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Departments of 1 Humoral Regulation and 3 Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; and 2 Department of Physiology, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The
electrophysiological properties of sinoatrial (SA) node pacemaker cells
vary in different regions of the node. In this study, we have
investigated variation of the 4-aminopyridine (4-AP)-sensitive current
as a function of the size (as measured by the cell capacitance) of SA
node cells to elucidate the ionic mechanisms. The 10 mM 4-AP-sensitive
current recorded from rabbit SA node cells was composed of transient
and sustained components
(Itrans and
Isus, respectively). The activation and inactivation properties
[activation: membrane potential at which conductance is
half-maximally activated (Vh) = 19.3 mV,
slope factor (k) = 15.0 mV;
inactivation: Vh = 
31.5 mV, k = 7.2 mV] as well as the density of
Itrans (9.0 pA/pF on average at +50 mV) were independent of cell capacitance. In contrast, the density of
Isus (0.97 pA/pF
on average at +50 mV) was greater in larger cells, giving rise to a
significant correlation with cell capacitance. The greater density of
Isus in larger
cells (presumably from the periphery) can explain the shorter action potential in the periphery of the SA node compared with that in the
center. Thus variation of the 4-AP-sensitive current may be involved in
regional differences in repolarization within the SA node.
electrophysiology; patch clamp; transient outward current; regional difference
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUS HISTOLOGICAL and electrophysiological studies have shown that the sinoatrial (SA) node is structurally and functionally heterogeneous (for review, see Ref. 29). In the center of the node (normally the leading pacemaker site), the cells are small and have "empty" cytoplasm with relatively few organelles and poorly organized myofilaments, whereas, in the periphery of the node near the crista terminalis, the cells are larger and more densely packed with mitochondria and well-organized myofilaments (4, 35). In the center of the SA node, action potentials are longer, the maximum diastolic potential is less negative, the upstroke velocity is lower, and the intrinsic pacemaker activity is slower than in the periphery (31, 32, 39, 40). We previously reported that the electrical activity of single pacemaker cells isolated from the rabbit SA node shows a similar heterogeneity (27): in small SA node cells (presumably from the center of the SA node), the action potential upstroke is slower, the diastolic and take-off potentials are more positive, and the pacemaker activity is slower than in large cells (presumably from the periphery). This work suggests that variations in membrane properties may underlie the regional differences in the electrical activity within the SA node. The more positive take-off potential, the slower action potential upstroke, and the slower pacemaker activity, in part at least, can be explained by lower densities of the tetrodotoxin (TTX)-sensitive Na+ current and the hyperpolarization-activated current (If) in smaller cells.
As mentioned above, the action potential in the center of the SA node is longer than that in the periphery. We recently showed (5) that this is part of a downward gradient in action potential duration along the conduction pathway in and around the SA node. This downward gradient in action potential duration is similar to, but more marked than, that elsewhere in the heart, e.g., from the ventricular subendocardium to the ventricular subepicardium (2, 34), and its function, as elsewhere in the heart, is presumably to help prevent reentry. Transient outward K+ current (Ito) may be responsible for the gradient in action potential duration from the center to the periphery of the SA node, because in the presence of 4-aminopyridine (4-AP; blocker of Ito) the gradient is reduced and no longer significant (7). The presence of 4-AP-sensitive Ito has been reported by various investigators in rabbit SA node pacemaker cells (15, 24, 25, 30, 37), as well as in the remaining part of the heart (for review, see Ref. 9). The present study was undertaken to investigate possible variation in the 4-AP-sensitive current (I4-AP) as a function of SA node cell size to understand the mechanisms underlying the regional differences in action potential duration within the SA node. The results obtained suggest that a lower density of the sustained component of I4-AP in smaller cells can explain the longer action potential in the center of the SA node. Thus variation of I4-AP may be involved in regional differences in repolarization in the SA node.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Single SA node pacemaker cells.
Single SA node cells were enzymatically isolated from adult rabbit
hearts by methods similar to those described previously (27, 28, 47,
48). In brief, a New Zealand White rabbit (6-10 wk old) weighing
1.0-1.5 kg was anesthetized with an intravenous injection of
pentobarbital sodium (30-40 mg/kg). Heparin (300-1,000 U/kg)
was injected at the same time. The chest was opened, and the heart was
rapidly excised into oxygenated Tyrode solution at 32°C. The SA
node region was then isolated and cut into several strips (0.5-1.0
mm in width) perpendicular to the crista terminalis. Atrial muscle and
fat tissue on the epicardial surface were carefully removed under a
dissecting microscope. The SA node tissue strips were digested with an
enzyme solution containing collagenase, elastase, and protease for 40 min at 36°C after a brief treatment with
Ca2+-free Tyrode solution for
5-10 min. The digested tissue specimens were placed in high
K+, low Cl
solution (Kraft-Brühe, K-B)
and gently triturated to produce a cell suspension. The
cells were kept at 4°C before they were used experimentally. In the
present study, only spindle-shaped cells showing regular spontaneous
activity were used. The morphological and electrophysiological
characteristics of the cells were similar to the characteristics of the
cells used in our previous studies (27, 28, 47, 48); these cells were
spindle shaped with no obvious or faint striations, showed regular
spontaneous beating when perfused with normal Tyrode solution, and
exhibited If
during hyperpolarizing voltage-clamp pulses (not shown) as well as a high input resistance (1.6-3.0 G
) at a holding potential of
60 mV. These characteristics suggest that these cells are SA
node pacemaker cells (29).
