Characterization of a transient outward
K+ current with inward
rectification in canine ventricular myocytes
Gui-Rong
Li,
Haiying
Sun, and
Stanley
Nattel
Department of Medicine, Montreal Heart Institute and University
of Montreal, Montreal, Quebec, Canada H1T 1C8
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ABSTRACT |
The threshold
potential for the classical depolarization-activated transient outward
K+ current and
Cl
current is positive to
30 mV. With the whole cell patch technique, a transient outward
current was elicited in the presence of 5 mM 4-aminopyridine (4-AP) and
5 µM ryanodine at voltages positive to the
K+ equilibrium potential in canine
ventricular myocytes. The current was abolished by 200 µM
Ba2+ or omission of external
K+
(K+o) and showed biexponential
inactivation. The current-voltage relation for the peak of the
transient outward component showed moderate inward rectification. The
transient outward current demonstrated voltage-dependent inactivation
(half-inactivation voltage:
43.5 ± 3.2 mV) and rapid,
monoexponential recovery from inactivation (time constant: 13.2 ± 2.5 ms). The reversal potential responded to the changes in
K+o concentration. Action potential clamp
revealed two phases of
Ba2+-sensitive current during the
action potential, including a large early transient component after the
upstroke and a later outward component during phase 3 repolarization.
The present study demonstrates that depolarization may elicit a
Ba2+- and
K+o-sensitive, 4-AP-insensitive, transient outward current with inward rectification in canine ventricular myocytes. The properties of this
K+ current suggest that it may
carry a significant early outward current upon depolarization that may
play a role in determining membrane excitability and action potential
morphology.
cardiac electrophysiology; transient outward potassium current; whole cell patch clamp; excitability; barium- and/or
potassium-sensitive transient outward peak current
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INTRODUCTION |
DEPOLARIZATION-ACTIVATED outward currents have been
distinguished based on differential time- and voltage-dependent
properties and pharmacological sensitivity (1), including
delayed-rectifier K+ currents (27)
and transient outward current
(Ito; see Ref. 8). Rapid and slow delayed rectifier K+ currents
(IKr and IKs) have been
demonstrated in guinea pig (29), canine (36), and human (19) cardiac
myocytes. Ultrarapid delayed rectifier K+ current
(IKur) has also been described in rat (4), canine (37), and
human (34) atrial cells.
Ito has been
recognized in cardiac Purkinje fibers since the 1960s (7, 8). Initially
believed to be a Cl
current
(8), it was subsequently shown in sheep Purkinje fibers to be due
predominantly to an increase in K+
conductance (16). The presence of
Ito was
demonstrated with whole cell voltage-clamp techniques in cardiac cells
from a wide range of species, including rat (15), rabbit (10, 11), dog (32, 33), elephant seal (24), ferret (5), and human (2, 11). Kenyon and
Gibbons (16) reported that 4-aminopyridine (4-AP) decreased
Ito in sheep
cardiac Purkinje fibers, and a 4-AP-resistant component of
Ito was reduced
by Cl
replacement (16).
4-AP has been subsequently used as a selective inhibitor of transient
outward K+ current, and
4-AP-sensitive and 4-AP-resistant components of Ito have been
reported in sheep Purkinje fibers (6), rabbit ventricular (38) and
atrial (39) cells, and canine ventricular (32) and atrial (36)
myocytes. The 4-AP-sensitive and 4-AP-resistant components are often
termed Ito1 and
Ito2,
respectively, after Tseng and Hoffman (32).
Ito2 has been
recognized (38, 39) to be a
Ca2+-activated transient outward
Cl
current
(ICl.Ca).
In canine cardiac myocytes, both delayed-rectifier
K+ currents and
Ito
(Ito1 and
Ito2) have been
demonstrated to play an important role in myocardial action potential
repolarization. We have recently described a 4-AP-resistant
Ito with inward
rectification in canine ventricular myocytes in a preliminary report
(20). The present study was designed to
1) characterize the voltage and time
dependence of the current, 2)
determine current-voltage
(I-V)
relation, and 3) assess whether this
Ito may be
significant during the action potential.
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MATERIALS AND METHODS |
Myocyte isolation. Left ventricular
tissues from isolated hearts were obtained via a left thoracotomy after
dogs were anesthetized with intravenous pentobarbital sodium (30 mg/kg). All hearts were initially placed in oxygenated Tyrode solution,
the left anterior descending coronary artery was cannulated, and
ventricular cells were enzymatically isolated with a procedure
described previously for human ventricular cell isolation by Li et al.
