Prepulse-induced mode 2 gating behavior with and without
-adrenergic stimulation in cardiac L-type Ca channels
Yuji
Hirano,
Takashi
Yoshinaga,
Mitsushige
Murata, and
Masayasu
Hiraoka
Department of Cardiovascular Diseases, Medical Research
Institute, Tokyo Medical and Dental University, Tokyo 113, Japan
 |
ABSTRACT |
Mode 2 gating of L-type Ca channels is characterized by high
channel open probability
(NPo) and long
openings. In cardiac myocytes, this mode is evoked physiologically in
two apparently different circumstances: membrane depolarization
(prepulse facilitation) and activation of protein kinase A. To examine
whether the phosphorylation mechanism is involved during
prepulse-induced facilitation of cardiac L-type Ca channels, we used
isolated guinea pig ventricular myocytes to analyze
depolarization-induced modal gating behavior under different basal
levels of phosphorylation. In control,
NPo measured at 0 mV was augmented as the duration of prepulse to +100 mV was prolonged
from 50 to 400 ms. This was due to the induction of mode 2 gating
behavior clustered at the beginning of test pulses. Analysis of open
time distribution revealed that the prepulse evoked an extra component,
the time constant of which is not dependent on prepulse duration. When
isoproterenol (1 µM) was applied to keep Ca channels at an enhanced
level of phosphorylation, basal NPo without
prepulse was increased by a factor of 3.6 ± 2.2 (n = 6). Under these conditions,
prepulse further increased
NPo by promoting
long openings with the same kinetics of transition to mode 2 gating
(
200 ms at +100 mV). Likewise, recovery from mode 2 gating, as
estimated by the decay of averaged unitary current, was not affected
after
-stimulation (
25 ms at 0 mV). The kinetic behavior
independent from the basal level of phosphorylation or activity of
cAMP-dependent protein kinase suggests that prepulse facilitation of
the cardiac Ca channel involves a mechanism directly related to
voltage-dependent conformational change rather than voltage-dependent phosphorylation.
prepulse facilitation; isoproterenol; phosphorylation
 |
INTRODUCTION |
THE VOLTAGE-DEPENDENT L-type Ca channels in heart play
important roles in the regulation of cardiac functions, including
pacemaker activity in the sinus node, conduction through the
atrioventricular node, and contraction of atrial and ventricular
myocytes. Because of their physiological and pharmacological
significance, mechanisms of the modulation of Ca channels have been
extensively studied (18). At the single-channel current level, changes
in gating behavior leading to the upregulation of L-type Ca channel
activity include not only increased availability and graded changes in open and closed times, but also the induction of "mode 2" gating behavior with unusually long openings. The mode 2 gating behavior was
initially described as the effect evoked by dihydropyridine Ca channel
agonists (11). This mode was then shown to work physiologically, inasmuch as it was evoked by
-adrenergic stimulation presumably acting via phosphorylation of the channel protein (26). The mode 2 gating was also elicited by repetitive or strong membrane depolarization (21). This effect can explain activity-dependent potentiation of the Ca channel in cardiac cells (17).
The potentiation or facilitation of Ca channels evoked by depolarizing
voltages is commonly observed in a variety of excitable tissues (6).
There are several distinct forms of prepulse-induced facilitation. In
Ca channels of several neuronal cells (N, P/Q, and neurosecretory L
type), the tonic inhibition mediated by G protein-coupled receptors can
be relieved by membrane depolarization (7), causing the increase in
current amplitude after prepulses. On the other hand, in L-type Ca
channels of chromaffin cells (1) and skeletal muscle (25), prepulse
facilitation was due to the voltage-dependent phosphorylation
mechanism. The prepulse-induced potentiation was also observed in
reconstitution systems of cloned L-type
1C and
1S Ca channels (2, 4, 16, 22,
24). The involvement of phosphorylation, however, is equivocal in the prepulse facilitation observed in expressed channels. The
voltage-dependent phosphorylation mechanism was unlikely in smooth
muscle
1S channels (16) and
neuronal
1C channels (2) but
was supported in cardiac
1C
channels expressed in Chinese hamster ovary (CHO) cells (24).