Electrophysiological measurements.
Membrane currents were recorded using the whole cell patch-clamp
technique at room temperature (24-26°C). Patch pipettes with a
resistance of 2-4 M were used. The pipette and cell capacitance (Cm) and the
series resistance (>80%) were electronically compensated, and the
current signal was filtered by a low-pass Bessel filter with a cut-off
frequency of 10 kHz (3 dB).
Cm, which is
proportional to the cell surface area, was obtained from the
capacitance compensation control of the amplifier after the whole cell
capacity current (in response to ±5-mV voltage-clamp pulses at
60 mV) had been eliminated. The accuracy of these values was
checked by integrating the area of the uncompensated capacity current
and fitting an exponential function to the decay of the uncompensated
capacity current.
Cm of the SA node
cells used in the present study ranged from 20.3 to 66.0 pF (mean ± SE = 44.2 ± 2.2 pF, n = 34). I4-AP was
obtained by subtracting currents in the presence of 4-AP from control
currents before 4-AP application. Ten millimolar 4-AP was usually used,
but one millimolar 4-AP was used in some experiments. I4-AP had
transient and sustained components
(Itrans and
Isus, respectively; see RESULTS):
I4-AP at the end
of 200-ms depolarizing clamp pulses was defined as
Isus, and the
difference of
I4-AP at its peak
and at the end of the pulse was defined as
Itrans. Because
Itrans did not
reach steady state at the end of 200-ms pulses (see Fig. 6),
Isus contains a
small fraction of the time-dependent component of
I4-AP. In these
experiments, 3 µM TTX and 300 µM Cd2+ were added to the perfusate
throughout the experiments to block Na+ current, L- and T-type
Ca2+ currents, and
Ca2+-activated transient outward
current (if present). In some experiments, 1 µM nifedipine (Sigma)
was used instead of 300 µM Cd2+
to block L-type Ca2+ current.
Membrane currents and membrane potential were recorded with an Axopatch
1C amplifier (Axon Instruments) and acquired on a personal computer
using a CED 1401 and Signal Averager or Voltage Clamp Software
(Cambridge Electronic Design) at a sampling rate of 4-5 kHz for
later analysis.
Solutions and drugs.
Normal Tyrode solution contained (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.3 NaH2PO4,
5 HEPES, and 10 glucose (pH 7.4).
Ca2+-free Tyrode solution was made
by simply omitting CaCl2 from the normal Tyrode solution. Enzyme solution consisted of
Ca2+-free Tyrode solution plus
collagenase (350-400 U/ml, Yakult), elastase (12-20 U/ml,
type IIA, Sigma), and protease (0.5 U/ml, type XIV, Sigma). K-B
solution contained (in mM) 20 taurine, 70 l-glutamic acid, 25 KCl, 10 KH2PO4,
3 MgCl2, 0.5 EGTA, 10 HEPES, and
10 glucose (pH 7.4). The pipette solution contained (in mM) 140 KCl, 5 MgATP, 0.4 Na2GTP, 11 HEPES, 1 CaCl2, and 11 EGTA (pCa 8, pH
7.2). The liquid junction potential between normal Tyrode solution and
the pipette solution was measured experimentally (4 mV) and was
corrected. A stock solution of 1 M 4-AP was prepared in
double-distilled water, and the pH was titrated to 7.4 with HCl. This
stock solution was added to the Tyrode solution to give a final
concentration of 4-AP.
Statistics. Data are presented as means ± SE (n = number of cells). All curve fitting was performed by a nonlinear least-squares method (see Figs. 4, 5C, and 6A) using Fig.P software (Fig.P Software). Statistical analysis was performed by a linear regression analysis (see Fig. 3) and Student's unpaired t-test (see Fig. 6, B and C). Values of P < 0.05 were considered to indicate significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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I4-AP in SA node cells.
In the majority of SA node cells studied (31 of 34 cells:
Cm 20.3-66.0
pF, mean ± SE = 44.2 ± 2.5 pF) membrane depolarization from a
holding potential of 60 or 80 mV activated transient outward current. Figure 1 shows
representative records of membrane current in response to 200-ms
depolarizing voltage-clamp pulses from 60 mV before (Fig.
1A, second
panel) and after (Fig.
1A, third
panel) the application of 10 mM 4-AP. Under control
conditions in the absence of 4-AP, the depolarization caused a rapid
activation of outward current that declined during the 200-ms pulse.