(19). Myocytes were isolated from the digested tissue, placed in a
high-K+ storage solution (19), and
kept in the medium for at least 1 h before use. A small aliquot of the
solution containing the isolated cells was placed in an open perfusion
chamber (1 ml) mounted on the stage of an inverted microscope.
Experiments were conducted at 36°C. Only quiescent rod-shaped cells
showing clear cross-striations were used.
Solutions. Tyrode solution contained
(in mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 2.0 CaCl2, 0.33 NaH2PO4,
10.0 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), with pH adjusted to 7.4 with NaOH. When
Na+ current
(INa) was
eliminated, NaCl in Tyrode solution was replaced by equimolar choline
chloride. The pipette solution contained (in mM) 20 KCl, 110 potassium
aspartate, 1.0 MgCl2, 10 HEPES, 5.0 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 0.1 GTP, 5 Mg2ATP, and 3 Na2-phosphocreatine, with pH
adjusted to 7.2 with KOH. Ryanodine (5 µM) was used to inhibit
ICl.Ca (or Ito2), 4-AP (5 mM) was used to block
Ito1, and
CdCl2 (300 µM) was used to block
the Ca2+ current
(ICa).
Electrophysiology and data analysis.
Membrane currents and/or action potentials were recorded with
the tight-seal whole cell patch-clamp techniques and with the use of an
Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Data were
acquired with command pulses that were generated by a 12-bit
digital-to-analog converter controlled by pClamp software (Axon
Instruments). Recordings were low-pass filtered at 2 kHz and were
stored on the hard disk of an IBM-compatible
computer.

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Fig. 1.
Depolarization-elicited currents. A:
representative tracings of depolarization-activated transient outward
currents in the voltage range of 70 to 0 mV (voltage protocol
shown in inset of
B) in a canine ventricular cell.
B:
Ba2+ at 200 µM fully inhibited
the transient outward current activated by depolarization and decreased
the holding current. C:
Ba2+-sensitive current obtained by
subtracting currents before and after application of Ba2+,
showing voltage-dependent inward rectification.
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Fig. 2.
Effect of omission of external K+
(K+o) on depolarization-elicited currents.
A: representative tracings of
depolarization-activated transient outward currents in the voltage
range of 70 to 0 mV (voltage protocol shown in
inset of
B) in a canine ventricular cell.
B: omission of
K+o abolished the transient outward current
activated by depolarization and decreased the holding current.
C:
K+o-sensitive current obtained by subtracting
currents before and after omission of K+o,
showing voltage-dependent inward rectification.
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Borosilicate glass (1.0 mm OD) pipettes were prepared with the use of a
Brown-Flaming puller (model P87) to produce a tip resistance of
2-3 M
when filled with the solution described above. Tip
potentials were compensated before the pipette touched the cell. A giga
seal was obtained, and the cell membrane was ruptured by gentle suction
to establish the whole cell configuration. The series resistance
(Rs) was
electrically compensated to minimize the duration of the capacitive
transient. After compensation, Rs was 2.1 ± 0.4 M
. Myocytes were current clamped to record action potentials
and/or were voltage clamped to record membrane currents with
the use of pClamp6 software. The liquid junctional potential was
determined by immersing the pipette into the bath filled with identical
pipette solution, and the voltage reading on the amplifier was set to
zero by adjusting the offset. The pipette was subsequently placed into
the external solution, and the voltage change gives the liquid junction
potential. The average junction potential (12 pipettes) was 10.5 ± 0.3 mV, which was not corrected in the experiments.
Nonlinear curve-fitting techniques (Clampfit in pClamp or Sigmaplot;
Jandel Scientific, San Rafael, CA) based on the Marquardt procedure
were used to fit equations to experimental data. Paired and unpaired
Student's t-tests were used as
appropriate to evaluate the statistical significance of differences
between two group means. Analysis of variance was used for multiple
groups. Values of P < 0.05 were
considered to indicate significance. Group data are expressed as
means ± SE.
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RESULTS |
Ito induced by depolarization in canine ventricular
myocytes. Outward currents were elicited by 300-ms depolarizing
voltage steps between
70 and 0 mV from a holding potential of
80 mV with 10-mV increments every 10 s.
INa was
suppressed by replacement of external
Na+ with equimolar choline.
