96
Subcellular Mechanism for Ca2+-Dependent
Enhancement of Delayed Rectifier K' Current
in Isolated Membrane Patches of Guinea
Pig Ventricular Myocytes
Jun-ichi Nitta, Tetsushi Furukawa, Fumiaki Marumo, Tohru Sawanobori, Masayasu Hiraoka
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Intracellular Ca2+ augments delayed rectifier K' current (IK)
in cardiac myocytes, which may play a major modulatory role
in repolarization of action potentials. We investigated subcellular mechanisms for Ca2`-induced enhancement of IK in
large-pipette inside-out membrane patches excised from isolated guinea pig ventricular myocytes. When [Ca 2+]` was raised
from 10`8 to 10`6 mol/L, the amplitude of IK measured at +80
mVwas increased from 12.0±2.2 to 19.5 ±3.3 pA (P<.01). The
enhancement of IK by Ca2+ was dose dependent, with an EC50
of 3.8x 10-8 mol/L. A calmodulin antagonist, W7 (50 ,umol/L),
calmidazolium (100 ,umol/L), or HT-74 (20 gtmol/L), added to
the intracellular solution abolished enhancement of IK by
Ca`2, whereas the inactive form of the W7 analogue, W5, had
no effect on IK. In the presence of a protein kinase inhibitor
with a relatively high specificity for protein kinase C (H7), for
protein kinase A (H8 or peptide-type inhibitor PKI), or for
calmodulin kinase II (KN-62) or a nonspecific inhibitor of
serine/threonine protein kinases (staurosporine), increases in
[Ca24], still enhanced lK. Ca 2+-induced enhancement of IK was
also observed when Mg 2+ and ATP were omitted from the
intracellular solution to delete exogenous phosphate donors
and when adenylylimidodiphosphate was added to preclude
trapped cytoplasmic substrates. Thus, cardiac lK was enhanced
by increases in [Ca 21]i at a physiological range via a calmodulin-dependent pathway, which did not involve a phosphorylation process. (Circ Res. 1994;74:96-104.)
Key Words * delayed rectifier K' current * calmodulin .
he calcium ions function as a ubiquitous intracellular signal mediator to influence ionic channel
activity, various enzymatic processes, and the
regulation of muscle contraction. In cardiac cells, cytosolic [Ca2+] ([Ca 21]i) rises after depolarization of membrane potential via Ca24 influx across the plasma membrane and subsequent release from the intracellular
stores.' Delayed rectifier K' current (IK), one of the
major outward currents conducting during the plateau
of action potentials in cardiac myocytes, is shown to be
the target of regulation by Ca2+.2-4 Enhancement of IK
by increased [Ca24]i may limit depolarization duration
and thus an amount of Ca2+ entry. This may act as a
negative-feedback mechanism protecting cardiac myocytes from excessive entry of Ca24. The rise in [Ca24]
also occurs under various pathological conditions including ischemia/reperfusion injury.5 In these pathological situations, the regulation of IK by Ca2+ may also act
as a self-protection mechanism, since the shortening in
action potential duration limits the amount of Ca2+
entry, diminishes contractile activity, and thus prevents
further loss of high-energy substrates.
Despite its possible pathophysiological importance,
the subcellular mechanism underlying regulation of IK
by Ca2' remains unclarified. The major difficulty comes
from the lack of methods for obtaining stable singlechannel recordings of IK in excised patches. The regulation of IK by Ca2+ has been studied exclusively in the
whole-cell clamp mode using dialyzed cells.2-4 However,
in the dialyzed whole-cell clamp configuration, [Ca24]i
cannot be controlled precisely, and it is nearly impossible to test numbers of different intracellular solutions
because of the limited efficacy of intracellular dialysis.
Recently, the major component of IK was reported to be
due to the activity of high-density extremely-low-conductance K4 channels, and this current could be recorded in excised membrane patches.6-8 By this method,
IK can be recorded under the conditions in which
various intracellular solutions can be changed rapidly
and easily. In the present study, we used the excised
membrane patch to explore the mechanism of subcellular regulation of IK by Ca24.
T
Ca2+
Materials and Methods
Preparation
Received December 24, 1992; accepted September 28, 1993.
From the Second Department of Internal Medicine (J.N.,
F.M.), Faculty of Medicine, and the Department of Cardiovascular Disease (T.F., T.S., M.H.), Medical Research Institute, Tokyo
Medical and Dental University, Yushima, Bunkyo-ku, Tokyo,
Japan.
Correspondence to Masayasu Hiraoka, MD, PhD, Department
of Cardiovascular Disease, Medical Research Institute, Tokyo
Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo 113, Japan.