Application of 4-AP resulted in a reduction of the outward current
mainly at the beginning of the depolarizing pulse. After the
application of 10 mM 4-AP, however, a small, rapidly decreasing
transient outward current remained. A higher concentration (20 mM) of
4-AP did not abolish this small transient outward current. The nature of the remaining current was not investigated in detail in the present
study, but it is possible that an outward tail of
If at positive
potentials could be responsible for a part of this current. I4-AP was
obtained by subtraction (Fig. 1A,
fourth panel). There were two
components of
I4-AP; it showed
a transient component (Itrans) with
rapid activation and inactivation and a small sustained component
(Isus) at
the end of the 200-ms pulse. The inactivation kinetics
of Itrans
were faster at more positive potentials. Figure 1B shows current-voltage relationships
of Itrans and
Isus as well as
I4-AP. The
threshold potential for activation was roughly the same (40
mV) for Itrans
and Isus (Fig.
1B).
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80 mV was
essentially the same as that from the holding potential of 60
mV. However, there was a marked activation of
If at a holding potential of 80 mV. A decrease of
I4-AP as a result
of rundown of the channel was minimal and negligible (<2% in 3 min).
One millimolar (rather than 10 mM)
I4-AP was also
made up of transient and sustained components, and its peak amplitude
was 59.3 ± 4.1 (10 mV), 62.8 ± 2.9 (at +20 mV), and 65.0 ± 3.9% (+50 mV) (n = 4) of that
of 10 mM I4-AP.
Among 34 SA node cells tested, 31 cells showed
I4-AP with
Itrans and
Isus components
as described above. In the remaining three cells
(Cm 40.8, 43.0, and 63.6 pF), however, there was no substantial
Itrans in
I4-AP; in these
cells membrane depolarization activated only Isus.
Heterogeneity of I4-AP.
Figure 2 compares
I4-AP in SA node
cells of different sizes. Figure 2A
illustrates superimposed records of
I4-AP in response to depolarizing pulses to various potentials from a holding potential of 60 mV from small, medium-sized, and large SA node cells
(Cm 20.3, 34.5, and 63.4 pF, respectively). The current-voltage relationships of
Itrans and
Isus are shown in
Fig. 2, B and
C, respectively. The amplitude of peak
I4-AP and
Itrans increased
with the increase of cell capacitance (Fig. 2,
A and
B). The amplitude of
Isus at the end
of 200-ms depolarizing pulses was minimal in the small cell
(Cm 20.3 pF) but
increased with the increase of
Cm (Fig. 2,
A and
C). The threshold potential for
activation of
Itrans was
similar in the three cells (Fig.
2B), as was that of
Isus (Fig.
2C).
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0.26,
P > 0.1, n = 19). In contrast, there is a
significant correlation between the density of
Isus at the end
of the 200-ms depolarization to +50 mV and
Cm
(r = 0.75, P < 0.0005, n = 19; Fig.
3B); the density of
Isus was greater
in larger SA node cells. Total charge movement by
I4-AP per unit
Cm was also
compared in SA node cells of different sizes. Charge movement was
calculated in each cell by integrating
I4-AP during a
200-ms depolarizing voltage-clamp pulse to +50 mV. The normalized
charge movement carried by
I4-AP was greater
in larger cells; there is a significant correlation between the
normalized charge movement and
Cm
(r = 0.54, P < 0.02, n = 19, Fig. 3C). These
results suggest that
I4-AP may play a
greater role in larger cells.
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Voltage dependence of activation and inactivation of
I4-AP.
Figure 4 shows activation curves for
I4-AP. In these
experiments I4-AP
was recorded in the presence of 300 µM
Cd2+ to block
Ca2+ current, and it is reported
in rat ventricular cells that extracellular divalent cations, including
Cd2+, cause a rightward shift of
voltage dependence of
Ito (1, 19). The
effect of 300 µM Cd2+ on
I4-AP in rabbit
SA node cells was evaluated first. In these experiments, 1 µM
nifedipine, instead of Cd2+, was
used to block L-type Ca2+ current.
The results obtained from rabbit SA node cells were inconsistent with
those from rat ventricular cells: voltage dependence of
I4-AP activation
was similar both in the presence (n = 6) and in the absence (n = 4) of
Cd2+. Therefore, quantitative
analysis was performed on data obtained in the presence of 300 µM
Cd2+.
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80 mV, and plotted against the test potential (Fig.
4A). In Fig.
4A, data obtained from three SA node
cells of different sizes
(Cm 20.3, 34.5, and 63.4 pF) are shown. Each set of data was fitted by the Boltzmann
equation
![<IT>G</IT> = <IT>G</IT><SUB>max</SUB>/[1 + exp(<IT>V</IT><SUB>h</SUB> − <IT>V</IT>)/<IT>k</IT>]](/content/vol276/issue4/fulltext/H1295/img001.gif)
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40 mV (Fig. 4B).
The voltage dependence of steady-state inactivation of
I4-AP was
examined with a double-pulse protocol: a conditioning 1-s prepulse to
various potential levels (from 80 to +10 mV in 10-mV steps) was
followed by a test depolarizing pulse to +50 mV from a holding
potential of 60 mV (Fig. 5). Figure
5A shows superimposed traces of
I4-AP during the
test pulse obtained from three SA node cells of different sizes
(Cm 23.6, 47.1, and 63.4 pF). The peak value of the outward current during the test
pulse is plotted against the prepulse potential in Fig.
