Two distinct inactivation processes related to phosphorylation
in cardiac L-type Ca2+ channel currents
Sayaka
Mitarai1,2,
Muneshige
Kaibara1,
Katsusuke
Yano2, and
Kohtaro
Taniyama1
1 Department of Pharmacology and 2 The Third
Department of Internal Medicine, Nagasaki University, School of
Medicine, Nagasaki 8528523, Japan
 |
ABSTRACT |
We
investigated the inactivation process of macroscopic cardiac L-type
Ca2+ channel currents using the whole cell patch-clamp
technique with Na+ as the current carrier. The inactivation
process of the inward currents carried by Na+ through the
channel consisted of two components >0 mV. The time constant of the
faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased
with depolarization, but the time constant of the slower inactivating
component (489 ± 21 ms at 0 mV) was not significantly influenced
by the membrane potential. The inactivation process in the presence of
isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents
and attenuated the effects of isoproterenol. In the presence of cAMP
(500 µM), the inactivation process consisted of a single slow
component. We propose that the faster inactivating component represents
a kinetic of the dephosphorylated or partially phosphorylated channel,
and phosphorylation converts the kinetics into one with a different
voltage dependency.
channel phosphorylation; whole cell patch clamp
 |
INTRODUCTION |
CALCIUM CHANNEL CURRENTS
ACTIVATE with membrane depolarization and inactivate over time.
The inactivating property has an important role in regulating
intracellular Ca2+ concentration and action potential
duration of cardiac cells. Inactivation of the currents is modulated by
at least three factors: 1) membrane potential, 2)
Ca2+, and 3) phosphorylation of the channel
(19). Attempts have been made to clarify relations between
the observed inactivation process of the currents and these three factors.
Inactivation of cardiac L-type Ca2+ channel currents is
accelerated by Ca2+ passing through the channel
(15, 17, 20) and by
intracellular Ca2+ released from the sarocplasmic reticulum
(25), hence the inactivation process is complicated.
Although Ca2+ channel currents carried by cations other
than Ca2+ show a relatively slow decline in the absence of
Ca2+-mediated inactivation (9,
15, 17, 20), it has been
reported that decay of these currents carried by Ba2+,
Sr2+, or Na+ is not fitted by a
single-exponential function (3, 6,
10, 15). Kass and Sanguinetti
(15) reported that decay of the currents carried by
Ba2+ or Sr2+ was best fitted by functions with
a two-exponential process. They proposed that the observed data might
be explained by two populations of Ca2+ channels with
different inactivation kinetics.
Stimulation of the
-receptor-cAMP cascade by isoproterenol and
effects of isoproterenol on the cardiac L-type Ca2+ channel
have been extensively studied (14). In single-channel studies, isoproterenol increases open-state probability by
increasing duration of the available state (4,
21) and prolonging open time of the channel
(27). These effects of phosphorylation on channel kinetics
were also evident using the phosphatase inhibitor okadaic acid
(23). Although it has been demonstrated that isoproterenol slows the decay of the outward current through the Ca2+
channel at high-membrane potential in frog ventricular heart cells
(2), these changes in kinetics of single-channel currents have not been thoroughly explored in the case of inward whole cell currents.
It has been suggested that some populations of the Ca2+
channels in cardiac myocytes are phosphorylated without stimulation of
cAMP production (14, 23). Consequently, in
whole cell recordings in the absence of exogenous stimulation of the
-receptor-cAMP cascade, the inactivation process of macroscopic
currents may reflect two populations of Ca2+ channels,
i.e., phosphorylated and dephosphorylated. We investigated the
inactivation process of macroscopic Na+ currents through
the cardiac L-type Ca2+ channel and the effects of
isoproterenol on this inactivation process. We report here that two
distinct inactivation processes are present in the case of the
Na+ currents through the channel and that isoproterenol
converts these two inactivation processes into a slow one. We also
found that
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase (5), attenuated the effects of isoproterenol.
 |
MATERIALS AND METHODS |
Ca2+ containing solution for whole cell current
recording contained (in mM) 144 NaCl, 0.33 NaH2PO4, 5.4 CsCl, 0.5 MgCl2, 1.8 CaCl2, 5.5 glucose, and 5 HEPES-NaOH buffer (pH 7.4).