Enzymatic dissociation of single ventricular myocytes from
guinea pig hearts was done using essentially the same method
as previously reported from our laboratory.9
Solutions
The internal (bath) solution contained (mmol/L) potassium
aspartate, 140; MgCl2, 1; CaCl2, 1; EGTA, 11; ATP, 5; and
HEPES, 5; the pH was adjusted to 7.3 with KOH. Concentrations of free Ca24 (pCa 9 to 5) and free Mg 24 (0.8 mmol/L) in
the internal (bath) solution were estimated on the basis of
Nitta et al IK Enhancement by Ca2
apparent stability constants for Ca2`-EGTA, K2-ATP, and
A
MgCl2 according to the calculation proposed by Tsien and
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Rink.'0 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), N-(6-aminohexyl)-5 chloro-1-naphthalenesulfonamide hydrochloride (W7), and
N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride
(W5) were purchased from Seikagaku Co, Tokyo, Japan; a
peptide-type inhibitor of protein kinase A (PKI) was purchased from Sigma Chemical Co, St Louis, Mo. They were
added to the bathing solution on each experimental day at final
concentrations as described in the text from stock solutions in
distilled water. 1-[N,O-Bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62, Seikagaku), staurosporine (Sigma), and 3-(2-benzothiazolyl)-4,5-dimethoxy-N[3-(4-phenylpiperidinyl)propyl]benzenesulfonamide (HT-74,
Takeda Chemical Industries LTD, Osaka, Japan) were added
to the bathing solution at final concentrations described in the
text from stock solutions in dimethyl sulfoxide (DMSO).
5'-Adenylylimidodiphosphate (AMP-PNP) was purchased
from Sigma. Glibenclamide, a gift of Hoechst Japan, Tokyo,
was dissolved as a 0.2 mmol/L stock solution in 2% DMSO and
diluted into the test solution to obtain the final concentration
indicated in the text. The final concentration of DMSO
contained in each test solution was less than 0.1%. The
external (pipette) solution contained (mmol/L) N-methylglucamine, 132; KCl, 4.8; MgCl2, 2; CaCl2, 1; glucose, 5; and
HEPES, 10; the pH was adjusted to 7.4 with HCI.
Recording Methods
Membrane currents were recorded in an inside-out patchclamp configuration described by Hamill et all' using glass
pipettes with a diameter of 3 to 4 gm. The resistance of the
pipette was 1 to 4 MU when filled with the internal solution.
The electrode was connected to the input stage of a patchclamp amplifier (Axopatch-1C, Axon Instruments, Inc, Foster
City, Calif) with a feedback resistance of 100 MU. Electrical
connection to the pipette and to the bath was made through a
Ag/AgCl half-cell electrode. The junction potential of each
electrode was adjusted to zero potential between the pipette
solution and the bath solution immediately before each cell
was approached and was checked again at the end of each
experiment. When the difference in junction potential between
two measurements was more than 2 mV, the value of the
membrane potential was corrected accordingly. The depolarizing test pulses were applied every 15 seconds. The current
signals were digitized on-line by a 12-bit resolution Labmaster
A/D converter (TecMar Scientific Solutions, Burlingame,
Calif) under the control of an IBM-PS/2 personal computer
and were stored on a hard disk. Data were analyzed using a
software program (pClamp, version 5.5.1, Axon Instruments).
Leak subtraction was made using the P/4 protocol on pClamp.
All experiments were performed at a temperature of 330 to
35`C.
Data Analysis
Data are expressed as mean+±SEM, and their statistical
significance was evaluated by Student's paired t test, where
appropriate. A value of P<.05 was considered significant.
Results
Characteristics Of IK in Excised Patches
To increase the likelihood of activating 'K channels,
long depolarizing voltage steps of 2.5-second duration
were applied to the positive potential. Fig 1A shows
superimposed currents that were elicited by a depolarizing test pulse to +80 mV, followed by repolarizing
pulses to various potentials between -100 and +50 mV
in 10-mV steps. Depolarizing pulses elicited a slowly
activating and noninactivating outward current. This
97
(PA)
50
40
30
20
10
a
0
-10
-t-
0
B
1
(sec)
3
2
c
5
0*
E
-
c
-50
-
0
a-
-a:
-100
10
External K
100
(mM)
FIG 1. Characteristics of macroscopic currents recorded in
excised inside-out membrane patches. A, Currents were recorded with 2.5-second depolarizing pulses from a holding
potential of -30 to +80 mV, followed by repolarizing pulses to
various potentials between -100 and +50 mV in 10-mV steps.
The currents are superimposed. B, Current-voltage relations of
tail currents were measured from seven patches using a voltagepulse protocol shown in A. C, Reversal potential of the delayed
rectifier K' current tail was measured at three different external
K' concentrations: 4.8, 24, and 48 mmol/L. The solid line was
obtained by fitting a linear regression to the data.
current was deactivated after repolarization with time,
displaying a slow decay of tail currents. Fig 1B illustrates
current-voltage relations of tail currents constructed
from seven patches. The reversal potential was
-73.0±2.7 mV. This value is close to the equilibrium
potential for K' in the condition in which [K']. was 4.8
mmol/L and [K+]i was 140 mmol/L, estimated by the
Nernst equation. The measurements of reversal potential were repeated at three different [K], values, 4.8,
24, and 48 mmol/L. In Fig 1C, the average reversal
potential is plotted against [K'],. The data were fitted
by a linear regression, resulting in a slope of approximately 50 mV per 10-fold change in [K].o These data
suggest K' as a main charge carrier for this current. We
next examined the time course of activation of this
current. In this and following analyses, we used data
obtained from slowly activating outward currents during
repolarizing pulses rather than from tail currents because the amplitude of IK tails was usually too small to
be analyzed in detail. After the leak subtraction was
made using the P/4 protocol, the activation of IK was
fitted by a single exponential function, and an example
at a membrane potential of +80 mV is shown in Fig 2A.