5B. In all three cells I4-AP during the
test pulse declined as the potential during the prepulse was made more
positive (Fig. 5, A and
B). However, in the large SA node
cell (Cm 63.4 pF), there was a substantial sustained outward current during the test
pulse that persisted even when the potential during the prepulse was
increased to +10 mV (Fig. 5, A and
B). The density of this sustained
current was lower in the medium-sized cell
(Cm 47.1 pF) and
minimal in the small cell (Cm 23.6 pF). This is consistent with the result that the density of Isus was
greater in larger SA node cells (Fig.
3B). The inactivating component of
I4-AP during the
test pulse was calculated by subtracting the noninactivating component
from the total
I4-AP during the test pulse (Fig. 5B). The
inactivating component of
I4-AP was normalized by the maximal current amplitude in each cell and plotted against the prepulse potential (Fig.
5C). In Fig.
5C, data were obtained from six SA
node cells of different sizes
(Cm
23.6-63.4 pF; mean ± SE = 44.0 ± 5.5 pF). There was no
significant cell size-dependent difference in the steady-state
inactivation curve, although a small variation was observed.
Inactivation was fully removed at potentials more negative than
70 mV, and the current was half-maximally inactivated around
30 mV. The combined data were fitted by the Boltzmann equation
![<IT>I</IT>/<IT>I</IT><SUB>max</SUB> = 1/[1 + exp(<IT>V</IT> − <IT>V</IT><SUB>h</SUB>)/<IT>k</IT>]](/content/vol276/issue4/fulltext/H1295/img002.gif)
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31.6
mV and k of 7.2 mV.
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Inactivation kinetics of I4-AP. The time course of the decline of I4-AP during depolarizing pulses was quantitatively analyzed in eight SA node cells (Fig. 6). The time-dependent decay of I4-AP at membrane potentials more positive than +10 mV was best fitted by a double-exponential function
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fast are the initial amplitude
and time constant of the "fast" phase of inactivation, and
Aslow and
slow are the corresponding
parameters for the "slow" phase.
A0 is a time-independent component. An example is shown in Fig.
6A; the top panel of Fig.
6A shows
I4-AP during a
depolarizing pulse to +40 mV and the bottom panel shows a
semilogarithmic plot of the current during the pulse. The filled
circles show the current minus the asymptote (i.e.,
A0). Most of
the points fall along a straight line with a of 91.4 ms. This
represents the slow phase of inactivation. However, at the start of the
pulse, the filled circles deviate from the straight line. The
difference between the filled circles and the line is plotted by the
open circles. The open circles fall along a straight line with a of
6.6 ms. This represents the fast phase of inactivation.
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fast and
slow show appreciable voltage
dependence at potentials between +10 and +60 mV (Fig. 6,
B and
C). The average fast and
slow values at +20 to +60 mV
were in the range of 7-9 ms and 60-90 ms, respectively, and
the values were somewhat longer (~14 and ~140 ms, respectively) at
+10 mV (Fig. 6, B and
C). The percentage of fast
inactivation was almost constant (0.65 to 0.75) at +10 to +60 mV.
Figure 6, B and
C, also shows that there was no
significant difference in fast
and slow between a group of small cells with a
Cm of <40 pF
(Cm 30.5 ± 3.5 pF, n = 4) and a group of large
cells with a Cm
of >40 pF (Cm
50.2 ± 4.0 pF, n = 4) at
potentials ranging from +10 to +60 mV.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrates that 4-AP-sensitive Ito exists in the majority (>90%) of rabbit SA node pacemaker cells. Although the presence of Ito in the SA node has been commented on by other investigators (15, 24, 25, 30, 37), the percentage of cells showing typical I4-AP in the present study (>90%) was much higher than in previous reports (15, 24, 25). Furthermore, Giles et al. (24, 25) reported that Ito was not recorded from rabbit SA node cells unless the cells were isolated from the most peripheral region of the node. In contrast, in the present study 4-AP-sensitive Ito could be recorded not only from large cells, which are presumably derived from a transitional or the peripheral region of the node, but also from small cells, which are presumably from the center of the node (27). There is no clear explanation for the discrepancy between previous reports and our present data, but it is possible that the presence of Ito in SA node cells was underestimated in previous studies, because in these studies 4-AP-sensitive current was not routinely recorded. In cell culture, Nathan (37) identified two morphologically distinct types of cells from the rabbit SA node (types I and II); the electrophysiological characteristics suggest that the type I cells possibly originated from the center and the type II cells were from the periphery. Both types of cells showed typical Ito (37).
The nature of I4-AP in SA node cells. The present study showed that I4-AP in most SA node cells is made up of two components, a component showing time-dependent inactivation (Itrans) and a sustained component (Isus). Ito and Ono (30) also reported the presence of time-dependent and sustained I4-AP in rabbit SA node cells. In the present study, in a small fraction of cells, there was no Itrans and only Isus existed. It is not known whether the two components are inactivating and noninactivating phases of current through a single type of 4-AP-sensitive channel or are separate currents through two types of 4-AP-sensitive channel.