Because a similar high-activity gating pattern was elicited by prepulse
and
-adrenergic stimulation, the phosphorylation mechanism might be
involved during prepulse-induced facilitation in the heart. This
hypothesis has not been tested in native cardiac myocytes, where
modulation by cAMP-dependent protein kinase A (PKA) stimulation or
positive prepulse is readily observable at the single-channel level. In
this study we examined the relationships of modal gating elicited by
PKA stimulation and by prepulse in guinea pig ventricular myocytes.
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MATERIALS AND METHODS |
Preparations.
Ventricular myocytes from guinea pig hearts were obtained by an
enzymatic dissociation procedure similar to that described previously
(12). Briefly, guinea pigs weighing 200-250 g were anesthetized
with pentobarbital sodium (40 mg/kg ip). The chest was opened, and the
aorta was cannulated in situ and perfused with Tyrode solution before
the heart was removed. Hearts were then retrogradely perfused with
low-Ca2+ (30 µM) Tyrode solution
with collagenase (0.4 mg/ml, type I; Sigma Chemical) for 30 min by use
of a Langendorff apparatus. After the enzyme was washed out, the cells
were dissociated in high-K+,
low-Cl
storage solution.
Single-channel recording.
Single L-type Ca channel currents
(ICa,L) were
recorded in cell-attached configuration (9) with use of an Axopatch 1D
amplifier at room temperature, as described in our previous study (13). The bath solution contained (in mM) 120 potassium aspartate, 20 KCl, 10 glucose, 2 EGTA, and 10 HEPES, with pH adjusted to 7.4 with KOH. The
pipette solution contained (in mM) 100 BaCl2 and 10 HEPES, with pH
adjusted to 7.4 with tetraethylammonium hydroxide. Pipettes were pulled
from capillary tubes in a two-step process, coated with insulating
varnish, and then fire polished. The electrode had a resistance of
5-10 M
when the pipette was filled with the Ba2+ solution. The membrane
potential of myocytes was depolarized to ~0 mV by
high-K+ solution. The electrode
potential was adjusted to give a zero current between the pipette
solution and the bath solution immediately before the seal formation.
Patch membranes were then depolarized at 1 Hz to 0-20 mV for 180 ms from the holding potential of
80 mV to check the presence and
the stability of channel activity in the pipette. The threshold for the
activation of L-type Ca channels was around
10 mV in our
recording conditions. Stability of basal Ca channel activity was
checked for 10 min before the pulse protocol was applied (see
RESULTS).
Current signals were filtered at 2-4 kHz (8-pole Bessel filter)
and sampled at a rate five times the filter frequency (10-20 kHz)
by use of a pCLAMP system (Axon Instruments) on a Pentium-based personal computer (Fujitsu FM/V). After digital subtraction for capacitive and leak components, idealized records obtained by standard
half-height criteria were used to calculate the channel open
probability (Po
or
NPo)
and to obtain averaged current traces. Most of the data analysis and
presentation were done by Origin (MicroCal). Fitting for open time
distribution with logarithmic binning was done by the
maximum-likelihood estimation method with use of pCLAMP software.
Statistics.
Where appropriate, numerical values are presented as means ± SD.
Differences in the numerical values between two groups were evaluated
using Student's t-test.
P < 0.05 was considered significant.
 |
RESULTS |
We compared the effect of a depolarizing prepulse on channel gating
behavior when basal phosphorylation levels of Ca channels are altered.
For this purpose, we applied isoproterenol (1 µM) to the bath
solution while the patch membrane was repeatedly depolarized at 0.2 or
0.5 Hz. The test potential was 0 or +10 mV without and with prepulses
of 50, 200, and 400 ms to +100 mV in this order. With use of this
standard protocol, the
NPo without
prepulse could be used as a guide for the basal phosphorylation level
of Ca channels.
Figure 1 illustrates current
records collected during the standard protocol delivered at 0.5 Hz.