The activation kinetics of IK in the excised patch could
be more complicated, as have been reported for the IK
in the whole-cell clamp configuration,'12-4 which was
also suggested by our own data in the slight deviation of
IK activation from a monoexponential fit at very early
Circulation Research Vol 74, No 1 January 1994
98
1
A
.
(pA)
A
7=332 1 msec
15
_
cotl
co
-
-nt
10 r
5 _Ba
0
5 pA
5mM
-
3
3
t
1
2
2
4
O
4
5
0
1
2
3
4
5 (sec)
(sec)
B
300 msec
B
10
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pCa6
pCa6
pCa8
C
---
40 L
40~~~~~~
8
---
c
SMw*
35
p 30
1t*
0.1
0
0.5
1.0
1.5
Time (sec)
FIG 2. Time course of activation of macroscopic delayed rectifier K+ current (IK). IK(X) indicates steady-state level of IK; IK(t),
amplitude of IK at time t. A, An example of the time course of IK
activation at a membrane potential of +80 mV and its fitting by a
single exponential function as a form of Equation 1 in the text are
shown. The time constant (Xr) for this case was 332.1 milliseconds. B, A logarithm of [IK(ct)-IK(t)I is plotted against the time
after the start of the depolarizing pulse (plot of the time course of
IK shown
in
A).
times (see Fig 2). Yet, we used a monoexponential
equation simply to characterize the major component of
the time course of 1K activation. Thus, the activation of
IK may be expressed as
(1)
IK(t)=IK()-A - exp(-t/i-)
where A represents the extrapolated amplitude at the
start of test pulse (t=O), X is the time constant, IK(t) is
the amplitude of IK at t, and IK(x) is the steady-state
25 _
0
20
40
Time (min)
60
80
FIG 3. A, Macroscopic delayed rectifier K+ current (IK) was
recorded in the absence and presence of 5 mmol/L Ba2+ in the
intracellular solution at pCa 6. In this and following figures,
membrane currents were recorded with a depolarizing pulse
from a holding potential of -30 to +80 mV for 2.5 seconds, and
current tracings are shown after leak subtraction was made. B,
Effects of changes in [Ca2+]j on macroscopic IK are shown.
Currents tracings recorded in the intracellular solution at pa 8
and pCa 6 are superimposed. C, Time course of the amplitude
Of IK after the membrane patch was excised is shown. Free Ca2+
concentration in the intracellular solution was changed from pCa
8 to pCa 6 or vice versa every 20 minutes. In this and: following
figures, closed circles represent IK amplitude elicited by depolarizing pulses from a holding potential of -30 to +80 mV
applied every 15 seconds, and the timing of changes in intracellular pCa and application of drugs are indicated by open bars
on the top of each figure. Please note that IK amplitude was not
signfficantly changed at a different sampling point when the pCa
was the same, suggesting a very small degree of rundown of
macroscopic IK
level of IK. To further confirm that the time course of IK
activation could be fitted by a single exponential function, we plotted a logarithm of [LK(oo) -IK(t)] against the
time after the start of the depolarizing pulse (Fig 2B).
The data clearly show that the time course of IK could be
expressed as a single exponential function. The mean
time constant (r) obtained from 13 patches was 341±29
milliseconds, which was in good agreement with the
data by Walsh et al.6 This current was blocked by
internally applied 5 mmol/L Ba`+ (Fig 3A) or 10
mmolIL tetraethylammonium (data not shown). When
these findings are taken together, it is reasonable to
assume that this current represents IK, probably its slow
component (IK,).12,13
Effects of Intracellular Ca2+ on IK
Fig 3B shows the effects of changing [Ca2]i on IK
elicited by a 2.5-second depolarizing pulse to +80 mV
from a holding potential of -30 mV. When [Ca'+]i was
increased from 10`8 to 10`6 mol/L, the amplitude of
slowly activating IK as well as the steady-state current
level at a holding potential of -30 mV was increased. At
a holding potential of -30 mV, the inward rectifier K+
current shows almost complete inward rectification, and
IK does not reach the threshold for its activation. Thus,
the change in steady-state current level may be due to
the change in other unspecified current(s), or the surface-charge screening effect by Ca'+ may also be involved. To minimize the influence of changes in steadystate current level, the amplitude of IK was measured
from the current level at the beginning of a 2.5 -second
depolarizing pulse to a steady-state current level. As
described in the whole-cell clamp mode,15 in guinea
pig ventricular myocytes developed slowly during a
'K
Nitta et al IK Enhancement by Ca2'
180 r
99
30
25 ~
160
pCa6
*
:
o
: pCa 8
|
20~
%
140
2C
0.
K
l
15
V/
o
10
/?
120
5
Q
100
0'
0
o
'
-
10
9
8
7
6
5
4
pCa
FIG 4. Dose-response relations between the increase in delayed rectifier K' current (IK) and [Ca2+]i. The amplitude of IK was
measured as a pseudo-steady-state level of IK [IK(X) in Equation
1 in the text] at fixed membrane potential of +80 mV, expressed
as a percentage of the value at pCa 9, and plotted against pCa
in the intracellular solution. A solid line was obtained by fitting of
the modified Hill equation (Equation 2 in the text) to the data.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
depolarizing pulse and did not reach a steady-state level
at the end of a 2.5-second depolarizing pulse. Thus, a
monotonically extrapolated level at 2.5 seconds was
taken as a steady-state level of IK [IK(K) in Equation 1].