The voltage dependence of activation and inactivation as well as inactivation kinetics of Itrans in SA node cells are similar to those of Ito in other types of cardiac cells (see below). Furthermore, in the present study 1 mM 4-AP blocked ~60-65% of I4-AP in rabbit SA node cells. Typical Ito in atrial and ventricular cells is reported to be blocked by 4-AP with an EC50 of 0.2-0.5 mM (12, 42), and therefore, the 4-AP dose dependence of I4-AP in SA node cells is comparable to that of Ito in atrial and ventricular cells. Kv4.2 and Kv4.3 channels are probably responsible for Ito (12, 17, 22), although it has also been suggested that Kv1.4 is responsible. We have recently cloned a Kv4.2 channel from a rabbit SA node cDNA library, and the channel when expressed in Xenopus oocytes is similar (but not identical) to I4-AP in the rabbit SA node (13). For example, for the cloned channel, Vh = +15 mV and k = 7 mV for activation and Vh =81 mV
and k = 16 mV for inactivation (13)
(see Table 1 for corresponding values for
I4-AP in rabbit SA node cells from present
study). The sensitivity of the cloned channel to 4-AP,
quinidine, and flecainide is less than that of the
Ito in rabbit SA
node cells (13, 33).
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60 mV (at which potential the 3 currents, if present, should be substantial), and this suggests that
IK,ATP,
IK1, and
IK,ACh (if
present) were not being affected. Furthermore,
IK,ATP should not
be present under normal conditions in the SA node, and
IK1 is absent
from the SA node (29).
In some cells (see, e.g., Fig. 6A),
there was an outward tail of
I4-AP current
after a pulse. Theoretically, this could be the result of block of
IK,r or
IK,s by 4-AP.
However, such outward tails of
I4-AP were not
seen in the majority of cells (see, e.g., Figs.
1A, fourth
panel, and 2A).
Furthermore, it is unlikely that IK,r was
substantially inhibited by 4-AP, because the current-voltage relationship of
Isus
(IK,r if
inhibited by 4-AP would contribute to
Isus) did not
show any evidence of inward rectification at positive potentials
(characteristic of
IK,r; Refs. 30,
45) in the present study (Figs. 1B and
2C).
IK,s is a small
current in the rabbit SA node; furthermore, it activates over a time
course of seconds, and, therefore, even if it is blocked by 4-AP
(whether it does is not known) it would not be expected to make a major contribution to
I4-AP during a
200-ms pulse typically used in the present study.
Under control conditions, outward tail current (residual
Ito,
IK,r,
IK,s) is
expected after a depolarizing pulse. However, under control conditions,
in all cells, there was an inward tail current after a pulse (see,
e.g., Fig. 1A). The inward tails
could be the result of activation of inward currents in response to repolarization (e.g., inward
Na+/Ca2+
exchange current). Alternatively, the inward tails could be explained by an outward current with a reversal potential of less than 60 mV. However, the reversal potential of
Ito, at least,
was more negative than 60 mV (data not shown).
4-AP-sensitive Ito in SA node cells compared with that
in other cardiac tissues.
The inactivation of
I4-AP in rabbit
SA node cells during depolarizing pulses was best approximated by a
double-exponential function. Inactivation of
Ito in other
cardiac tissues has also been reported to be best fit by a
double-exponential function in rabbit atrial and ventricular cells as
well as atrioventricular node (11, 26, 36, 49). The inactivation
kinetics of I4-AP in SA node cells are comparable to those reported for rabbit atrial and
ventricular cells (26, 49) as well as rat and ferret ventricular cells
(3, 10). However, faster or slower inactivation has also been reported
in rabbit atrial cells (11, 49) (Table 1). The transient component of
I4-AP
(Itrans) in
rabbit SA node cells was half-maximally inactivated at 31.5 mV
(with a slope factor of 7.2 mV) in the present study (Table 1). These
values are similar to those reported for
Ito in rabbit
atrial and crista terminalis cells (11, 24, 49) as well as rabbit, rat,
and canine ventricular cells (3, 26, 34) (Table 1). In contrast, more
positive Vh
values (approximately 14 mV) have been reported for
Ito in human
atrial (42) and ferret ventricular (10) cells (Table 1). The activation
curve for I4-AP
obtained from SA node cells in the present study showed a weaker
voltage dependence (k 15.0 mV) and a
more positive midpoint
(Vh +19.3 mV)
than that for Ito
in other cardiac tissues (11, 24, 42) with the exception of rabbit and
ferret ventricular cells (10, 26) (Table 1). This apparent difference
could be the result of the different methods used to determine the
activation parameter; in the present study it was calculated as the
chord conductance from peak
I4-AP during
depolarizing test pulses, whereas in other studies it was obtained from
the amplitude of tail current in response to repolarization after
depolarizing test pulses. An apparent more shallow voltage dependence
in association with an apparent more positive
Vh can arise when
using the chord conductance method if the instantaneous current-voltage
relationship is nonlinear (43).