Traces were selected to show the effect of prepulse and isoproterenol,
separately or combined, on unitary
ICa,L. The
prepulse duration was 400 ms for the data taken here. In the control
without prepulse, channel openings were short and appeared randomly
distributed during 180-ms test pulses. Prepulse to +100 mV evoked long
openings clustered at the beginning of the test pulses. Channel
reopenings could be of long duration (Fig. 1A,
bottom, trace 3). Besides the effect on channel open
times, prepulse affected the proportion of sweeps with channel activity
("availability"). Although this was not a single-channel patch,
there were five blank sweeps (of 100 sweeps) in the control without
prepulse. The number of blank sweeps was increased to 17 after prepulse
application. These effects of prepulse were essentially the same after
isoproterenol was applied to increase the channel activity. In this
patch, isoproterenol increased basal NPo (without
prepulse) by a factor of 2.2. Channel openings were more frequent with
slightly prolonged duration, and they were almost uniformly distributed
during 180-ms test pulses. Then prepulse evoked long openings clustered
mainly at the beginning of test pulses. During the application of
isoproterenol, prepulse also increased the number of blank sweeps from
3 to 18 in this case. Unitary current amplitude was not affected by
prepulse and/or isoproterenol application (~1.8 pA at 0 mV).

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Fig. 1.
Effect of prepulse and/or isoproterenol on unitary Ca channel current
before (A) and after
(B) isoproterenol application. Cells
were depolarized to 0 mV without prepulse
(top) and after prepulse to 100 mV
for 400 ms (bottom). Four
consecutive sweeps are shown. Voltage-clamp pulses (in
top and
bottom) were delivered at 0.5 Hz.
Patch contained 2 Ca channels, as judged from maximum number of
stacked openings.
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Figure 2 shows a set of diaries or temporal
profiles of NPo
during the experiment with use of the standard protocol, from which
traces in Fig. 1 were taken. Inasmuch as the diaries were arranged
according to the prepulse duration, the actual chronological order was
as follows; sweep 1 of
a, b, c, and
d and then sweep 2 of a, b, c, and
d and so on. This arrangement
clarified a general tendency of the channel behavior; i.e.,
NPo was larger as
duration of the prepulse was extended and returned to the control value when no prepulse was applied. As shown in Fig. 1, this was due to an
increase in long openings accumulated at the beginning of test pulses.
The increase in
NPo (averaged
over total sweeps) was not hampered by the increase in the number of
blank sweeps. The stationarity of
NPo values in
four rows suggests that holding the patch at
80 mV for
1.4-1.8 s is sufficient to remove the effect imposed by the
previous prepulse. Figure 2B shows a
set of NPo
diaries in an identical format from the same patch after basal
NPo was increased
by isoproterenol. Through induction of long openings, prepulse again
increased NPo as
its duration was prolonged. Change in gating behavior (and hence
NPo) produced by prepulse to ~100 mV was a consistent finding for the entire period
of single-channel recording in all (>20) cases examined.

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Fig. 2.
Stationarity of prepulse facilitation in control and after
isoproterenol application. A: temporal
profile of channel open probability
(NPo) in
control. B: temporal profile of
NPo after
isoproterenol application. Depolarizations without
(a) and with prepulses of 50-, 200-, and 400-ms duration (b, c, and
d, respectively) were delivered
sequentially in this order, but profiles are shown separately according
to prepulse duration. Insets: schemes
of voltage protocol and position of current trace at test potential of
0 mV.
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We then analyzed the changes in channel open times induced by prepulse
and/or isoproterenol application. Figure 3
shows open time distributions in two different formats from the data
shown above. With use of a conventional binning mode, the control
distribution (with no prepulse) appeared to be approximated with a
single-exponential component. This situation was similar after
isoproterenol application, although there were few long openings that
could not be covered by a single component (10 of ~6,000 events in
this case). Thus the effect of isoproterenol on channel open time was
characterized by a slight prolongation of the open time constant in
this case (see Ref. 14 for multiple types of responses to
-adrenergic stimulation). On the other hand, the distribution after
prepulse extended to several tens of milliseconds and clearly required additional components. To cover these openings, we used a logarithmic binning method (23) (Fig. 3,
insets). Because complicated and time-consuming voltage protocol was required in this study, the number
of events was often insufficient for reliable multiple (triple or
more)-exponential fittings. As a first approximation, we tentatively
fitted the open time distributions with no prepulse as a single
exponential and the distribution with prepulse as double exponentials.