The increase in [Ca'+], from 10`8 to 10`6 mol/L enhanced the amplitude of IK from 12.0+2.2 to 19.5 +3.3
pA (n=7, P<.01).
It is widely observed that IK in the whole-cell clamp
configuration shows rundown after starting intracellular
dialysis, which might interfere with the data analysis for
relatively long experimental protocols. To evaluate the
magnitude of rundown for macroscopic IK in the excised-patch configuration, we monitored the amplitude
of IK every 15 seconds for 1 or 2 hours at pCa 8 and pCa
6. Fig 3C shows a typical example of such experiments.
Unlike IK in the whole-cell clamp configuration, macroscopic IK recorded in the excised-patch configuration
showed only a minimum degree of rundown. We performed a total of six experiments for this protocol, and
the degree of rundown was less than 10% in each
experiment.
Fig 4 displays dose-response relations for Ca2-induced enhancement of IK, in which the amplitude of IK
at a fixed membrane potential of + 80 mV was measured
at each [Ca2+]i and was normalized to the value at pCa
9. Data were fitted by a modified Hill equation in the
following form:
I= 100 * {I+Ima.* [Ca2+]h/(kh+[Ca 2+]h)}
(2)
where I represents the amplitude of IK expressed as a
percentile of the amplitude of IK at pCa 9, Ima, is the
maximal IK, h is the Hill coefficient, and k is the pCa
level causing half-maximal enhancement. The intracellular Ca2+ increased the amplitude of IK in a dosedependent manner, and the increase in IK amplitude
reached a plateau at pCa 6. The Hill coefficient was 1.4,
and the [Ca2]i that causes half-maximal enhancement
was 3.8x10`8 mol/L.
Effects of intracellular Ca2 on steady-state activation
Of IK was studied. Membrane potential was depolarized
from a holding potential of -30 mV to various test
potentials between -100 and + 100 mV in 10-mV steps
for 2.5 seconds. Fig 5 illustrates steady-state activation
Of IK as a function of the test potential. The threshold
-100
-50
0
50
100
Vm (mV)
FIG 5. Plot showing the effects of [Ca2+], on the steady-state
activation of delayed rectifier K' current (IK). The amplitude of IK
was obtained as a pseudo-steady-state level of IK [IK(') in
Equation 1 in the text]. Vm indicates membrane voltage.
for the activation of this current was approximately -30
to -20 mV at pCa 6, and this current increased in
amplitude with increasing membrane potential. At neither pCa 6 nor pCa 8 did the activation of IK reach the
maximum plateau after a 2.5-second depolarization to
+ 100 mV.
Intracellular Mechanisms Involved in
Enhancement of IK by Increased [Ca2+]i
It has been reported that lK was modulated by protein
kinases A and C.2-4,7,15-17 To examine whether or not
phosphorylation by protein kinase A or C was involved
in Ca2+-induced enhancement of IK, the effects of H7, a
general protein kinase inhibitor having a high specificity
especially for protein kinases A and C, or H8, a relatively specific protein kinase A inhibitor, were studied.
Fig 6 shows a representative experiment studying the
effect of change in [Ca2+i] on IK in the presence of 20
gmol/L H7. This concentration of H7 (20 ,umol/L) is
more than six times higher than the Ki (3 ,umol/L) for
protein kinase A and more than three times higher than
the Ki (6 ,umol/L) for protein kinase C.18 In the presence
of H7, the elevation of [Ca2+]i from 10`8 to 10`6 mol/L
also caused an increase in IK. In the presence of H7 (20
,umol/L), the averaged amplitude of IK at pCa 8 was
13.2+ 1.0 pA and that at pCa 6 was 22.2+ 1.5 pA (n=5,
P<.01). This magnitude of enhancement of IK
(68+11%) was similar to that in the absence of H7
(63+28%) (P=NS).
Similar results were obtained in the presence of 10
,gmol/L H8, a concentration more than eight times
higher than the K, (1.2 gmol/L) for protein kinase A.18
In the presence of H8, the average amplitude of IK at
pCa 8 was 11.9+1.6 pA and that at pCa 6 was 19.4+1.5
pA (P<.01). The magnitude of enhancement of IK
(63+13%) was also similar to that in the absence of H8
(63+28%) (P=NS). We also tested the effects of other
types of protein kinase inhibitors. In the presence of 5
,mol/L PKI, a peptide-type inhibitor of protein kinase
A, and 5 ,umol/L staurosporine, a nonspecific inhibitor
of serine/threonine protein kinases, the magnitude of
enhancement of IK was not significantly different from
that occurring in the absence of any protein kinase
inhibitors (68±24% [n=3] and 61+19% [n=3], respectively). PKI is known to inhibit protein kinase A competitively, with a Ki of 2.3 nmol/L.19 Staurosporine is
100
Circulation Research Vol 74, No 1 January 1994
zCc6
oCc3
b
(PA)
A
30
H 7 2OuM
20
0~~~~~0
10 _
20
o
0
5
15
10
Time
20
25
3
2
0
15
5
4
(sec)
'2
(min)
FIG 6. Plot showing the effects of H7, a protein kinase inhibitor
with relatively high sensitivity for protein kinases A and C, on the
enhancement of delayed rectifier K' current (IK) by intracellular
Ca2l. [Ca2i1) was increased from 10-8 to 10-6 mol/L in the
presence of 20 ,umol/L H7. The enhancement of IK by raising
[Ca2+1J, was observed even in the presence of 20 ,mol/L H7.