Ito1 was blocked
by 5 mM 4-AP,
Ito2 was inhibited with 5 µM ryanodine, and
ICa was blocked
by 300 µM Cd2+. Figure
1A displays current
responses in a representative cell to voltage steps to values between
70 and 0 mV (protocol shown in Fig.
1B,
inset). Figure
1B shows that the
depolarization-activated current was fully blocked by the addition of
200 µM Ba2+. The
Ba2+-sensitive current is shown in
Fig. 1C. Both original and
Ba2+-sensitive currents show a
transient outward behavior and voltage-dependent inward rectification.
The Ba2+-sensitive
Ito was carried
by neither Ito1
(since it was 4-AP insensitive) nor by
Ito2 (seen in the
presence of ryanodine and absence of
ICa).
Figure 2 shows the effect of the omission of external
K+ (K+o)
on depolarization-induced
Ito. Figure
2A displays currents elicited in a
representative cell by the voltage-clamp protocol shown in Fig.
2B,
inset. Figure
2B shows that the omission of
K+o abolished the depolarization-elicited
outward current. K+o omission-sensitive
(K+o-sensitive) current is displayed in Fig.
2C. As was the case for the
Ba2+-sensitive current, the
K+o-sensitive current also shows
voltage-dependent inward rectification. In subsequent experiments, the
current properties were analyzed by the use of currents sensitive to
Ba2+ and/or
K+o omission.
Voltage dependence of
Ito sensitive to
Ba2+ or
K +o omission.
To study voltage dependence of the depolarization-activated
Ito, myocytes
were depolarized to more positive potentials using the same
voltage-clamp protocol as in Fig. 1B.
Because the activation of the
Ito was too fast
to separate from membrane capacitance at more positive potentials,
Ba2+-sensitive and/or
K+osensitive current was used to
determine the
I-V
relation for membrane currents. Figure 3 shows Ba2+-sensitive currents from
a representative cell at depolarization voltages between
70 and
+40 mV from a holding potential of
80 mV. Figure
3A displays current recordings at
potentials between
70 and
40 mV. The
Ito was seen at
70 mV in this cell, just positive to the resting potential of
71 mV, as indicated by the step from the holding current. The
Ito was augmented
as the depolarization potential was made more positive. The current
showed voltage-dependent inward rectification in the voltage range
30 to 0 mV (Fig. 3B) and
reached a steady-state level at potentials between +10 and +40 mV (Fig.
3C).

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Fig. 3.
Voltage dependence of depolarization-activated
Ba2+-sensitive current.
Ba2+-sensitive currents were
obtained by subtracting currents before and after the addition of 200 µM Ba2+ as in Fig.
1C. Current tracings are shown at
potentials between 70 and 40 mV
(A), between 30 and 0 mV
(B), and between +10 and +40 mV
(C).
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Figure 4A
illustrates schematically the method of current measurement in a
Ba2+-sensitive current recording
and indicates the measurement for the total current
(Itotal), the
time-dependent current peak
(Ipeak), the
steady-state current
(Iss), and the
holding current
(Ih). For
analyses of average currents, the measured currents were normalized to
cell capacitance to evaluate current density and thereby control for
variations in cell size.

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Fig. 4.
Current-voltage
(I-V)
relations of depolarization-elicited currents.
A: schematic showing current
measurement in a Ba2+-sensitive
current tracing recorded with 300-ms step to 30 mV from a
holding potential of 80 mV.
Itotal, total
current; Ipeak,
time-dependent peak current;
Iss, steady-state
current; Ih,
holding current. B:
I-V
relations of Ba2+-sensitive
component (n = 20) for
Itotal ( ),
Ipeak ( ),
Iss ( ), and
Ih ( ),
extrapolated to the
I-V
curves. C:
I-V
relations of K+o-sensitive component
(n = 15).
Ba2+- or
K+o-sensitive currents were obtained as in
Figs. 1C or
2C. Data are means ± SE. TP, test
potentials.
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Figure 4B shows mean density-voltage
relations for Ba2+-sensitive
currents (n = 20 cells).
Itotal and
Ipeak density
increased to reach a maximum at about
10 and +10 mV,
respectively, and then showed inward rectification. The
I-V
curve for Iss
displayed strong inward rectification positive to
50 mV, similar
to that of inwardly rectifying K+ current
(IK1), suggesting that
Iss may be
IK1 or a
component of IK1.