When analyzed in this simplified manner, the effect of prepulse was
induction of an extra component with a time constant ~10 times that
of components without prepulse in both conditions. Figure
4 illustrates sets of time constants in
representative cases. In the control and after isoproterenol application, the duration of prepulse had not much affected the value
of the time constant, but it increased the proportion of the extra
component as it was prolonged.

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Fig. 3.
Prepulse-induced changes in open time distribution in control
(A) and after isoproterenol
application (B).
Top: without prepulse;
bottom: after 400-ms prepulse to +100
mV. Insets: open time distribution
obtained by logarithmic binning. Vertical axis, number of events after
square-root transformation. Solid line, exponential fit to data by
maximum-likelihood estimation method. Values indicate fitted time
constants with single (top) and
double exponentials (bottom). These
time constants are also used to fit conventionally binned distributions
(thick lines).
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Fig. 4.
Effect of prepulse duration on open time constants in control
(left) and after isoproterenol
(right) in patches
1 (A) and
2 (B). Open time distribution obtained
by logarithmic binning was fitted by single (without prepulse) and
double exponentials (50-, 200-, and 400-ms prepulse). Results from 2 representative cases are presented. Open bars, time constant for data
without prepulse and shorter time constant for data with prepulses;
gray bars, newly induced time constants for data with prepulses. Solid
lines above histograms, contribution by extra component to total
events.
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Thus prepulse-induced changes in kinetic behavior were essentially the
same in control and after isoproterenol application: prepulse evoked a
gating pattern characterized by long-lasting openings, the induction of
which was dependent on prepulse duration. These results suggest that
the kinetic scheme proposed by Pietrobon and Hess (21) is applicable
also for prepulse-induced modulation after isoproterenol application.
In their scheme of voltage-dependent potentiation, two distinct kinetic
modes (activity with brief openings and activity with long openings)
are connected by first-order forward
(kf)
and backward
(kb)
rate constants, which were given as the function of membrane potential.
We next analyzed the time course of the change in gating behavior
induced by isoproterenol based on this scheme.
Figure 5A
compares dependence of averaged single-channel current on prepulse
duration in control and after isoproterenol application from the same
data shown above. Following the method employed by Pietrobon and Hess
(21), averaged current or
NPo was obtained over nonblank sweeps in Figs. 5 and 6. This
is to diminish the effects evoked by the prepulse-induced decrease in
the availability. Because basal channel activity with no prepulse was
greater after isoproterenol application, the amount of prepulse-induced
increase in NPo
was also larger after isoproterenol (Fig.
5B). However, when
NPo was
normalized to the value without prepulse (Fig.
5C), the relative increase was
generally greater in the control and the time courses of potentiation
were similar in the two conditions. According to the first-order
kinetic scheme, the increase in
NPo follows an
exponential rise when it is due to the transition to a
high-Po gating
mode. We pooled data and summarized them in Fig. 6. The time course of
the onset of prepulse-induced potentiation (measured at +100 mV) was
not significantly altered after isoproterenol-induced potentiation of
channel activity.

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Fig. 5.
Effect of prepulse duration on
NPo.
A: averaged unitary current traces
from sweeps with various prepulses superimposed according to prepulse
duration as indicated above trace. a:
Control; b: after isoproterenol
application. B: absolute value of
NPo plotted
against prepulse duration. C:
NPo normalized to
value without prepulse plotted against prepulse duration.
"Average" was taken over nonblank sweeps to diminish effects
evoked by prepulse-induced decrease in availability.
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Fig. 6.
Summary of effect of prepulse duration on
NPo.
NPo was
normalized to value without prepulse. Averaged data from 4 patches were
plotted against prepulse duration. Thin lines, exponential rise
{1 + A × [1 exp( t/ )]} with
a time constant of 198 ms for both cases. "Average" was taken
over nonblank sweeps to diminish effects evoked by prepulse-induced
decrease in availability.
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We then evaluated the time course of recovery or exit from the
high-activity mode. At normal test potentials, channels return to the
normal mode with very little chance to enter the
high-Po mode
again during the rest of the test pulse. Therefore, the time constant
of the decay of the prepulse-induced current is a good parameter to
estimate the backward rate constant (
1/kb).