B
pCa8
FI
pCa6
pCa6
pCc8
W7 50MM
W5 50,4M
40
b
C
¢-}
_R
30
4sy
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
known to inhibit protein kinases A20 and C,21 Ca2+/
calmodulin-dependent protein kinase II (CaM kinase
II),22 and other serine/threonine protein kinases with
IC50 values of less than 10 nmol/L. These data suggest
that phosphorylation by neither protein kinase A nor C
may be involved in the enhancement of IK by intracellular Ca2`.
We next tested a possible involvement of calmodulin
in the Ca2+-induced enhancement of IK. Fig 7 shows a
representative experiment studying the elevation of
[Ca 2+], on IK in the presence of calmodulin antagonist
W7 (50 ,umol/L) or W5 (50 ,mol/L), which has less
specificity for inhibiting the calmodulin effect. This
concentration of W7 is approximately twice as high as
the IC5o value (approximately 30 ,mol/L) for calmodulin, and the concentration of W5 is approximately a
quarter of the IC50 value (approximately 210 gmol/
L).2324 The enhancement of IK by the elevation of
[Ca2+]i was almost completely abolished in the presence
of W7, whereas it was still clearly observed in the
presence of W5. Fig 8 shows summarized data obtained
from six experiments. In the presence of W7 (50 gmol/
L), the amplitude of IK was not enhanced by the
elevation of [Ca2+]i from pCa 8 (10.1+2.0 pA) to pCa 6
(11.2+2.1 pA) (P=NS), whereas it was increased from
17.1+2.8 pA at pCa 8 to 25.0±4.7 pA at pCa 6 (P<.01)
in the presence of W5 (50 ,mol/L). Furthermore, we
also tested the effects of different classes of calmodulin
antagonists, calmidazolium (100 nmol/L, n=5)25 and
HT-74 (20 ,tmol/L, n=4).26 In the presence of calmidazolium (100 nmol/L) or HT-74 (20 ,mol/L), the Ca2+induced enhancement of IK was abolished. In the presence of calmidazolium (100 nmol/L), the amplitude of
IK was 10.9±2.9 pA at pCa 8 and 11.3±2.6 pA at pCa 6
(P=NS); the corresponding values in the presence of
HT-74 (20 ,mol/L) were 13.0±1.9 and 13.4+±2.3 pA,
respectively (P=NS).
We took a different approach to test the hypothesis
that the Ca2+-induced enhancement of IK was mediated
by a Ca2+/calmodulin-dependent pathway; we perfused
the intracellular aspects of membrane patches for 15
minutes with the solution containing no Ca2' and a high
concentration of the Ca2+-chelating agent EGTA (11
mmol/L). In this way, endogenous calmodulin attached
*
a
%
20
5
10
5
10
15
20
25
20
25
0
10
1
15
Time
30
35
(min)
FIG 7. Effects of calmodulin antagonists W7 and W5 on the
enhancement of delayed rectifier K' current (IK) by increases in
[Ca2+]J. A, Current recordings of macroscopic IK are shown in
the presence of 50 ,mmol/L W7 at pCa 6 (denoted by a), in the
absence of calmodulin antagonists at pCa 6 (denoted by b), and
in the presence of 50 ,mol/L W5 at pCa 6 (denoted by c). B,
Time course of changes in IK amplitude is shown when [Ca2+]i
was increased from 1 -8 to 10-6 mol/L in the preserce of W7 (50
,umol/L) or W5 (50 ,tmol/L). Ca2+-induced enhancement of IK
was suppressed when 50 ,umol/L W7 was present in the intracellular solution, whereas the IK enhancement was still observed
in the presence of 50 ,mol/L W5, an inactive form of the W7
analogue. An arrow associated with a, b, or c indicates the timing
of membrane current recording shown in A.
to the intracellular aspect of a patch membrane could
be detached and washed away.27 Fig 9 shows the effect
of intracellular Ca21 in this condition. The amplitude of
IK in the solution at pCa 6 after exposure to Ca2+-free
solution was less than that in the solution at pCa 8,
although slightly higher than that in the Ca2+-free
solution. Similar findings were observed in all six
patches tested. We also examined whether exogenously
applied calmodulin had any effects on IK; however,
enhancement of IK by exogenously applied calmodulin
(1 to 3 ,mol/L, purchased from Sigma) was not appreciable at pCa values from 8 to 6 (n=6, data not shown).