Extrapolating the
I-V
curves to the voltage axis, the reversal potentials
(VRev) for
Itotal,
Ipeak, and
Iss were between
70 and
74 mV. Correcting for the average 10.5-mV liquid
junction potential, the
VRev would be
80.5 to
84.5 mV, very close to the calculated
K+ equilibrium potential
(EK,
84
mV). This suggests that the depolarization-induced Ba2+-sensitive current is carried
by K+. Figure
4C displays mean density-voltage
relations of K+o-sensitive currents obtained
as in Fig. 2C. The
I-V
relations obtained with K+o-sensitive
currents (n = 15 cells) were similar to those obtained with
Ba2+-sensitive currents,
suggesting that the concept that
Ba2+-sensitive or
K+o-sensitive currents are the same.
K+ o
dependence and
Ba2+
concentration-dependent inhibition of the depolarization-induced
current.
The K+ selectivity of
Ba2+-sensitive
Ito was further
assessed by evaluating the response of the
VRev to various
K+o concentrations
([K+]o). The
VRev of the
Ito was
determined by extrapolating the time-dependent peak current to
Ih. A 10-fold
change in K+o led to a 52 ± 2 mV/decade
(n = 6) shift in the
VRev value (Fig.
5A), in
agreement with the prediction of the Nernst equation (60 mV/decade). To
determine whether
Ipeak and
Iss have similar
sensitivity to Ba2+, the 50%
inhibitory concentration (IC50)
of Ba2+ on
Ipeak and
Iss was assessed
in six cells using 300-ms steps to
20 mV from a holding
potential of
80 mV. IC50
was 5.0 ± 0.2 µM for
Ipeak and 4.9 ± 0.2 µM for
Iss
(P = not significant; Fig. 5B), indicating identical and very
high Ba2+ sensitivity.

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Fig. 5.
A: relation between
Ipeak reversal
potential (RP) and extracellular
K+ concentration
([K+]o;
means ± SE, n = 6 at each
[K+]o).
Best-fit regression line (shown) had a slope of 51.5 mV/decade change
in
[K+]o.
B: concentration-dependent effect of
Ba2+ on depolarization-elicited
currents. Concentration-response curves shown are fitted by the
equation E = Emax/[1 + (K/C)n], where E is the effect at a
concentration C, Emax is the
maximum effect, K is the concentration for half-maximum action, and
n is the Hill coefficient. No
significant difference in the 50% inhibitory concentration of
Ba2+ on
Ipeak and
Iss was observed
with the use of 300-ms steps to 20 mV from a holding potential
of 80 mV. Data are means ± SE
(n = 6).
[Ba2+],
Ba2+ concentration.
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Figure 6 shows the voltage dependence of
Ipeak
(A) and
Iss
(B) in the presence of different
[K+]o
(2.7, 5.4, and 10.8 mM). The
I-V
curves for Ipeak
and Iss shifted to the right with increased
EK. The
VRev of
Iss shifted with
[K+]o
like that of
Ipeak; however,
the maximum amplitude of
Iss increased as
[K+]o
increased. This behavior suggests increased conductance at higher
[K+]o,
behavior typical of
IK1.

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Fig. 6.
Effects of the change of
[K+]o
(2.7, 5.4, and 10.8 mM) on the voltage dependence of
K+o-sensitive currents.
A: voltage dependence of
Ipeak.
I-V
relation curves shifted to the right along with the change of the
K+ equilibrium potential
(EK).
B: voltage dependence of
Iss.
I-V
relation curves shifted to the right, and conductance of the current
increased along with the alteration of
EK. Data are
means ± SE; n = 7, 10, and 7, respectively, for 2.7, 5.4, and 10.8 mM
K+o.
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The results indicate that the addition of
Ba2+ and the omission of
K+o strongly suppress the
depolarization-elicited Ito. Clearly,
Ba2+- and/or
K+o-sensitive current is not classical 4-AP-sensitive
Ito1, because
1) the threshold voltage of
activation for
Ito1 is
30
to
20 mV in canine ventricular myocytes (21), and
2) experiments for the determination
of the current are conducted in the presence of 5 mM 4-AP, which fully
blocks Ito1. We
will refer to the Ba2+-
and/or K+o-sensitive transient
outward peak current
(Ipeak) with
inward rectification as
Ito.ir.
Kinetics of time-dependent
Ito.ir.