Ca2+-free solution was prepared by omitting
CaCl2 from the Ca2+ solution and adding 0.1 mM
EGTA. The Tyrode solution contained (in mM) 144 NaCl, 0.33 NaH2PO4, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 5.5 glucose, and 5 HEPES-NaOH buffer (pH 7.4). The
pipette solution contained (in mM) 110 cesium aspartate, 20 CsCl, 2 MgCl2, 3 MgATP, 10 EGTA, 5 HEPES, 0.5 NaGTP, and 5 NaCl,
the pH was adjusted to 7.1. In some experiments, 500 µM cAMP was
added to the pipette solution. H-89 was stored in 3 vol% ethanol
solution at a concentration of 10 mM at
30°C. Single
ventricular cells were isolated from guinea pig hearts, using a
modification of a reported method (13). In brief, guinea
pigs weighing under 250 g were anesthetized with pentobarbital
sodium, the dissected hearts were mounted on a Langendorff apparatus,
and the hearts were perfused at 37°C first with the Tyrode solution
and then with CaCl2-omitted Tyrode solution. Finally, the
hearts were perfused with CaCl2-omitted Tyrode solution
containing collagenase. Thereafter, single ventricular cells were
preserved in storage solution (12).
Membrane currents from single ventricular cells were recorded using the
whole cell patch-clamp method (7) with an EPC-7 patch-clamp amplifier. The glass microelectrodes we used had a tip
resistance ranging from 1.5 to 2.5 M
. The liquid junction potential
between the pipette solution and the Tyrode solution, ~
12 mV, was
canceled in each experiment. K+ currents were blocked with
intracellular and extracellular Cs+ substituted for
K+. Na+ channel current and T-type
Ca2+ channel current were inactivated by setting the
holding potential at
40 mV. Other currents were subtracted using
currents recorded in the presence of 2 µM nifedipine and 500 µM
CdCl2. Series resistance was compensated up to 75%.
Currents and voltage signals were analyzed using a personal computer.
Inactivation time course of nifedipine/Cd2+-sensitive
currents was fitted with exponential functions using least squares
(Kaleida Graph, version 3.08, Synergy Software). Comparisons were made
using a paired or unpaired Student's t-test where
appropriate and one-way ANOVA complemented by Dunn's procedure as a
multiple comparison procedure. All data are presented as means ± SE. All experiments were done at 35-36°C.
 |
RESULTS |
Inactivation process of Na+ currents
through the cardiac L-type Ca2+ channel.
L-type Ca2+ channel currents were recorded in the whole
cell voltage-clamp configuration at +10 mV from the holding potential of
40 mV at 5-s intervals in the presence of external
Ca2+ and subsequently in the absence of external
Ca2+. Peaks of the inward currents are
consecutively plotted against time in Fig.
1A. Application of
Ca2+-free solution resulted in a decrease of inward
currents, then stable inward currents were observed ~3 min after the
application. Stable inward currents are carried mainly by
Na+ after extracellular Ca2+ has been decreased
with EGTA (6, 9, 18). The inward
currents were abolished by nifedipine (2 µM) and CdCl2
(500 µM). Subtracted current traces, i.e., nifedipine/Cd-sensitive
current traces, are shown in Fig. 1, B and C. In
Ca2+-free solution (Fig. 1C), decay of the
current was much slower than observed in the case of the
Ca2+ current (Fig. 1B), and the tail inward
current was observed. Inactivation process of the current was best
fitted by functions with a two-exponential process (Fig.