Ca2'/calmodulin complexes are known to mediate
diverse biologic reactions through a pathway involving
phosphorylation by CaM kinase II. Therefore, we next
tested the effect of a selective inhibitor of CaM kinase
II, KN-62 (3 gmol/L).28 As shown in Fig 10, in the
presence of KN-62 (3 ,umol/L), the elevation of [Ca2+]
clearly enhanced the amplitude of IK. From four
patches, in the presence of KN-62 (3 ,umol/L) the mean
amplitude of IK was increased from 9.0+1.6 pA at pCa
8 to 20.8+5.2 pA at pCa 6 (P<.01). The finding that
Nitta et al IK Enhancement by Ca2'
A
101
B
25
3gM
KN62
20 1k
20
15
5V
CL
1-
CL 15
C)
*
1-
10
z
pCoe
pCaS
+ W7
+ W7
(SOAM)
pCa6
pCaOS
pC6
+ W5
+ WS
5h
pCa6
(5Qsjm)
(50UM)
(50oM)
0
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FIG 8. Bar graphs showing the effects of calmodulin antagonists W7 (A) and W5 (B) on the Ca2'-induced enhancement of
delayed rectifier K+ current (IK). The amplitude of IK was measured from six experiments in the presence or absence of 50
cmol/L W7 or 50 ,umol/L W5 at pCa 8 or pCa 6. Results are
expressed as mean+SEM. Please note that, when pCa was
changed from 8 to 6, the amplitude of IK was not increased in the
presence of W7, whereas it was increased in the presence of W5
with a magnitude similar to that in the absence of calmodulin
antagonists. *P<.05.
staurosporine, a nonspecific protein kinase inhibitor
with a specificity for CaM kinase II, could not attenuate
Ca2`4induced IK enhancement further argued against
the role of CaM kinase II in IK enhancement by Ca2`.
To further support the idea that a Ca2+/calmodulin
complex can enhance IK via a phosphorylation-independent pathway, we examined the effect of intracellular
Ca2` on IK in the solution containing no ATP or Mg 2+.
Even in this condition in which no exogenously applied
substrate for protein kinases was present, Ca2' could
enhance the amplitude of IK, and calmodulin antagonist
W7 (50 ,umol/L) attenuated the Ca2'-induced enhancement of IK (Fig 11). Similar findings were obtained in all
five patches tested. Even in the absence of ATP or Mg 2+
in the intracellular solution, the possible involvement of
endogenous ATP trapped to the intracellular side of the
excised membrane patch may be possible. To exclude
this possibility, we examined the effect of changing in
[Ca2+]i in the presence of AMP-PNP (3 mmol/L). Ca2'
could enhance the amplitude of IK even in the presence
of AMP-PNP (3 mmol/L) (Fig 12). Thus, the enhance-
0
15
20
25
ment of IK by Ca2' was mediated by calmodulin, but a
phosphorylation process might not be involved.
Discussion
Previous studies using the whole-cell clamp technique
have shown that IK in guinea pig ventricular myocytes is
enhanced by an increase in [Ca2]1i,2-4 but the intracellular mechanism by which Ca2+ regulated IK has not
been fully understood. In the present study, we confirmed by using excised cell-free patches that IK in
guinea pig ventricular myocytes was indeed enhanced by
increased intracellular Ca21 in the range of physiological levels. The novel finding in the present study is that
Ca2+-induced enhancement of IK occurs via a calmodulin-dependent pathway, but the action seems not to be
mediated via a phosphorylation reaction.
Macroscopic lK Recorded in Excised
Membrane Patches
Recent reports6-8 suggested that the current analysis
using excised membrane patches was an invaluable tool
in the study of regulatory mechanisms of IK independent
ATK
2S
_25-W
-
10
Time (min)
effect
of KN-62, a Ca2+/calmodulinFIG 10. Plot showing the
dependent protein kinase 11 antagonist, on the Ca2+-induced
enhancement of delayed rectifier K' current (IK). The increase in
[Ca2+], from pCa 8 to pCa 6 enhanced macroscopic IK even in
the presence of 3 gmol/L KN-62.
pCa6
- Cc- free
5
-ee
W7
5C.U
@C0
*0
i ..
CL
1-
2
I.I
IL
0
1,
,0
0
+I
00
u
0.
0
00
0
0*
0
0
5
10
Time
.b
5
^
0,00
*0
4P
0
00
sr
a
0
2;
25
(min)
FIG 9. Plot showing the effects of increases in [Ca 2+], on
delayed rectifier K+ current (IK) amplitude after the intracellular
aspect of the membrane was perfused with the solution containing no Ca2+ and a high concentration of Ca2+-chelating agent
EGTA (11 mmol/L) for approximately 20 minutes. The amplitude
Of IK was decreased in the Ca2+-free solution, and IK was not
enhanced by increasing [Ca2+1] to pCa 6.
Time (minr)
FIG 11. Plot showing the effects of deleting exogenous phosphate donors on Ca2l-induced enhancement of delayed rectifier
K+ current (IK). [Ca2+1J was increased from pCa 8 to pCa 6 in the
intracellular solution containing no Mg 2+ or ATP. The intracellular solution also contained 1 ,umol/L glibenclamide to inhibit the
activity of ATP-sensitive K+ current. Please note that the elevation of pCa from 8 to 6 augmented IK even in the absence of
Mg 2+ or ATP, and the application of calmodulin antagonist W7
(50 gcmol/L) attenuated the Ca2+-induced IK enhancement.
