faster activation kinetics of
IKr suggest that it participates more than
IKs. In addition to differences in activation
kinetics and sensitivity to different potassium-channel blockers,
IKr and IKs differ in
sensitivity to 
-adrenergic stimulation. IKr is not affected by -adrenergic stimulation, whereas
IKs is enhanced (11). Therefore, under some
physiological or pathological conditions in which the sympathetic tone
is elevated, the participation of IKs on
repolarization may be more important (1).
Activation kinetics of IKr and IKs is
similar in these age groups.
The activation kinetics of IKr and
IKs is similar in neonatal and adult ventricular
myocytes. In a previous work (12), it was reported that activation of
IK in cat ventricular myocytes is slowly activated
in a monoexponential way, with a kinetics similar to the
IKs reported in the present work. We do not have a
clear explanation for these differences.
Deactivation kinetics of IKr is faster in neonatal
myocytes.
The deactivation kinetics is faster in neonatal myocytes. Two
exponential components adequately describe the IKr
and IKs deactivation time course. The main
difference between these age groups is in deactivation of
IKr. The s of deactivation of
IKr at membrane potentials positive to 50 mV
is about three times slower in adult than neonatal cells.
Species-dependent differences in the IKr deactivation time course have been found. In guinea pig ventricular myocytes, f and s at 40 mV are 117 and 632 ms, respectively (3), and in AT-1 cells, f and
s are 80 and 400 ms, respectively (23). However, in dog
ventricular myocytes f 
470 ms and s 5 s (14), and in adult cat ventricular myocytes, f
279 ms and s 3,334 ms (present study).
The difference in deactivation kinetics of IKr
between adult and neonatal ventricular myocytes predicts differences in
the contribution of IKr to repolarization at high
stimulation rates in addition to differences in rate-dependent response
to IKr- and IKs-specific
blockers. Increases in the contribution of IKs to
repolarizing currents at higher stimulation frequencies have been
attributed to the accumulation of IKs resulting
from its slow deactivation kinetics. In guinea pig ventricular
myocytes, the reverse use-dependent effects of class III antiarrhythmic drugs, which specifically block IKr, have been
attributed to IKs accumulation resulting from its
slow deactivation kinetics (10). In contrast to the results obtained in
guinea pig myocytes, we have found that IKr
deactivation is slower than IKs deactivation in cat
ventricular myocytes. From our results in adult cat ventricular myocytes, IK accumulation of activation may be
mainly attributed to the slower deactivation kinetics of
IKr, principally at potentials positive to the
normal resting potentials of these cells (80 mV). On the other
hand, in neonatal myocytes, deactivation of both current components is
similarly fast, predicting little accumulation at high stimulation
rates. Our prediction has been experimentally confirmed. In adult
myocytes accumulation of activation is observed during a train of
pulses applied at a frequency of 2 Hz, using a holding potential of
40 mV. However, when the train is applied from a holding
potential of 80 mV, closer to the normal resting potential, no
accumulation of activation is observed. These results suggest that
under physiological conditions, in normally polarized cells,
IK accumulation of activation may not be relevant.
In neonatal myocytes accumulation of activation was not observed at
holding potentials of 40 or 80 mV. The main limitation of
this protocol is that we have used square waveform voltage-clamp pulses
instead of action potential waveform voltage-clamp pulses, and a single (2 Hz) stimulation frequency. Furthermore, the increase in current during the train of pulses applied from a holding potential of 40 mV could be caused by cumulative activation from using a
holding potential above the activation threshold. The I-V
relationships given in Fig. 1 do not necessarily contradict such an
interpretation because isochronal measurements were performed.
The genes that encode the major subunits of IKs and
IKr have been cloned. The minK subunit coassembles
with an 
-subunit, KvLQT1, to form IKs channels
(18, 2), and the ether-à-go-go-related gene (ERG) subunit
coassembles with minK-related peptide 1 (MiRP1) to form
IKr (1, 19, 21). ERG has
electrophysiological properties similar but not identical to
IKr. The time constants of deactivation of ERG are
much slower than those of native IKr, as expressed in guinea pig and mouse myocytes (20, 14). A novel ERG isoform with a
shorter and divergent NH2-terminal cytoplasmic domain that is abundantly expressed in the heart has been identified in humans and
mice (13, 15). Expression of this isoform induces a current that has
rapid deactivation kinetics and coassembles with the longer isoform.