Role of I4-AP in action potential in SA node. In atrial, Purkinje, and ventricular cells, block of Ito by 4-AP slows the initial phase of repolarization (phase 1), abolishes the notch, elevates the plateau, and prolongs the action potential (16, 23, 24, 26). In canine atrial cells, block of IK,ur by 4-AP results in a prolongation of the action potential (49). We have studied the effects of 4-AP in small ball-like preparations of tissue from different regions of the rabbit SA node (6, 7). In small balls of tissue from the periphery of the SA node, 4-AP had all of these actions: it slowed the initial phase of repolarization and abolished the notch if present, elevated the plateau, and prolonged the action potential (6, 7). In small balls of tissue from the center of the SA node, the effects of 4-AP were smaller: a smaller elevation of the plateau and a smaller increase in action potential duration (in the center, the action potential does not have a notch) (6, 7). The dose dependence of the 4-AP-induced action potential prolongation in the small balls of tissue was similar to that of I4-AP in the present study and Ito in other studies (6, 7).
The greater effects of 4-AP on tissue from the periphery of the SA node than on tissue from the center can be explained by the results from the present study. In the periphery of the SA node the cells are larger than those in the center (4). In the present study, the density of Isus was greater in larger cells and the charge carried by I4-AP normalized for Cm was greater in larger cells (Fig. 3). The role of I4-AP is expected, therefore, to be greater in the larger cells from the periphery of the SA node. There is a second reason why I4-AP is expected to play a more important role in the periphery of the SA node compared with the center. In the periphery of the SA node, the voltage-dependent inactivation of Itrans is expected to be less (because the diastolic potential is more negative in the periphery of the SA node), and thus greater Ito is expected to be activated during the action potential. In conclusion, I4-AP is present in the majority of SA node cells but is less important in smaller cells as a result of a smaller density of the sustained component of I4-AP. The heterogeneity of I4-AP in SA node cells helps explain why the action potential is longer in the center of the SA node than in the periphery, a protective mechanism to help prevent reentry.| |
ACKNOWLEDGEMENTS |
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This work was supported by the British Heart Foundation, the Ministry of Education, Science, and Culture of Japan, and the Japan Society for the Promotion of Science.
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FOOTNOTES |
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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: H. Honjo, Dept. of Humoral Regulation, Research Institute of Environmental Medicine, Nagoya Univ., Nagoya 464-8601, Japan.
Received 14 May 1998; accepted in final form 3 December 1998.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Agus, Z. S.,
I. D. Dukes,
and
M. Morad.
Divalent cations modulate the transient outward current in rat ventricular myocytes.
Am. J. Physiol.
261 (Cell Physiol. 30):
C310-C318,
1991
2.
Antzelevitch, C.,
S. Sicouri,
S. H. Litovsky,
A. Lukas,
S. C. Krishnan,
J. M. Di Diego,
G. A. Gintant,
and
D.-W. Liu.
Heterogeneity within the ventricular wall. Electrophysiology and pharmacology of epicardial, endocardial, and M cells.
Circ. Res.
69:
1427-1449,
1991
3.
Apkon, M.,
and
J. M. Nerbonne.
Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes.
J. Gen. Physiol.
97:
973-1011,
1991
4.
Bleeker, W. K.,
A. J. C. Mackaay,
M. Masson-Pévet,
L. N. Bouman,
and
A. E. Becker.
Functional and morphological organization of the rabbit sinus node.
Circ. Res.
46:
11-22,
1980
5.
Boyett, M. R.,
I. Kodama,
H. Honjo,
M. Yamamoto,
M. Uzzerman,
M. R. Nikmaram,
and
R. Niwa.
A second line of defence for the sinoatrial node of the rabbit (Abstract).
J. Physiol (Lond.)
504:
70P-71P,
1997.
6.
Boyett, M. R.,
I. Kodama,
R. Suzuki,
and
H. Honjo.
A 4-aminopyridine-sensitive current controls action potential duration in the rabbit sinoatrial node (Abstract).
J. Physiol. (Lond.)
497:
44P-45P,
1996.
7.
Boyett, M. R.,
H. Honjo,
M. Yamamoto,
M. R. Nikmaram,
R. Niwa,
and
I. Kodama.
Regional differences in effects of 4-aminopyridine within the sinoatrial node.
Am. J. Physiol.
275 (Heart Circ. Physiol. 44):
H1158-H1168,
1998
8.
Boyle, W. A.,
and
J. M. Nerbonne.
A novel type of depolarization-activated K+ current in isolated adult rat atrial myocytes.
Am. J. Physiol.
260 (Heart Circ. Physiol. 29):
H1236-H1247,
1991
9.
Campbell, D. L.,
R. L. Rasmusson,
M. B. Comer,
and
H. C. Strauss.
The cardiac calcium-independent transient outward potassium current: kinetics, molecular properties, and role in ventricular repolarization.