Figure 7 illustrates the averaged
single-channel record at +20, 0, and
40 mV after prepulse to
+100 mV for 400 ms. Isoproterenol increased NPo or amplitude
of ensemble-averaged current but did not significantly alter the decay
time constant of the averaged current at each test potential. Figure
8 shows the summary of decay time constants in control and after isoproterenol application at various test potentials. The time constants at 0 mV were 25.4 ± 5.1 ms
(n = 7) in control and 26.3 ± 4.9 ms (n = 6) after isoproterenol
application, which were not statistically different.

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Fig. 7.
Decay of prepulse-facilitated current: +20
(a), 0 (b), and 40 mV
(c).
Top: voltage protocol to obtain
averaged current traces at various test voltages
(Vtest).
A: average traces with
single-exponential fitting in control. Fitted time constants are
indicated. B: average traces after
isoproterenol application. Calibration bars are the same for both
conditions. Patch contained 6 Ca channels, as judged from maximum
number of stacked openings.
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Fig. 8.
Voltage dependence of decay time course. Time constants of decay in
average current at various test voltages are presented for control
(A) and after isoproterenol
application (B). Lines are drawn
with assumption of an exponential voltage dependence for backward rate
constant,
kb.
1/kb = 1/[0.04 × exp( 0.081 × V)]
for both conditions, where V is
voltage.
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 |
DISCUSSION |
We analyzed prepulse-induced transition to mode 2 gating behavior of
cardiac L-type Ca channels with continuous monitoring of basal channel
activity without prepulse. We found that the kinetics of
prepulse-induced transition into and recovery from the high-activity
mode were not affected after basal
NPo was increased by
-adrenergic stimulation. The kinetic behavior independent from
the levels of PKA activity or phosphorylation state suggests that
prepulse facilitation of cardiac Ca channels involves a mechanism other
than phosphorylation.
Prepulse facilitation of L-type Ca channels.
Several different forms of prepulse-induced facilitation are observed
among Ca channels of different excitable tissues (6). In L-type
1C and
1S Ca channels, the mechanisms
of prepulse facilitation include a voltage-dependent phosphorylation.
This was demonstrated for the L-type channel in chromaffin cells (1) and skeletal muscle (15, 25). The phosphorylation mechanism is
supported also in some reconstitution systems of cloned L-type Ca
channels. The cardiac
1C
subunit expressed without auxiliary subunits in CHO cells was subject
to voltage-dependent potentiation, when it was partially activated by
an exogenous catalytic subunit of PKA (24). Prepulse potentiation was
occluded, however, when the system was maximally phosphorylated by
prolonged dialysis of PKA or addition of a phosphatase inhibitor. This
observation is consistent with the mechanism of voltage-dependent
phosphorylation. Prepulse facilitation was also observed for the
skeletal
1S channel expressed
alone in CHO cells (16). However, the expressed
1S channel was not modulated by
phosphorylation, raising the possibility that the mechanism is
different in this case. When the neuronal
1C subunit was expressed in the
Xenopus oocyte, prepulse facilitation was observed only when the
-subunit (except type
2a) was coexpressed (2, 4).
Bourinet et al. (2) observed that basal current amplitude and the
degree of facilitation were not affected by the agents that stimulate
PKA activity but were suppressed by PKA inhibitors. They therefore
suggested that a certain level of phosphorylation might be essential
for the channel to show prepulse facilitation but that the
1C complex might be fully phosphorylated under basal conditions in
Xenopus oocytes. The presence of
prepulse facilitation under fully phosphorylated circumstances implies
that voltage-dependent phosphorylation is an unlikely mechanism of
facilitation in
1C channels
expressed in the Xenopus oocyte. In
our experiments using native cardiac myocytes, prepulse facilitation
could still be evoked after basal channel activity was increased by
isoproterenol. If our system was saturated in terms of phosphorylation
during isoproterenol application, this observation would indicate that
prepulse facilitation involved a mechanism other than phosphorylation.
The expression of cloned Ca channel subunits in reconstitution systems
allows the functional characterization of individual channel subtypes
and the roles for regulatory subunits. The modulation of recombinant Ca
channels by phosphorylation, however, is often difficult to reproduce.