Therefore, it is possible that native IKr results from a mixture of both isoforms forming homo- or heteromultimers. A
novel potassium gene has been cloned. This gene encodes a MiRP1 that
assembles with HERG to form a channel with characteristics closer to
those of IKr than channels formed solely by HERG
subunits (1). MiRP1-HERG complexes deactivate threefold more rapidly than channels formed only by HERG subunits (1).
A possible explanation for our findings could be that there exist
species- and age-dependent differences in the ratio of expression of
the ERG isoforms, both the isoform with the longer
NH2-terminal cytoplasmic domain and the isoform with the
shorter domain. It is probable that changes in the ratio of expression
of both isoforms may alter the kinetics of the endogenous
IKr (13, 15). Another possible explanation for our
findings may be species- and age-dependent IKr
channel composition. Some channels could be composed of the MiRP1-ERG
complex and some of them of the ERG subunit alone. Channels composed of
the MiRP1-ERG complex may be expressed at higher levels in cardiac
myocytes that show a more rapid IKr deactivation.
However, to confirm or reject these and/or other hypotheses, more
direct evidence should be obtained using molecular biology approaches.
| |
ACKNOWLEDGEMENTS |
The authors thank Dr. Paul Bennet for critical reading of the
manuscript, Gusti Gould de Pineda for editorial assistance, M. S. Gabriela Ramírez for important cooperation, and Juan Fernando Fernández for technical assistance.
| |
FOOTNOTES |
This work was supported by a grant from Consejo Nacional de Ciencia y
Technologia (Mexico; no. 3729P-M) (J. A. Sánchez-Chapula) and from FOMES 96-97 (Subsecretaria de
Educacion Superior e Investigacion Cientifica-Secretaria de
Educacio
Publica, Mexico).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. A. Sánchez-Chapula, Universidad de Colima, Apdo. Postal 199, CP
28000, Colima, Col. México, México (E-mail:
sancheza{at}cgic.ucol.mx).
Received 25 May 1999; accepted in final form 26 August 1999.
| |
REFERENCES |
1.
Abbott, G. W.,
F. Sesti,
I. Splawsky,
M. E. Buck,
M. H. Lehmann,
K. W. Thimothy,
M. T. Keating,
and
S. A. Goldstein.
MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia.
Cell
97:
175-187,
1999[ISI][Medline].
2.
Barhanin, J.,
F. Lesage,
E. Guillemare,
M. Fink,
M. Lazdunski,
and
G. Romey.
K(v)LQT1 and IsK (minK) proteins associate to form the IKs cardiac potassium channel.
Nature
384:
78-80,
1996[Medline].
3.
Chinn, K.
Two delayed rectifiers in guinea pig ventricular myocytes distinguished by tail current kinetics.
J. Pharmacol. Exp. Ther.
264:
553-560,
1993[Abstract/Free Full Text].
4.
Elizalde, A.,
H. Barajas-Martinez,
R. Navarro-Polanco,
and
J. A. Sánchez-Chapula.
Frequency dependent effects of 4-aminopyridine and almokalant on action-potential duration of adult and neonatal rabbit ventricular muscle.
J. Cardiovasc. Pharmacol.
33:
352-359,
1999[ISI][Medline].
5.
Follmer, C. H.,
and
T. J. Colatsky.
Block of delayed rectifier potassium current, IK, by flecainide and E-4031 in cat ventricular myocytes.
Circulation
82:
289-293,
1990[Abstract/Free Full Text].
6.
Furukawa, T.,
S. Kimura,
N. Furukawa,
A. L. Bassett,
and
R. J. Myerburg.
Potassium rectifier currents differ in myocytes of endocardial and epicardial origin.
Circ. Res.
70:
91-103,
1992[Abstract/Free Full Text].
7.
Gintant, G. A.
Two components of delayed rectifier current in canine atrium and ventricle. Does IKs play a role in the reverse rate dependence of class III agents?
Circ. Res.
78:
26-37,
1996[Abstract/Free Full Text].