The time dependence of
Ito.ir was
assessed as illustrated in Fig.
7A, which
shows representative 200 µM
Ba2+-sensitive current obtained
during a 300-ms voltage step from
80 to
30 mV. The raw
data were best fitted by a biexponential equation with time constants
shown. Mean data for inactivation time constants in 12 cells are shown
in Fig. 7B. At all voltages, inactivation of the current was well fitted by a biexponential relation
and poorly fitted by a monoexponential function. The fast inactivation
time constant showed significant voltage dependence (P < 0.01), indicating that rapid
inactivation is faster when the test potential is positive to
10
mV. However, the slow inactivation time constant showed no significant
voltage dependence (P > 0.05).

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Fig. 7.
Time-dependent inactivation of
Ba2+- and/or
K+o-sensitive transient outward peak current
(Ito.ir).
A: inactivation of
Ba2+-sensitive
Ito.ir on 300-ms
depolarization from 80 to 30 mV was fitted by a
biexponential function (curve shown as solid line, points are raw
data). B: rapid and slow time
constants ( 1 and
2, respectively) of
Ito.ir
inactivation obtained with the approach illustrated in
A (n = 12). Ba2+-sensitive current was
obtained as in Fig. 1C. Data are means ± SE.
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Voltage dependence of
Ito.ir
inactivation was determined as illustrated in Fig.
8A.
Prepulses of 300-ms duration were applied to conditioning potentials
between
120 and 0 mV, and then currents were recorded during
300-ms test pulses to +10 mV before and after 200 µM
Ba2+. The inactivation variable of
Ba2+-sensitive
Ito was
determined as
Ito.ir at a given
prepulse potential divided by the maximum
Ito.ir in the
absence of a prepulse. Figure 8B shows
mean results obtained from analysis of voltage-dependent inactivation.
Inactivation reached a maximum at 0 mV and was incomplete. Mean data
are represented with filled circles, and the curve is the best-fit
Boltzmann distribution. The half-inactivation voltage (V0.5) averaged
43.5 ± 3.2 mV (n = 11), whereas the slope factor averaged 12.6 ± 0.7 mV.

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Fig. 8.
Voltage-dependent inactivation of
Ito.ir.
A: protocol and representative
Ba2+-sensitive current tracings
used to assess voltage-dependent inactivation of
Ito.ir.
B: mean voltage-dependent inactivation
relation for
Ito.ir.
Inactivation was assessed with the protocol shown in
A. Data were fit to Boltzmann relation
of
I/Imax = [1 + exp(V-V0.5)/K)] 1,
where I/Imax is the inactivation
variable of Ito.ir, V is
the membrane potential,
V0.5 is the
membrane potential for half-maximal inactivation, and
K is a slope factor. The
Ba2+-sensitive current was
obtained as in Fig. 1C. Data are means ± SE. CP, conditioning potentials.
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The time dependence of reactivation of
Ba2+-sensitive
Ito.ir from
inactivation was studied with the paired-pulse protocol illustrated in
Fig. 9A.
Identical 300-ms pulses (P1 and P2) from the holding potential
(
80 mV) to
20 mV were delivered every 10 s, with varying P1-P2 intervals. The current during P2
(I2) relative
to the current during P1
(I1) was
determined as a function of the P1-P2 recovery interval. The curve in
Fig. 9B shows a nonlinear exponential
curve fit to mean data from 10 cells. The reactivation curve of
Ito.ir was well
fitted in individual experiments by monoexponential functions with time
constants averaging 13.2 ± 2.5 ms.

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Fig. 9.
Time-dependent reactivation of
Ito.ir.
A: representative
Ba2+-sensitive current tracings
(obtained as in Fig. 1C) for
assessing reactivation of
Ito.ir from
inactivation obtained with 2 identical pulses shown in
inset (300 ms from the holding
potential 80 to 20 mV), P1 and P2, with varying P1-P2
interval. B: reactivation curve for
Ito.ir was best
fitted by a monoexponential function with inactivation time constant of
12.5 ms (n = 10). Data are means ± SE. I1, current
during P1; I2,
current during P2; t, change in
time of P1 and P2 interval.
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Effects of intracellular
Mg2+ and
spermine on Ito.ir.