Circulation Research Vol 74, No 1 January 1994
102
pcos
3pCo
pCoB
AMP-PNP 3mM
50
a
1c
*0
40 _
0
m
1-
0
30 _
20
t
0
5
tO
15
20
25
Time (min)
FIG 12. Plot showing the effect of the presence of adenylylimidodiphosphate (AMP-PNP) in the intracellular solution on Ca24induced delayed rectifier K' current (IK) enhancement. Even in
the presence of 3 mmol/L AMP-PNP, the change in [Ca42+], from
pCa 8 to pCa 6 still enhanced IK amplitude.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
of cytoplasmic components. Before discussing the modulation of IK by intracellular Ca24 in this method, we have
to consider whether the slowly activating outward current
observed in excised membrane patches indeed represents IK recorded in the whole-cell clamp experiments,
and if so, which component(s) of IK this current represents. This current had a threshold for activation at
approximately -30 to -20 mV, displaying slowly activating outward currents during depolarizing pulses without
inactivation. The current increased in amplitude with
membrane depolarization but did not reach full activation after a 2.5-second depolarization even up to +100
mV. On repolarization, the current decayed with time,
thus exhibiting slowly deactivating tail currents. The
reversal potential of tail currents at [K+]o of 4.8 mmol/L
was -73 mV on average, and the value at different levels
of [K+]o revealed a slope of 50 mV per a 10-fold change
in [K+10, indicating that the main charge carrier of this
current was K+. These results are in good agreement with
the properties of IK recorded from guinea pig ventricular
myocytes with the whole-cell clamp configuration from
various laboratories.2-4,12,13,15-17,29,30 It has been suggested
that IK in guinea pig ventricular myocytes is the composite of at least two components, rapidly activating IK (IKr)
and slowly activating IK (IK).1213 Recently, it was reported that another component, IKP, which activated very
rapidly and was conductive at plateau potential, might
exist in guinea pig ventricular myocytes.14 IK, may not be
the main constituent of the slowly activating outward
current in excised membrane patches, because this slowly
activating outward current did not show significant inward rectification and the time course of current activation was much faster for than for the slowly activating
'Kr
outward current. This current is also unlike IKP because
its activation kinetics were much slower than those of IKp
(less than 10 milliseconds). Furthermore, it was reported
that IKp did not require intracellular Ca24 for its activation. Since the time course and voltage dependence of its
activation are similar to those of IKS in guinea pig
ventricular myocytes, it is most likely that the slowly
activating outward current recorded in excised membrane patches mainly represents IKS in whole-cell clamp
experiments, as suggested by Walsh et al,6 although
contamination by 'Kr, IKP, or other unspecified components could not be completely excluded. It was also
reported that modulations of the slowly activating outward current in excised membrane patches by protein
kinases A and C and GTP-binding protein were similar
to those in IKs in guinea pig ventricular myocytes.7,8
Characteristics of the slowly activating outward current in excised membrane patches, however, are not
totally identical to those of IK. It is widely known that IK,
in the whole-cell clamp configuration shows rundown
after starting intracellular dialysis, whereas the slowly
activating outward current in excised membrane
patches did not show significant rundown. Because of
similar characteristics between these two currents discussed above, it may be more likely that this difference
in the magnitude of rundown is due to different experimental conditions rather than to the fact that these two
currents represent totally different components of IK.
Duchatelle-Gourdon et a131 reported that the rundown
Of IK in the whole-cell clamp configuration depended on
[Mg 2+]i. The basal free [Mg 2+]i was estimated to be 0.8
mmol/L, and the amplitude of IK showed rundown when
the concentration of Mg 2+ in the pipette solution was
greater than 0.8 mmol/L, and it showed runup when
Mg 2+ in the pipette solution was less than 0.8 mmol/L.
In our experiment, we used a free concentration of
Mg 2+ of 0.8 mmol/L in the intracellular solution. This
may be one possible explanation for the lack of significant rundown in the slowly activating outward current in
our experiment. However, we cannot exclude other
possible explanations, such as the possibility that some
cytosolic components related to rundown were washed
away in the excised membrane patch; thus, further
experiments are needed to determine the precise reason
for the lack of rundown-in this current.
Effects of Intracellular Ca2' on IK
The elevation of [Ca2+]i augmented the amplitude of
IK in the excised membrane patches. These findings
were also consistent with the data in the whole-cell
clamp experiments.4 Kass32 reported that the delayed
rectifier outward current (Ix) recorded using a conventional two-microelectrode voltage-clamp arrangement
from the cardiac Purkinje fiber of calf or dog was not
activated by intracellular Ca2`. His conclusion was
drawn from the finding that, in the presence of a Ca 2+
channel blocker, nisoldipine, Ca22+ channel current and
contractile activity were abolished, whereas the amplitude of outward current tails that follow the depolarizing pulses was not suppressed. In his study, in the
presence of nisoldipine, no slowly activating outward
currents were observed during depolarizing pulses,
leaving outward currents without clear time dependence, which was apparently different in current kinetics from the slowly activating outward current in the
excised patch. Thus, in the cardiac Purkinje fiber of calf
or dog, the main component(s) of outward current
might differ from that in guinea pig ventricular myocytes. Another possible explanation for the lack of Ca42+
sensitivity in Ix was that [Ca2+]i below the level for
inducing muscle contraction could affect lx. It was
reported that, in chemically skinned rat ventricular
muscles, the threshold for skinned fiber contraction was
at a pCa of approximately 6.5.33 At this concentration,
intracellular Ca 2+ enhanced IK to almost a maximum
degree. Thus, it might be possible that in his experiment
Nitta et al IK Enhancement by Ca2+
[Ca2"]l did not decrease to the level at which the
amplitude of IK would be modulated.