8.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[ISI][Medline].
9.
Ho, W. K,
I. Kim,
C. O. Lee,
J. B. Youm,
S. H. Lee,
and
Y. E. Earm.
Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations.
Biophys. J.
76:
1959-1971,
1999[Abstract/Free Full Text].
10.
Jurkiewicz, N. K.,
and
M. C. Sanguinetti.
Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent. Specific block of rapidly activating delayed rectifier K+ current by dofetilide.
Circ. Res.
72:
75-83,
1993[Abstract/Free Full Text].
11.
Kass, R. S.
Delayed potassium channels: cellular, molecular, and regulatory properties.
In: Cardiac Electrophysiology: From Cell to Bedside, edited by D. Zipes,
and J. Jalife. Philadelphia, PA: Saunders, 1995, p. 54-82.
12.
Kleiman, R. B.,
and
S. R. Houser.
Outward currents in normal and hypertrophied feline ventricular myocytes.
Am. J. Physiol. Heart Circ. Physiol.
256:
H1450-H1461,
1989[Abstract/Free Full Text].
13.
Lees-Miller, J. P.,
C. Kondo,
L. Wang,
and
H. J. Duff.
Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts.
Circ. Res.
81:
719-726,
1997[Abstract/Free Full Text].
14.
Liu, D. W.,
and
C. Antzelevitch.
Characteristics of the delayed rectifier current (IKr and IKs) in canine ventricular epicardial, midmyocardial and endocardial myocytes. A weaker IKs contributes to the longer action potential of the M cell.
Circ. Res.
76:
351-365,
1995[Abstract/Free Full Text].
15.
London, B.,
M. C. Trudeau,
K. P. Newton,
A. K. Beyer,
N. G. Copeland,
D. J. Gilbert,
N. A. Jenkins,
C. A. Satler,
and
G. A. Robertson.
Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current.
Circ. Res.
81:
870-878,
1997[Abstract/Free Full Text].
16.
Navarro-Polanco, R.,
and
J. A. Sanchez-Chapula.
4-Aminopyridine activates potassium currents by activation of a muscarinic receptor in feline atrial myocytes.
J. Physiol. (Lond.)
498:
663-678,
1997[ISI][Medline].
17.
Salata, J. J.,
N. K. Jurkiewicz,
B. Jow,
K. Folander,
P. J. Guinosso, Jr.,
B. Raynor,
R. Swanson,
and
B. Fermini.
IK of rabbit ventricle is composed of two currents: evidence for IKs.
Am. J. Physiol. Heart Circ. Physiol.
271:
H2477-H2489,
1996[Abstract/Free Full Text].
18.
Sanguinetti, M. C.,
M. E. Curran,
A. Zou,
J. Shen,
P. S. Spector,
D. L. Atkinson,
and
M. T. Keating.
Coassembly of K(v)LQT1 and minK (IsK) proteins to form cardiac IKs potassium channel.
Nature
384:
80-83,
1996[Medline].
19.
Sanguinetti, M. C.,
C. Jiang,
M. E. Curran,
and
M. T. Keating.
A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel.
Cell
81:
299-307,
1995[ISI][Medline].
20.
Sanguinetti, M. C.,
and
N. K. Jurkiewicz.
Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents.
J. Gen. Physiol.
96:
195-215,
1990[Abstract/Free Full Text].
21.
Trudeau, M. C.,
J. W. Warmke,
B. Ganetzky,
and
G. A. Robertson.
HERG, a human inward rectifier in the voltage-gated potassium channel family.
Science
269:
92-95,
1995[Abstract/Free Full Text].
22.
Wang, Z.,
B. Fermini,
and
S. Nattel.
Rapid and slow components of delayed rectifier current in human atrial myocytes.
Cardiovasc. Res.
28:
1540-1546,
1993.
23.
Yang, T.,
M. S. Wathen,
A. Felipe,
M. M. Tamkun,
D. J. Snyders,
and
D. M. Roden.
K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells).
Circ. Res.
75:
870-878,
1994[Abstract/Free Full Text].
Am J Physiol Heart Circ Physiol 278(2):H484-H492
0363-6135/00 $5.00
Copyright © 2000 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