Intracellular Mg2+
(Mg2+i) and
polyamines, such as spermine, have been reported to be related to
inward rectification property of
IK1
and/or cloned channels with inward rectification (25). We
therefore omitted
Mg2+i or
included 5 µM spermine in the pipette solution to observe whether
Ito.ir was
changed after cell dialysis for 10 min. The omission of
Mg2+i did not
induce a significant change in
Ito.ir (Fig.
10). Similarly, spermine inclusion did
not significantly change the current amplitude or
I-V
relation of
Ito.ir either. At
a voltage step to
40 mV from
80 mV,
Ito.ir was 3.9 ± 0.8 (n = 8), 3.8 ± 0.7 (n = 4), and 4.1 ± 1.1 (n = 4) pA/pF, respectively, under
control,
Mg2+i-free,
and spermine-inclusion conditions.

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Fig. 10.
Effect of omitting intracellular
Mg2+
(Mg2+i) on
Ito.ir.
A: current recordings in a
control cell. B: current recordings in
another cell dialyzed with a Mg2+-free pipette solution for
10 min. Ba2+-sensitive
Ito.ir was
obtained as in Fig. 1C.
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Comparison between 4-AP-sensitive
Ito1 and
K+osensitive
Ito.ir.
All of the above studies of Ba2+-
or K+o-sensitive
Ito.ir were
performed in the presence of 5 mM 4-AP, excluding the participation of
classical 4-AP-sensitive
Ito1. It was of interest to compare directly some of the properties of 4-AP-sensitive Ito1 with
Ito.ir sensitive
to K+o omission. The two components were
separated by the application of 5 mM 4-AP and subsequent omission of
K+o in the presence of 5 µM ryanodine.
Membrane currents were elicited by 300-ms voltage steps to between
70 and +40 mV from a holding potential of
80 mV, with
10-mV increments applied every 10 s. Figure
11, A-E,
displays control currents (A),
currents after 4-AP application (B),
4-AP-sensitive Ito1
(C), currents after
K+o omission
(D), and K+o omission-sensitive currents
(E) in a representative cell.
4-AP-sensitive
Ito1
(C) is clearly different in time
course and voltage dependence from the
K+o-sensitive component
(E). The
I-V
relations of the time-dependent peak current for 4-AP-sensitive and
K+o-sensitive components are shown in Fig.
11F. 4-AP-sensitive
Ito1 shows a
linear (ohmic) I-V
relation and an activation threshold potential at about
20 mV,
whereas the K+o-sensitive
Ito.ir shows an I-V
relation with inward rectification and a threshold voltage at about
70 mV.

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Fig. 11.
Difference between 4-aminopyridine (4-AP)-sensitive
Ito1 and
4-AP-insensitive, K+o-sensitive
Ito.ir.
A: membrane current in control.
B: recordings after the application of
5 mM 4-AP for 10 min. C:
4-AP-sensitive current obtained by subtracting currents before and
after the application of 4-AP. D:
recordings after the omission of K+o in the
same cell. E:
K+o-sensitive current obtained by subtracting
currents before and after the omission of
K+o. F:
I-V
relation curves of 4-AP-sensitive
Ito1 and
K+o-sensitive
Ito.ir in the
same 5 cells; data are means ± SE. Experiments were conducted with
external choline solution in the presence of 300 µM
Cd2+ and 5 µM ryanodine.
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Figure 12 displays
I-V
relations of 4-AP-sensitive
Ito1 and
K+o-sensitive
Ito.ir at holding potentials
of
80,
70, and
60 mV.
Ito1 was not
affected by the alteration of holding potentials (Fig.
12A), indicating that
Ito1 is not
inactivated over the holding potential range tested, consistent with
previous observation (21). However,
K+o-sensitive
Ito.ir was
significantly decreased by changing holding potentials from
80
to
70 and
60 mV (Fig.
12B;
n = 6, P < 0.01), suggesting that this
current, unlike
Ito1, may undergo
inactivation at holding potentials between
80 and
60 mV.
These findings are consistent with the voltage dependence of
Ito.ir
inactivation shown in Fig. 8 and point out the differences in basic
biophysical properties between 4-AP-sensitive
Ito1 and
Ba2+- or
K+o-sensitive
Ito.ir.

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Fig. 12.
Effects of the holding potential
(Vh) on
4-AP-sensitive
Ito1 and
K+o-sensitive
Ito.ir (obtained
as in Fig. 11) in the same cells. A:
I-V
relation curves of 4-AP-sensitive
Ito1 at holding
potentials of 80, 70, and 60 mV. No significant
difference in
Ito1 was observed
in the holding potentials tested. B:
I-V
relation curves of K+o-sensitive
Ito.ir at holding
potentials of 80, 70, and 60 mV.