1C).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Currents through cardiac L-type Ca2+ channels
in the absence of extracellular Ca2+. A: plot of
peak inward currents elicited by a pulse to +10 mV against time after
the start of whole cell current recording. The membrane potential was
held at 40 mV, and 300-ms pulses were applied at 5-s intervals. Below
the plot is given the composition of the external solution. The inward
currents were blocked by 2 µM nifedipine (nif) and 500 µM
CdCl2. B: nifedipine/Cd-sensitive current
observed in 1.8 mM CaCl2 containing solution. C:
nifedipine/Cd-sensitive current observed in Ca2+-free
solution is shown (top). Semilogarithmic plots of the
current are shown (bottom). During the 300-ms test pulse,
the first points deviate from the straight line (time constant of 445 ms, R = 0.947) and the difference is shown by lower set
points, which can be fitted by a second exponential function with a
time constant of 21 ms (R = 0.994). Each current was
obtained from the experiment shown in A.
|
|
Figure 2A shows a typical
example of the subtracted current traces recorded in
Ca2+-free solution, at selected potentials.
Nifedipine/Cd-insensitive currents in each potential are presented in
Fig. 2A. During depolarization, the inward currents
began to activate at
20 mV (17 ± 9 pA), peaked at +10 mV or +20
mV (628 ± 38 pA for +10 mV, 605 ± 39 pA for +20 mV,
n = 12), and reversed near +40 mV (see Fig.
6A). The reversal potential is similar to that observed by
other researchers (18). The faster inactivating
component was not remarkable up to
10 mV, and it appeared at 0 mV. At
10 mV, the decay of the current was best fitted by a function with a
single-exponential process. Above 0 mV, the decay of the currents was
best fitted by functions with a two-exponential process (Fig.
2B). Time constants of the faster inactivating component
decreased with depolarization of membrane potentials (P < 0.008); 30.5 ± 2.2 ms at 0 mV, 23.1 ± 1.0 ms at +10 mV,
21.4 ± 1.0 ms at +20 mV, and 17.0 ± 1.5 ms at + 30 mV
(n = 12; Fig. 3). There
were no statistical significant differences between time constants at
+10 mV and at +20 mV. Time constants of the slower inactivating
component were not significantly affected by the membrane
potential; 548 ± 15 ms at
10 mV, 489 ± 21 ms at 0 mV,
534 ± 27 ms at +10 mV, 598 ± 27 ms at +20 mV, and 530 ms ± 30 ms at +30 ms (n = 12; Fig.
3).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Inactivation properties of currents through cardiac
L-type Ca2+ channels in the absence of extracellular
Ca2+. A: nifedipine/Cd-insensitive superimposed
current traces recorded at membrane potentials of 30 mV-0 mV
(upper left) and +10 to +30 mV (upper right).
Nifedipine/Cd-sensitive superimposed current traces recorded at
membrane potentials of 30 mV-0 mV (lower left) and
+10 to +30 mV (lower right). These current traces were
elicited by 300-ms test pulses at 5-s intervals from a holding
potential of 40 mV in Ca2+-free solution. B:
semilogarithmic plots of the currents shown in A. At 10
mV, the points were fitted by a straight line (time constant of 456 ms). Above +10 mV, in each panel, the first points deviate from the
straight line, and the difference is shown by the lower set points,
which can be fitted by a second exponential function.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Mean values of the time constant of the faster
inactivating components ( ) and the slower inactivating
components ( ) at presented membrane potentials in
Ca2+-free solution. The left vertical axis is for the time
constant of the slower inactivating component, and the right vertical
axis is for the time constant of the faster inactivating component.
Data points are means ± SE (n = 12).
|
|
Effects of isoproterenol on Na+
currents through the cardiac L-type Ca2+
channel.
Application of isoproterenol (10 nM) increased peaks of the currents
recorded at +10 mV, as shown in Fig.
4A. During the voltage-clamp step, the increase in peak current on isoproterenol was associated with
a marked slowing of the time course of inactivation (Fig. 4B). As shown in Fig. 4B, isoproterenol slightly
slowed the time course of activation. In the inactivation process,
isoproterenol increased the slower inactivating component and decreased
the faster inactivating component (Fig. 4C). We obtained
similar results in two other experiments. To confirm these effects of
isoproterenol on the inactivating process, we used 100 nM
isoproterenol.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of isoproterenol (10 nM) on currents through cardiac L-type
Ca2+ channel in Ca2+-free solution.