In: Cardiac Electrophysiology: From Cell to Bedside, edited by D. P. Zipes,
and J. Jalife. Philadelphia, PA: Saunders, 1995, p. 83-96.
10.
Campbell, D. L.,
R. L. Rasmusson,
Y. Qu,
and
H. C. Strauss.
The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis.
J. Gen. Physiol.
101:
571-601,
1993
11.
Clark, R. B.,
W. R. Giles,
and
Y. Imaizumi.
Properties of the transient outward current in rabbit atrial cells.
J. Physiol. (Lond.)
405:
147-168,
1988
12.
Comer, M. B.,
D. L. Campbell,
R. L. Rasmusson,
D. R. Lamson,
M. J. Z. Y. Morales,
and
H. C. Strauss.
Cloning and characterization of an Ito-like potassium channel from ferret ventricle.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H1383-H1395,
1994
13.
Conley, E.,
H. O'Beirne,
J. M. Owen,
P. M. Hopkins,
M. R. Boyett,
and
J. C. Hancox.
Cloning and expression of a Kv4.2 channel cloned from the rabbit sinoatrial node (Abstract).
J. Physiol. (Lond.)
511:
149P-150P,
1998.
14.
Davies, N. W.,
A. I. Pettit,
R. Agarwal,
and
N. B. Standen.
The flickery block of ATP-dependent potassium channels of skeletal muscle by internal 4-aminopyridine.
Pflügers Arch.
419:
25-31,
1991[Medline].
15.
Denyer, J. C.,
and
H. F. Brown.
Rabbit sino-atrial node cells: isolation and electrophysiological properties.
J. Physiol. (Lond.)
428:
405-424,
1990
16.
Di Diego, J. M.,
Z.-Q. Sun,
and
C. Antzelevitch.
Ito and action potential notch are smaller in left vs. right canine ventricular epicardium.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H548-H561,
1996
17.
Dixon, J. E.,
W. Shi,
H.-S. Wang,
C. McDonald,
H. Yu,
R. S. Wymore,
I. S. Cohen,
and
D. McKinnon.
Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current.
Circ. Res.
79:
659-668,
1996
18.
Dobrzynski, H.,
Z. Henderson,
Y. Takagishi,
S. Rothery,
N. J. Severs,
H. Honjo,
M. M. Tamkun,
I. Kodama,
and
M. R. Boyett.
Expression and localization of the Kv1.5 K+ channel in the guinea-pig sinoatrial node (Abstract).
J. Physiol. (Lond.)
506:
54P,
1998.
19.
Dukes, I. D.,
and
M. Morad.
The transient K+ current in rat ventricular myocytes: evaluation of its Ca2+ and Na+ dependence.
J. Physiol. (Lond.)
435:
395-420,
1991
20.
Fedida, D.,
B. Wible,
Z. Wang,
B. Fermini,
F. Faust,
S. Nattel,
and
A. M. Brown.
Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.
Circ. Res.
73:
210-216,
1993[Abstract].
21.
Feng, J.,
B. Wible,
G.-R. Li,
Z. Wang,
and
S. Nattel.
Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes.
Circ. Res.
80:
572-579,
1997
22.
Fiset, C.,
R. B. Clark,
Y. Shimoni,
and
W. R. Giles.
Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle.
J. Physiol (Lond.)
500:
51-64,
1997[Medline].
23.
Giles, W. R.,
and
Y. Imaizumi.
Comparison of potassium currents in rabbit atrial and ventricular cells.
J. Physiol. (Lond.)
405:
123-145,
1988
24.
Giles, W. R.,
and
A. C. G. van Ginneken.
A transient outward current in isolated cells from the crista terminalis of rabbit heart.
J. Physiol. (Lond.)
368:
243-264,
1985
25.
Giles, W.,
A. van Ginneken,
and
E. F. Shibata.
Ionic currents underlying cardiac pacemaker activity: a summary of voltage clamp data from single cells.
In: Cardiac Muscle: the Regulation of Excitation and Contraction, edited by R. D. Nathan. New York: Academic, 1986, p. 1-27.
26.
Hiraoka, M.,
and
S. Kawano.
Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes.
J. Physiol. (Lond.)
410:
187-212,
1989
27.
Honjo, H.,
M. R. Boyett,
I. Kodama,
and
J. Toyama.
Correlation between electrical activity and the size of rabbit sinoatrial node cells.
J. Physiol. (Lond.)
496:
795-808,
1996[Medline].
28.
Honjo, H.,
I. Kodama,
W.-J. Zang,
and
M. R. Boyett.
Desensitization to acetylcholine in single sinoatrial node cells isolated from the rabbit heart.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H1779-H1789,
1992
29.
Irisawa, H.,
H. F. Brown,
and
W. Giles.
Cardiac pacemaking in the sino-atrial node.
Physiol. Rev.
73:
197-227,
1993
30.
Ito, H.,
and
K. Ono.
A rapidly activating delayed rectifier K+ channel in rabbit sinoatrial node cells.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H443-H452,
1995
31.
Kodama, I.,
and
M. R. Boyett.
Regional differences in the electrical activity of the rabbit sinus node.