This might be related to levels of phosphorylation of the expressed
channels under basal conditions and also to the difference in cellular
environments, including the availability of anchor proteins (8).
Therefore, to elucidate the mechanism of prepulse facilitation of
cardiac L-type Ca channels, analysis of the data on the native cardiac
myocyte is important. With use of this preparation, PKA-dependent
modulation of Ca channels is regularly observed and well characterized.
Modal gating behavior of cardiac L-type Ca channels.
In cardiac L-type Ca channels, "mode 2" or
high-Po gating
behavior is elicited by dihydropyridine application (11),
-adrenergic stimulation (26), and strong depolarization (21). Our
main concern in this study is the interrelationship of mode 2 behavior elicited by
-adrenergic stimulation and strong depolarization.
Mode 2 gating elicited by
-adrenergic stimulation is determined by
phosphorylation and dephosphorylation processes (10, 20). From the
analysis of kinetic behavior, we previously proposed that enhancement
of cardiac L-type Ca channels during
-adrenergic stimulation
involves multiple functional modulatory sites, which might be
phosphorylated independently (14). Inasmuch as several putative
phosphorylation sites are identified in
1- and
-subunits, mode 2 gating may be induced by full occupancy of multiple phosphorylation sites or the phosphorylation of a difficult-to-phosphorylate site (26).
On the other hand, the prepulse-induced facilitation was described by a
first-order kinetic scheme by Pietrobon and Hess (21)
In
their work, forward
(kf)
and backward
(kb)
rate constants were obtained as a function of voltage. The
prepulse-induced changes in modal gating appear to coexist with
conventional voltage-dependent activation and inactivation. During
depolarizing prepulses, channels are voltage inactivated. This effect
was observed in this study as an increase in the number of blank
sweeps, consistent with the previous report (3). However, in our
experimental conditions, effects of voltage-dependent inactivation on
total channel activity were slight compared with those evoked by
prepulse-induced long openings (Fig. 2). The second voltage-dependent
equilibrium of channel gating implies a second and reluctant voltage
sensor or transduction of the change in voltage to more than one
conformational change.
On the basis of these schemes, a view emerges that likely explains the
interaction of prepulse facilitation and the phosphorylation mechanism:
prepulse may produce a conformational change in the cardiac Ca channel
that makes it a better substrate for phosphorylation (1, 25). In this
case, forward
(kf)
and backward
(kb)
constants are not only the function of voltage, but also of kinase
activity and phosphatase activity. At a given test potential, an
increase in kinase activity accelerates the forward transition and,
conversely, phosphatase activity stimulates backward development.
We compared prepulse-induced transition between modes with and without
isoproterenol application in the same patch. The increased NPo after
isoproterenol application, as observed, was due not only to increased
PKA activity, but also to decreased phosphatase type I activity (10,
19). The application of isoproterenol then would alter forward
(kf)
and backward
(kb)
rate constants of modal gating and, therefore, affect the time course
of onset of the increase in
NPo [
= 1/(kf + kb)]
(Fig. 6) and decay of the average current (
1/kb)
(Fig. 8). This was not the case in this study. These results suggest
that at physiological levels of phosphorylation of Ca channels, where
channel activity was readily observable and strongly regulated by PKA
activity, prepulse facilitation is not directly due to a
voltage-dependent phosphorylation.
Although the concept of modal gating has been successfully applied to
describe the complex gating behavior of cardiac Ca channels, function-structure relationships of modal gating remain to be clarified. When mechanisms underlying mode 2 gating evoked by phosphorylation and prepulse are different, physical and molecular entities of changes in channel conformation should not be
the same. However, it is still difficult to discriminate different types of mode 2 gating behavior from the analysis of kinetics within a
mode itself. Further studies are needed to clarify the complex nature
of gating and its relation to molecular structure.
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ACKNOWLEDGEMENTS |
This work was supported by a grant from the Ministry of Education,
Science, Sport, and Culture of Japan to Y. Hirano.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Hirano, Dept.
of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical
and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
(E-mail: hirano.card{at}mri.tmd.ac.jp).
Received 28 September 1998; accepted in final form 4 March 1999.
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