A: plot of peak inward currents elicited by a pulse to +10
mV. The membrane potential was held at 40 mV, and 300-ms pulses were
applied at 5-s intervals. Below the plot is given the composition of
the external solution and the timing of application of isoproterenol.
The inward currents were blocked by 2 µM nifedipine and 500 µM
CdCl2. B: at left, superimposed
current traces were obtained during the application of isoproterenol.
At right, current in the absence of isoproterenol
(normalized control) was scaled to a similar peak current amplitude
recorded in the presence of isoproterenol. Nifedipine/Cd-insensitive
currents were not subtracted in the case of these currents.
C: at left, the isoproterenol
increased-nifedipine/Cd-sensitive current. Semilogarithmic plots of the
current are shown (right). During the 300-ms test pulse, the
first points deviate from the straight line (time constant of 703 ms),
and the difference is shown by the lower set points, which can be
fitted by a second exponential function with a time constant of 25 ms.
In B and C, each current trace was obtained from
the experiment shown in A.
|
|
Figure 5A shows a typical
example of the subtracted current traces recorded in the presence of
100 nM isoproterenol, at selected potentials. Nifedipine/Cd-insensitive
currents in each potential are presented in Fig. 5A;
isoproterenol increased the nifedipine/Cd-insensitive currents. During
depolarizations, the inward current began to activate at
20 mV,
peaked at 0 mV or +10 mV, and reversed near +40 mV (also see Fig.
6A). Figure 6A
shows the peak current-voltage relationship of nifedipine/Cd-sensitive
currents obtained in the absence and presence of isoproterenol.
Enhancement of the currents was prominent, with weak depolarization.
The relative values for the mean peak currents were 12.3, 4.2, 1.3, 1.4, 1.4, and 1.4 at
20 mV,
10 mV, 0 mV, +10 mV, +20 mV, and +30
mV, respectively (n = 12 for control, n = 8 for isoproterenol). Isoproterenol did not change the reversal
potential of the currents. In the presence of 100 nM isoproterenol, the
faster inactivating component was never observed at any membrane
potential (Fig. 5, A and B). At
20 mV, during
the 300-ms depolarization, the current did not inactivate (Fig.
5A). Although isoproterenol remarkably slowed the time
course of activation in the cases of weak depolarizations, the effect
of isoproterenol on activation at a higher membrane potential was not
remarkable. Time course of the inactivating component observed in the
presence of isoproterenol tended to slow with depolarization of
membrane potentials; 591 ± 54 ms at
10 mV, 538 ± 60 ms at 0 mV, 642 ± 59 ms at +10 mV, 684 ± 38 ms at +20 mV,
and 696 ± 65 ms at +30 mV (n = 8; Fig.
6B). There was no statistical significance between these
values, and those at each test potential were not significantly
different from those in the case of the slower inactivating components
observed in the absence of isoproterenol.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of isoproterenol (100 nM) on inactivation properties of
currents through cardiac L-type Ca2+ channel in
Ca2+-free solution. A: nifedipine/Cd-insensitive
superimposed current traces recorded at membrane potentials of 30
mV-0 mV (upper left) and +10 to +30 mV (upper
right). Nifedipine/Cd-sensitive superimposed current traces
recorded at membrane potentials of 30 mV-0 mV (lower
left) and +10 to +30 mV (lower right), respectively.
These current traces were elicited by 300-ms pulses at 5-s intervals
from a holding potential of 40 mV in Ca2+-free solution
with 100 nM isoproterenol. B: semilogarithmic plots of the
currents shown in A. At each potential, the points during
the whole of the 300-ms pulse can be fitted by a single exponential
function.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 6.