Pflügers Arch.
404:
214-226,
1985[Medline].
32.
Kodama, I.,
M. R. Nikmaram,
M. R. Boyett,
R. Suzuki,
H. Honjo,
and
J. M. Owen.
Regional differences in the role of the Ca2+ and Na+ currents in pacemaker activity in the sinoatrial node.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H2793-H2806,
1997
33.
Lei, M.,
and
M. R. Boyett.
Inhibition of transient outward current, ito, by flecainide and quinidine in rabbit isolated sinoatrial node cells.
J. Physiol. (Lond.)
511:
78P-79P,
1998.
34.
Liu, D.-W.,
G. A. Gintant,
and
C. Antzelevitch.
Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle.
Circ. Res.
72:
671-687,
1993
35.
Masson-Pévet, M. A.,
W. K. Bleeker,
E. Besselsen,
B. W. Treytel,
H. J. Jongsma,
and
L. N. Bouman.
Pacemaker cell types in the rabbit sinus node: a correlative ultrastructural and electrophysiological study.
J. Mol. Cell. Cardiol.
16:
53-63,
1984[Medline].
36.
Nakayama, T.,
and
H. Irisawa.
Transient outward current carried by potassium and sodium in quiescent atrioventricular node cells of rabbits.
Circ. Res.
57:
65-73,
1985
37.
Nathan, R. D.
Two electrophysiologically distinct types of cultured pacemaker cells from rabbit sinoatrial node.
Am. J. Physiol.
250 (Heart Circ. Physiol. 19):
H325-H329,
1986
38.
Navarro-Polanco, R. A.,
and
J. A. Sanchez-Chapula.
4-Aminopyridine activates potassium currents by activation of a muscarinic receptor in feline atrial myocytes.
J. Physiol. (Lond.)
498:
663-678,
1997[Medline].
39.
Nikmaram, M. R.,
M. R. Boyett,
I. Kodama,
R. Suzuki,
and
H. Honjo.
Variation in effects of Cs+, UL-FS-49, and ZD-7288 within sinoatrial node.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H2782-H2792,
1997
40.
Opthof, T.,
A. C. G. van Ginneken,
L. N. Bouman,
and
H. J. Jongsma.
The intrinsic cycle length in small pieces isolated from the rabbit sinoatrial node.
J. Mol. Cell. Cardiol.
19:
923-934,
1987[Medline].
41.
Paulmichl, M.,
P. Nasmith,
R. Hellniss,
K. Reed,
W. A. Boyle,
J. M. Nerbonne,
E. G. Peralta,
and
D. E. Clapham.
Cloning and expression of a rat cardiac delayed rectifier potassium channel.
Proc. Natl. Acad. Sci. USA
88:
7892-7895,
1991
42.
Shibata, E. F.,
T. Drury,
H. Refsum,
V. Aldrete,
and
W. Giles.
Contributions of a transient outward current to repolarization in human atrium.
Am. J. Physiol.
257 (Heart Circ. Physiol. 26):
H1773-H1781,
1989
43.
Snyders, D. J.,
M. M. Tamkun,
and
P. B. Bennett.
A rapidly activating and slowly inactivating potassium channel cloned from human heart: functional analysis after stable mammalian cell culture expression.
J. Gen. Physiol.
101:
513-543,
1993
44.
van Bogaert, P.-P.,
and
D. J. Snyders.
Effects of 4-aminopyridine on inward rectifying and pacemaker currents of cardiac Purkinje fibres.
Pflügers Arch.
394:
230-238,
1982[Medline].
45.
Verheijck, E. E.,
A. C. G. van Ginneken,
J. Bourier,
and
L. N. Bouman.
Effects of delayed rectifier current blockade by E-4031 on impulse generation in single sinoatrial nodal myocytes of the rabbit.
Circ. Res.
76:
607-615,
1995
46.
Wang, Z.,
B. Fermini,
and
S. Nattel.
Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents.
Circ. Res.
73:
1061-1076,
1993
47.
Watanabe, E.-I.,
H. Honjo,
T. Anno,
M. R. Boyett,
I. Kodama,
and
J. Toyama.
Modulation of pacemaker activity of sinoatrial node cells by electrical load imposed by an atrial cell model.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H1735-H1742,
1995
48.
Watanabe, E.-I.,
H. Honjo,
M. R. Boyett,
I. Kodama,
and
J. Toyama.
Inactivation of the calcium current is involved in overdrive suppression of rabbit sinoatrial node cells.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H2097-H2107,
1996
49.
Yamashita, T.,
T. Nakajima,
H. Hazama,
E. Hamada,
Y. Murakawa,
K. Sawada,
and
M. Omata.
Regional differences in transient outward current density and inhomogeneities of repolarization in rabbit right atrium.
Circulation
92:
3061-3069,
1995
50.
Yue, L.,
J. Feng,
G.-R. Li,
and
S. Nattel.
Characterization of an ultrarapid delayed rectifier potassium channel in canine atrial repolarization.
J. Physiol. (Lond.)
496:
647-662,
1996[Medline].
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