Ito.ir
significantly decreased as the holding potential changed to 70
and 60 mV from 80 mV.
** P < 0.01 compared with the
previous holding potential in all test potentials. Data are means ± SE; n = 6.
|
|
Contribution of Ito.ir
to the action potential.
We evaluated the possible contribution of
Ito.ir to the
action potential with the use of the action potential clamp technique. The action potential was first recorded in current-clamp mode in normal
Tyrode solution and was then loaded as a voltage-clamp waveform to
record the membrane currents in choline solution in the presence of 5 mM 4-AP, 5 µM ryanodine, and 300 µM Cd2+. The membrane
current during the action potential was obtained by subtracting
membrane currents before and after the addition of 200 µM
Ba2+. Similarly, a ramp protocol
was used in the same cell.
Figure 13 shows representative recordings
from a canine ventricular cell. Figure
13A shows the action potential
waveform, and Fig. 13B displays
current tracings recorded with action potential clamp in the absence
(control) and presence of 200 µM
Ba2+. Figure
13C displays
Ba2+-sensitive current obtained by
subtracting the current in the presence of
Ba2+ from the control current. Two
components of Ba2+-sensitive
currents were revealed during the action potential, one transient
outward component evident immediately after depolarization and another
component during phase 3 repolarization corresponding to classical
IK1. Little
current was present during the plateau of the action potential. In the
same cell, Ba2+-sensitive current
was assessed by a ramp protocol (Fig.
13D). Membrane currents
activated by the ramp protocol are shown in Fig.
13E in the absence and presence of
Ba2+. Similarly, two components
of Ba2+-sensitive currents were also
elicited by the ramp protocol (Fig. 13F). Similar results were obtained
with both of the protocols in a total of six cells.

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|
Fig. 13.
Contribution of
Ito.ir to action
potential. A: action potential from a
canine ventricular myocyte was used to clamp the same cell.
B: membrane currents
activated by the action potential waveform before (control) and after
exposure to 200 µM Ba2+.
C:
Ba2+-sensitive current during the
action potential, obtained by subtracting membrane currents before and
after the addition of Ba2+.
D: ramp protocol used to clamp the
same cell. E: membrane currents were
recorded by the ramp protocol before (control) and after the addition
of 200 µM Ba2+.
F:
Ba2+-sensitive current during the
ramp protocol obtained by subtracting membrane currents before and
after the addition of Ba2+.
|
|
 |
DISCUSSION |
In the present study, we have demonstrated that
1) depolarization elicits a
4-AP-resistant
Ito at
physiological temperature in canine ventricular myocytes under
conditions that prevent intracellular Ca2+ transient,
2) the depolarization-induced
current inactivates quickly, 3) the
current is fully suppressed by the application of
Ba2+ (200 µM) or the removal of
K+o, and
4) the
VRev of the
Ba2+- and
K+o-sensitive current responds to changes in
[K+]o.
This novel transient outward component
(Ito.ir) shares
some features (sensitivity to Ba2+
addition and K+o removal, inward
rectification) with
IK1. Some
properties of
Ito.ir are close
to those of outward current carried by the recently cloned human
TWIK-1 channel (18).
Comparison with previously reported
Ito.
Two types of Ito
have been reported in mammalian cardiac myocytes (32, 38, 39). One is
4-AP-sensitive
(Ito1), and the another is a Ca2+-activated
Cl
current
(Ito2). The
threshold potential for
Ito1 activation
is
30 to
20 mV, and the
I-V
relation curve is linear (Fig. 10F; see Ref. 21).
Ito2 is activated
at
30 mV, corresponding to the activation of
ICa and
intracellular Ca2+ transient, and
the
I-V
relation curve is bell shaped (38, 39). Ito.ir is
apparent at
70 to
60 mV, just positive to the current reversal potential (e.g.,
EK), and the
I-V
relation curve is neither linear nor bell shaped.
Ito.ir is present
under conditions (5 mM 4-AP, 5 µM ryanodine, and 300 µM
Cd2+) that fully suppress
Ito1 and
Ito2 and has
biophysical properties that are clearly different from the two latter,
well-described currents.