A: voltage dependence of peak nifedipine/Cd-sensitive
currents in the absence ( ) and presence ( ) of
100 nM isoproterenol. Depolarizations of 300-ms duration were present
in 10-mV increments at 5-s intervals from a holding potential of 40
mV in Ca2+-free solution. Data points are means ± SE
(n = 12 for control, n = 8 for
isoproterenol). B: mean values of the time constant of the
inactivating components in the presence of 100 nM isoproterenol at
selected potentials. Data points are means ± SE
(n = 8).
|
|
Effects of a protein kinase inhibitor and cAMP on
Na+ currents through the cardiac L-type
Ca2+ channel.
Application of H-89 (10 µM) for 4 min decreased the currents recorded
at +10 mV, as shown in Fig.
7A. The peak amplitude of the
inward currents decreased to 52 ± 4% (n = 7) of
the control value during application of H-89 for 4 min. The time course
of inactivating components was not significantly affected by H-89. The
time constants of the faster inactivating components were 27.6 ± 1.8 ms and 27.1 ± 2.7 ms for control and H-89, and the time
constants of the slower inactivating components were 723 ± 68 ms
and 652 ± 88 ms for control and H-89, respectively. The magnitudes of the faster inactivating component and the slower inactivating component showed different sensitivities to H-89. Initial
amplitudes of the faster inactivating component and the slower
inactivating component decreased to 72 ± 6% and 39 ± 7% of control values, respectively. In the presence of H-89, the effects
of isoproterenol (100 nM) were observed. The peak amplitude of inward
currents increased by 1.15 over that observed just before the
application of isoproterenol in the presence of H-89 (n = 5). As shown in Fig. 7A, effects of isoproterenol
in the presence of H-89 on the inactivating process differed from those
observed in the absence of H-89. The inactivating process was fitted by functions with a two-exponential process. The time course of the slower
inactivating component was accelerated with isoproterenol (346 ± 31 ms, n = 5, P < 0.01), but the time
course of the faster inactivating component did not change
significantly (30.5 ± 0.7 ms).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of a protein kinase inhibitor, H-89 (10 µM), and cAMP
(500 µM), on inactivation properties of currents through cardiac
L-type Ca2+ channel in Ca2+-free solution.
Current traces were elicited by 300-ms pulses at 5-s intervals from a
holding potential of 40 mV in Ca2+-free solution.
A: at left, superimposed current traces were
obtained during the sequential application of H-89 and isoproterenol
(100 nM)/H-89 in a cell. Each current trace was obtained before H-89
(control), 4 min after 10 µM H-89, and at maximal effects of 100 nM
isoproterenol in the presence of H-89. Isoproterenol was applied 4 min
after the application of H-89. B: current trace obtained in
500 µM cAMP in the pipette solution is shown (left).
Semilogarithmic plots of the current are shown (right). The
points during the whole of the 300-ms pulse can be fitted by a single
exponential function. C: current trace obtained in case of 5 µM isoproterenol is shown (left). Semilogarithmic plots of
the current are shown (right). The points during the whole
of the 300-ms pulse can be fitted by a single exponential function. In
A-C, nifedipine/Cd-insensitive currents were subtracted.
D: bar graph compares effects of 100 nM isoproterenol in the
presence of 10 µM H-89 (n = 5), 100 nM isoproterenol
(n = 8), and 500 µM cAMP (n = 6) on
relative initial amplitude of the slower inactivating component.
Relative initial amplitude of the slower inactivating component:
initial amplitude of slow-inactivating component/(initial amplitude of
fast-inactivating component + initial amplitude of
slow-inactivating component). Data are means ± SE.
|
|
As shown in Fig. 7B, in the presence of 500 µM cAMP in the
pipette solution, a large inward current with a single inactivating process was observed at +10 mV. The inactivating process was best fitted by a single-exponential function having a time constant of
1,343 ± 32 ms (n = 6). This value was
significantly larger than that observed in the presence of
100 nM isoproterenol (Fig. 6B) and similar to that observed
in the presence of 5 µM isoproterenol (Fig. 7C, 1,277 ± 116 ms, n = 3).