Subcellular Mechanism for Ca2`-Induced
IK Enhancement
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The fact that Ca2'-induced enhancement of IK OCcurred in excised patches may imply that intracellular
Ca2+ acts directly on channel protein or via some
components bound to the intracellular side of cell
membrane. Many biologic reactions dependent on Ca2'
occur through a pathway involving protein kinase C or
the Ca2'-binding protein calmodulin. The finding that
the presence of calmodulin antagonists, W7, calmidazolium, or HT-74, abolished Ca2'-induced enhancement of IK raises a possibility that a calmodulin-dependent pathway is important for the modulation of IK. It
was reported in nerve tissue, however, that W7 per se
inhibited a K' current and a Ca2' current independent
of a calmodulin pathway.34 Inability of W5 to suppress
Ca2'-induced augmentation of 'K may support a calmodulin-dependent pathway rather than a nonspecific
action of W7, because W5 lacking chlorine in its molecule has less specificity for inhibiting the effect of
calmodulin. We also found that the Ca2'-induced enhancement of IK did not occur after the intracellular
side of the membrane patches was perfused for 15
minutes with solution containing no Ca2' and a high
concentration of EGTA. The application of a high
concentration of Ca2'-chelating agent EGTA for a
relatively long duration has been used to detach the
membrane-bound form of endogenous calmodulin.26
Thus, this finding appears to provide additional evidence that Ca24-induced 'K was mediated by endogenously present calmodulin. We also tested whether
exogenously applied calmodulin could mimic the Ca24induced enhancement of IK but found no augmentation
of Ca24 action on lK by exogenously applied calmodulin
up to 3 ,gmol/L (data not shown). Therefore, we speculate that calmodulin can mediate the Ca2'-induced lK
enhancement only if calmodulin is present adjacent to
the IK channel, and exogenously applied calmodulin
could not approach the channel in the biologic membrane. Alternatively, it is also possible that calmodulin
has different actions in different tissues and that the
calmodulin we used (harvested from bovine brain) does
not work in the action that takes place in cardiac cells.
It should also be noted that our data did not argue
against the modulation of 'K by protein kinases A and
C.2-4,7,14,15,30 We actually found that application of H7 or
H8 diminished the amplitude of 'K at a given concentration of Ca24 (data not shown).
It is intriguing to study how calmodulin enhances the
amplitude of IK, because numerous biologic processes
are known to be mediated by a Ca24 /calmodulin complex. The most attractive candidate, perhaps, is CaM
kinase II, because this is shown to modulate several
sarcolemmal ion channels; recently, it was reported that
the slowly activating IK-like outward current (Isk) expressed by microinjecting neonatal mouse cardiac
poly(A)+ or complementary RNA encoding this channel was enhanced by intracellular Ca2+ via CaM kinase
11.35 It was also reported that the opening of a delayed
K4 current in Euhadra neurons was mediated by CaM
kinase 11.36 Therefore, we studied this aspect by examining the effects of a specific inhibitor of CaM kinase II
103
(KN-62), a nonspecific protein kinase inhibitor with a
specificity for CaM kinase II (staurosporine), or, in the
absence of ATP, a substrate for protein kinases on
Ca2 -induced IK activation. The elevation of [Ca2"]i still
increased the amplitude of IK despite of these treatments, indicating that a phosphorylation via CaM kinase II was not involved. These results suggest that a
Ca 2+ /calmodulin complex directly modulates IK, probably via an allosteric interaction with the channel protein
itself or channel-associated protein. Direct interactions
between channels and Ca 2+ /calmodulin complexes have
been reported; a Ca24 /calmodulin complex appears to
directly activate the Ca24-dependent sodium channel in
excised membrane patches of Paramecium37 and to
directly inactivate the Ca24-release channel of sarcoplasmic reticulum.38
Physiological Importance of Ca24-Induced
Enhancement of IK
Could the Ca24-induced enhancement of IK work
under physiological conditions and what might be the
physiological role of this regulation? Our data showed
that intracellular Ca 2+ augmented IK at a concentration
of 10`8 mol/L or higher and reached a steady state at
approximately 5 x 10-7 mol/L. The [Ca 24]i that causes
half-maximal enhancement was 3.8x 10-8 mol/L. Considering the fact that intracellular Ca24 activity in
diastole in myocardium is in the range between 10-8 and
7x 10-8 mol/L and it rises to approximately 5x10`6
mol/L when cells are activated,3940 the Ca24-induced
enhancement of IK appears to take place in the range
where [Ca 24]i varies during a course of cell contraction
and relaxation. Thus, under physiological conditions,
Ca24 that enters through voltage-dependent Ca24 channels enhances IK, helping to terminate action potential
repolarization and to eliminate further Ca24 influx.
Thus, the regulation of lK by Ca24 may work protectively
for the heart under various pathological conditions. For
example, the augmentation of IK by increased [Ca24]i
shortens the action potential duration, thus reducing
the amount of Ca2+ entry, thereby maintaining energy
consumption at a minimum by suppressing muscle contraction and eliminating Ca24 overload in cells.
Acknowledgments
We wish to thank Noriko Fujita for excellent secretarial
assistance and Yoko Sugimoto for superb technical assistance.
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Subcellular mechanism for Ca(2+)-dependent enhancement of delayed rectifier K+
current in isolated membrane patches of guinea pig ventricular myocytes.
J Nitta, T Furukawa, F Marumo, T Sawanobori and M Hiraoka
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Circ Res. 1994;74:96-104
doi: 10.1161/01.RES.74.1.96
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