Martin et al. (22) reported a 3,4-diaminopyridine-insensitive,
Ca2+-independent transient outward
K+ current in feline cardiac
ventricular myocytes (22). The current was not activated upon initial
whole cell patch formation, but activation depended on patch duration,
and it was therefore named patch duration-dependent
K+ current
[IK(PDD)].
IK(PDD) shares
some properties with
Ito.ir described
in the present paper, such as 4-AP insensitivity,
Ba2+ sensitivity, time-dependent
recovery, and voltage-dependent inactivation. However, the
Ito.ir that we
studied was developed upon membrane rupture, and its
I-V
relation (with inward rectification) is clearly different from that of
IK(PDD), which is
linear in the voltage range of
40 to +60 mV.
Comparison with previously reported outward currents
with inward rectification.
IKr has been
demonstrated to show inward rectification and to have a bell-shaped
I-V
relation, but the current is not transient (29). We were able to detect
a large Ito.ir in
the presence of 5 µM E-4031 (n = 5, data not shown), excluding the participation of
IKr.
It is known that
IK1 can exhibit
time and voltage dependence (17); however, time- and voltage-dependent
phenomena are well recognized only at membrane potentials negative to
the normal resting potential (12). The
I-V
relation of depolarization-induced instantaneous
IK1 was reported
to be linear under high K+o (14-40 mM);
however, the monoexponential inactivation time constant of
instantaneous IK1
is in the range of 1.1 (14) to 7.7 (17) ms. The inward rectification of
IK1 has been
thought to be related to the action of
Mg2+i (17)
and polyamines (25) on this channel and/or intrinsic, voltage-dependent gating or closing of the
IK1 channel (17). Therefore, the
IK1 channel has
been considered to act as a diode (23) active only on hyperpolarization
of the membrane (26). The single channel conductance is nearly ohmic in
the inward direction (17, 23, 27), and it is believed that very little
or no current passes through the channel in the outward direction under physiological conditions (23, 33). Shimoni et al. (30) reported that
IK1 was
inactivated during the upstroke and plateau phases of the action
potential and is consequently available for repolarization only during
phase 3.
The Ito.ir that
we studied shares some features (sensitivity to
Ba2+ addition and
K+o removal, inward rectification) with
IK1 but was not
affected by omitting
Mg2+i or
including spermine in the pipette solution. This may be related to the
possibility that submillimolar endogenous polyamines mask the action of
Mg2+i removal
or micromolar spermine inclusion. Although studies from Tourneur et al.
(31) and Ibarra et al. (13) suggested that IK1 may also play
an active role during action potential depolarization, the kinetics and
I-V
relation of depolarization-activated outward IK1 have not been
directly analyzed experimentally.
Ito.ir could be
carried by IK1, a
component thereof, or by a previously unidentified channel that shares
many feature with
IK1.
Ito.ir shows
features that are consistent with the idea of an intrinsic
voltage-dependent inactivation mechanism (25). The different response
of Ipeak (Ito.ir) and
Iss to changes in
[K+]o
(Fig. 6) suggests that they may not be carried by the same mechanism,
with the Iss
responding more like classical
IK1. We have
observed Ito.ir
in ventricular myocytes from guinea pig, rabbit, and human hearts
(unpublished data), indicating widespread expression in mammalian
hearts.
Potential significance of our
observation. Cell excitability has generally been
associated with the ability of inward currents to generate an action
potential upstroke. More recent studies suggest that
IK1 may also play
a role in the excitability of cardiac cells (13) by stabilizing the
resting potential (25). Because significant outward currents carried by
Ito.ir can be
elicited by depolarization over a time course comparable to
INa, this outward component may play a role in cell excitability, especially when INa is reduced,
as in myocardial ischemia. Recent work suggests that
voltage-dependent changes in maximal velocity with increased [K+]o
are poorly explained by changes in
INa (35). This
discrepancy may be due to a participation of
Ito.ir in
determining maximal velocity, which becomes particularly important when
INa is reduced.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Heart and Stroke Foundation of
Québec, and Fonds de la Recherche en Santé du Québec
(FRSQ).
 |
FOOTNOTES |
G.-R. Li is a research scholar of FRSQ.
Address for reprint requests: G.-R. Li, Montreal Heart Institute, 5000 Belanger St. East, Montreal, Quebec, Canada H1T 1C8.
Received 6 August 1997; accepted in final form 14 November 1997.
 |
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