The effects of isoproterenol, cAMP, and H-89 on the inactivating
process at +10 mV are summarized in Fig. 7D. The relative initial amplitudes of the slower inactivating component [initial amplitude of slow-inactivating component/(initial amplitude of fast-inactivating component + initial amplitude of
slow-inactivating component)] were 0.37 ± 0.1 (n = 12), 0.43 ± 0.12 (n = 5), 1.0 (n = 8), and 1.0 (n = 6) for control,
100 nM isoproterenol with 10 µM H-89, 100 nM isoproterenol, and 500 µM cAMP, respectively.
 |
DISCUSSION |
Our results clearly show that the inactivation process of inward
currents, carried by Na+ through the cardiac L-type
Ca2+ channel in the absence of external Ca2+,
consisted of two components. We first investigated the nature of the
inactivating process of the two components. The inward currents began
to activate at
20 mV, and the faster inactivating component appeared
>0 mV. The time course of the faster inactivating component
accelerated with depolarizing test potentials. On the other hand, time
course of the slower inactivating component was not significantly
affected by test potentials. Faster inactivating components have been
observed in Ba2+ or Na+ currents through the
cardiac L-type Ca2+ channel in whole cell recordings
(3, 10); the voltage dependency and
underlying mechanism were not given. We observed Na+
currents through the Ca2+ channel in the presence of 0.5 mM
MgCl2, resulting in relatively small inward currents due to
the blocking effect of Mg2+ (6,
9, 18). The Mg2+ block cannot
explain the nature of the two inactivation processes, because the rates
of Mg2+ block and unblock are very fast (16).
Isoproterenol increased the slower inactivating component and decreased
the faster inactivating component, and a protein kinase inhibitor
attenuated these effects. These results indicate that phosphorylation
of the channel converts the faster inactivating component into the
slower one. In single-channel studies, phosphorylation of the channel
via the
-receptor-cAMP cascade modulates slow-gating kinetics and
prolongs duration of the available state, in which the channel can open
with membrane depolarization (4, 21, 27). Yue et al. (27) found that the
stimulation of the
-receptor-cAMP cascade also remarkably prolonged
open time of the channel, indicating that phosphorylation modulates
rapid-gating kinetics. These effects on the channel result in an
increase in the current and slowdown in inactivation of the macroscopic
Ca2+ channel current (19). The faster
inactivating component and the slower inactivating component showed
different sensitivities to a protein kinase inhibitor in that the
slower inactivating component was decreased more remarkably by H-89
than was the faster inactivating component. Similar findings were noted
in the case of Ca2+ currents in guinea pig ventricular
myocytes where a chemical phosphatase decreased the slower inactivating
component rather than the faster inactivating one (1).
Reduction in the faster inactivating component suggests that the
component represents kinetics of a partly phosphorylated channel.
Although it has been reported that the Ca2+ channel cannot
open without phosphorylation (14, 24), some investigators have reported that phosphorylation is necessary for
channel opening activity (8, 22). In the
presence of intracellular cAMP or a relatively high concentration of
isoproterenol, inactivation of the currents consisted of a single slow
process with a very slow time course. Ono and Fozzard (22)
have reported that a high concentration (16 µM) of isoproterenol
induced channel openings with long open time.
The above arguments lead to the hypothesis that these three components
(the faster inactivating component, the slower inactivating component,
and the very slow inactivating component) correspond to the degree of
phosphorylation of the channel: the minimally phosphorylated channel
shows faster inactivation, and the intermediately phosphorylated
channel shows slower inactivation. Finally, the maximally
phosphorylated channel shows very slow inactivation. This hypothesis is
consistent with proposals concerning single-channel studies
(8, 22).
Application of isoproterenol increased the peak currents to a greater
extent with weak depolarizations, findings compatible with data in the
case of Ba2+ currents in rat cardiac myocytes
(26) and in frog ventricular cells (2). The
leftward shift of activation properties probably contributes to this
voltage-dependent enhancement (2, 26). According to our present data, difference in the activation threshold between the faster inactivating component and the slower inactivating one may be responsible for the voltage-dependent effect of
isoproterenol: isoproterenol may convert the faster inactivating
component, which has a high threshold of activation, into the slower
inactivating component, which has a low threshold of activation, the
result being a large enhancement in weak depolarization. These
arguments are compatible with the shift in cardiac Ca2+
channel gating currents by isoproterenol, as noted in embryonic chick
heart cells (11). In the case of inactivating properties, the effects of isoproterenol cannot be explained by the shift, because
time constants of the faster inactivating components (17 ms ~ 30.5 ms) are not comparable to those of the inactivating components in
the presence of 100 nM isoproterenol (538 ms ~ 696 ms). We found
that isoproterenol slowed the time course of activation in the case of
weak depolarizing test potentials, an event noted in frog ventricular
cells and in cultured neonatal rat ventricular cells (2).
We fitted the inactivation time course of the faster component after
fitting that of the slower component. To some extent, this procedure
affects the time constant of the faster inactivating component. For a
more accurate time constant of inactivating component, it will be
necessary to use longer test pulses for currents reaching a
steady-state level.
Our data are in agreement with previous observations of single-channel
studies and whole cell studies, and present a new insight into
inactivating kinetics of macroscopic currents through phosphorylated and dephosphorylated channels. On the basis of our results, it will be
possible to identify phosphorylation sites responsible for changing the
inactivation process, using heterologously expressed cardiac L-type
Ca2+ channel subunits with site-directed mutagenesis.
 |
ACKNOWLEDGEMENTS |
We thank Drs. M. Kameyama and K. Yamaoka for helpful discussion.
 |
FOOTNOTES |
This work was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports, and Culture, Japan.
Address for reprint requests and other correspondence: M. Kaibara, Dept. of Pharmacology, Nagasaki Univ., School of Medicine, 1-12-4 Sakamoto, Nagasaki 8528523, Japan (E-mail:
mkaibara{at}alpha.med.nagasaki-u.ac.jp).
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.
Received 3 May 1999; accepted in final form 16 March 2000.
 |
REFERENCES |
1.
Allen, TJA,
and
Chapman RA.
The effects of a chemical phosphatase on single calcium channels and the inactivation of whole-cell calcium current from isolated guinea-pig ventricular myocytes.
Pflügers Arch
430:
68-80,
1995[ISI][Medline].
2.
Bean, BP,
Nowycky MC,
and
Tsien RW.
-Adrenergic modulation of calcium channels in frog ventricular heart cells.
Nature
407:
371-375,
1984.
3.
Boyett, MR,
Honjo H,
Harrison SM,
Zang WJ,
and
Kirby MS.
Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes.
Pflügers Arch
428:
39-50,
1994[ISI][Medline].
4.
Brum, G,
Osterrieder W,
and
Trautwein W.
-Adrenergic increase in the calcium conductance of cardiac myocytes studied with patch clamp.
Pflügers Arch
401:
111-118,
1984[ISI][Medline].
5.
Chijiwa, T,
Mishima A,
Hagiwara M,
Sano M,
Hayashi K,
Inoue T,
Naito K,
Toshioka T,
and
Hidaka H.
Inhibition of forskolin-induced neutrite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma.
J Biol Chem
265:
5267-5272,
1990[Abstract/Free Full Text].
6.
Hadley, RW,
and
Hume JR.
An intrinsic potential-dependent inactivation mechanism associated with calcium channels in guinea-pig myocytes.
J Physiol (Lond)
389:
205-222,
1987[Abstract].
7.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
392:
85-100,
1981.
8.
Herzig, S,
Patil P,
Neumann J,
Staschen CM,
and
Yue DT.
Mechanisms of
-adrenergic stimulation of cardiac Ca2+ channels revealed by discrete-time Markov analysis of slow gating.
Biophys J
65:
1599-1612,
1993[Abstract].
9.
Hess, P,
and
Tsien RW.
Mechanism of ion permeation through calcium channels.
Nature
309:
453-456,
1984[ISI][Medline].
10.
Josephson, IR,
Sanchez-Chapula J,
and
Brown AM.
A comparison of calcium currents in rat and guinea-pig single ventricular cells.
Circ Res
54:
144-156,
1984[Abstract].
11.
Josephson, IR,
and
Sperelakis N.
Phosphorylation shifts the time-dependence of cardiac Ca2+ channel gating currents.
Biophys J
60:
491-497,
1991[Abstract].
12.
Kaibara, M,
and
Kameyama M.
Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of guinea-pig.
J Physiol (Lond)
403:
621-640,
1988[Abstract].
13.
Kaibara, M,
Mitarai S,
Yano K,
and
Kameyama M.
Involvement of Na+-H+ antiporter in regulation of L-type Ca2+ channel current by angiotensin II in rabbit ventricular myocytes.
Circ Res
75:
1121-1125,
1994[Abstract].
14.
Kameyama, M,
Hescheler J,
Hofmann F,
and
Trautwein W.
Modulation of Ca current during the phosphorylation cycle in the guinea pig heart.
Pflügers Arch
407:
123-128,
1986[ISI][Medline].
15.
Kass, RT,
and
Sanguinetti MC.
Inactivation of calcium channel current in calf cardiac Purkinje fiber.
J Gen Physiol
84:
705-726,
1984[Abstract].
16.
Lansman, JB,
Hess P,
and
Tsien RW.
Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+.
J Gen Physiol
88:
321-347,
1986[Abstract].
17.
Lee, KA,
Marban E,
and
Tsien RW.
Inactivation of calcium channels in mammalian heart cells: joint dependence on membrane potential and intracellular calcium.
J Physiol (Lond)
364:
395-411,
1985[Abstract].
18.
Matsuda, H.
Sodium conductance in calcium channels of guinea-pig ventricular cells induced by removal of external calcium ions.
Pflügers Arch
407:
465-475,
1986[ISI][Medline].
19.
McDonald, TF,
Pelzer S,
Trautwein W,
and
Pelzer DJ.
Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells.
Physiol Rev
74:
365-507,
1994[Free Full Text].
20.
Mentrad, D,
Vassort G,
and
Fischmeister R.
Calcium-mediated inactivation of the calcium conductance in cesium-loaded frog heart cells.
J Gen Physiol
83:
105-131,
1984[Abstract].
21.
Ochi, R,
and
Kawashima Y.
Modulation of slow gating process of calcium channels by isoprenaline in guinea-pig ventricular cells.
J Physiol (Lond)
424:
187-204,
1990[Abstract].
22.
Ono, K,
and
Fozzard HA.
Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells.
J Physiol (Lond)
454:
673-688,
1992[Abstract].
23.
Ono, K,
and
Fozzard HA.
Two phosphatase sites on the Ca2+ channel affecting different kinetic functions.
J Physiol (Lond)
470:
73-84,
1993[Abstract].
24.
Rosen, RIP,
Hess P,
Tsien RW,
Reeves JP,
and
Smilowitz H.
Cardiac calcium channels in planar bilayers: insights into mechanisms of ion permeation and gating.
Science
245:
1115-1118,
1986.
25.
Sham, JSK,
Cleemann L,
and
Morad M.
Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.
Proc Natl Acad Sci USA
92:
121-125,
1995[Abstract].
26.
Tiaho, F,
Nargeot J,
and
Richard S.
Voltage-dependent regulation of L-type cardiac Ca channels by isoproterenol.
Pflügers Arch
419:
596-602,
1991[ISI][Medline].
27.
Yue, DT,
Herzig S,
and
Marban E.
-Adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes.
Proc Natl Acad Sci USA
87:
753-757,
1990[Abstract].
Am J Physiol Cell Physiol 279(3):C603-C610
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society