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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:681–687, 2002
Vol. 300, No. 1
4139/958717
Printed in U.S.A.
The Role of Muscarinic K⫹ Channels in the Negative
Chronotropic Effect of a Muscarinic Agonist
MITSUHIKO YAMADA
Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan
Received May 15, 2001; accepted September 15, 2001
This paper is available online at http://jpet.aspetjournals.org
Parasympathetic regulation of the heart rate is mediated
by acetylcholine (ACh) (Loewi and Navratil, 1926; Löffelholz
and Pappano, 1985). ACh activates M2 muscarinic receptors
and the heterotrimeric Gi-2, Gi-3 and/or Go proteins in cardiac
myocytes (Luetje et al., 1988). The ␣ subunits of the Gi
proteins inhibit the adenylyl cyclase (Sunahara et al., 1996),
whereas the ␤␥ subunits of the Gi proteins directly activate
the inwardly rectifying muscarinic K⫹ (KACh) channel in
sinoatrial (SA), atrial, and atrioventricular (AV) nodes, and
Purkinje myocytes (Logothetis et al., 1987; Sowell et al.,
1997; Yamada et al., 1998).
KACh channels cause the negative chronotropic effect by
hyperpolarizing SA node cells (del Castillo and Katz, 1955;
Hutter and Trautwein, 1955; Noma and Trautwein, 1978;
Sakmann et al., 1983). On the other hand, the suppression of
adenylyl cyclase decreases the heart rate by inhibiting such
inward currents in SA node cells as the hyperpolarizationactivated (If) current, the L-type calcium current, and the
sustained inward current, all of which are activated by cAMP
This work was supported by Grant 12670715 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, research grants for
cardiovascular disease (11C-1 and 12C-7) from the Ministry of Health, Labor
and Welfare of Japan, and the Program for Promotion of Fundamental Studies
in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.
effect of CCh. The KACh channel in atrial myocytes was also
activated by this range of concentrations of CCh as measured
with the patch-clamp method. The tertiapin-insensitive component was induced by 1 to 300 nM CCh in a concentrationdependent manner and accounted for ⬃25% of the maximum
effect of CCh. The sinus rate in the presence of 1 ␮M CCh and
300 nM tertiapin was similar to that in the presence of 2 mM
CsCl, a blocker of the hyperpolarization-activated If current.
Furthermore, no tertiapin-insensitive component existed in the
presence of 2 mM CsCl. Therefore, the negative chronotropic
effect of ⱖ300 nM CCh is mainly mediated by KACh channels,
whereas that of ⱕ100 nM CCh may result from suppression of
the If current.
or cAMP-dependent protein kinase, especially in the presence of ␤-adrenergic stimulation (Irisawa et al., 1993). It is
unknown to what extent each of the signal transduction
pathways is responsible for the negative chronotropic effect
of ACh. This is partly because there have been no selective
inhibitors of every signal transduction pathway evoked by
muscarinic stimulation.
A peptidyl honeybee toxin tertiapin was recently found to
selectively inhibit KACh channels in cardiac myocytes (Jin
and Lu, 1998; Drici et al., 2000; Kitamura et al., 2000).
Tertiapin fully inhibits ACh-induced whole-cell KACh channel currents in rabbit atrial myocytes in a concentrationdependent manner in a range of concentrations between 10
pM and 10 ␮M through ⬃1:1 stoichiometry (Kitamura et al.,
2000). The half-maximum IC50 of tertiapin is ⬃8 nM independent of the membrane potential. Tertiapin also inhibits
the KACh channel current activated by a hydrolysis-resistant
GTP analog applied to the cytosol, indicating that the effect
of tertiapin is not mediated by a muscarinic receptor. At the
single-channel level, tertiapin inhibits the KACh channel
from the outside of the membrane by reducing the NPo (N is
the number of functional channels, and the Po is the open
probability of each channel) without affecting the singlechannel conductance or fast open-close kinetics. Thus, tertiapin is a slow blocker of the KACh channel.
ABBREVIATIONS: ACh, acetylcholine; CCh, carbachol; If currents, hyperpolarization-activated currents; SA, sinoatrial; AV, atrioventricular; KACH,
muscarinic K⫹.
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ABSTRACT
Acetylcholine causes bradycardia through M2 muscarinic receptors in sinoatrial node cells. I examined with electrocardiogram how the muscarinic K⫹ (KACh) channel participates in the
sinus bradycardia induced by a muscarinic agonist in the Langendorff preparation of rabbit hearts. In the presence of 100 nM
propranolol, 1 nM to 10 ␮M carbachol (CCh) induced sinus
bradycardia in a concentration-dependent manner. Tertiapin
(100 or 300 nM), which selectively blocks KACh channels in
cardiac myocytes, significantly inhibited the effect of ⱖ300 nM
but not ⱕ100 nM CCh. The effect of CCh was divided into
tertiapin-sensitive and -insensitive components. The former
component was induced by ⬎100 nM CCh in a concentrationdependent manner and accounted for ⬃75% of the maximum
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In this study, I examined the effect of tertiapin on the sinus
bradycardia caused by carbachol (CCh) in the presence of a
␤-adrenergic blocker in the Langendorff preparation of rabbit
hearts with ECG. I divided the effect of CCh into the tertiapin-sensitive (i.e., KACh current-dependent) and -insensitive
(i.e., KACh current-independent) components. The tertiapinsensitive component was ⬃10 times less sensitive to CCh but
⬃3 times more effective than the tertiapin-insensitive component in decreasing the sinus rate. The tertiapin-insensitive
component seemed to be mediated by inhibition of If currents
probably through suppression of the basal cAMP level.
Materials and Methods
Results
Concentration-Dependent Effect of Carbachol on
the Sinus Rate of a Rabbit Heart.I first examined the
concentration-dependent effect of CCh on the sinus rate in
the Langendorff preparation of a rabbit heart by monitoring
the ECG (Fig. 1). At the beginning of each experiment, a
␤-adrenergic blocker propranolol (100 nM) was applied to
eliminate the effect of residual catecholamine in the heart
(Fig. 1A). Propranolol (100 nM) decreased the sinus rate to
85.7 ⫾ 1.9% of the control on average and increased the PQ
interval significantly (Table 1). After the sinus rate became
stable (from recordings 1 and 2), CCh was applied in a cumulative manner. CCh decreased the sinus rate in a concentration-dependent manner in the range of concentration between 10 nM and 1 ␮M (recordings 3– 6). CCh caused sinus
arrest in eight of nine hearts at 1 ␮M and in all four hearts
at 10 ␮M. The PQ interval slightly but significantly increased
from 51.12 ⫾ 2.78 ms in controls to 59.22 ⫾ 1.98 ms in the
presence of 300 nM CCh (n ⫽ 9). However, CCh did not cause
the second or third degree of AV conduction block before
inducing the sinus arrest in 39 of 40 hearts examined. Thus,
I did not further analyze the effect of CCh and/or tertiapin on
the AV conduction.
As shown in Fig. 1, tertiapin (100 nM) applied in the
presence of 1 ␮M CCh, reversed the sinus rate to 59% of the
controls (recording 7). From a previous study (Kitamura et
al., 2000), 100 nM tertiapin inhibits ACh (1 ␮M) induced
KACh channel currents by ⬃89% in rabbit atrial myocytes.
Thus, KACh channels might not be entirely responsible for
the bradycardiac effect of 1 ␮M CCh. Atropine (10 ␮M) applied at the end of the experiment almost completely restored
the sinus rate to the value before application of CCh (recording 8).
Effect of Tertiapin on the Sinus Bradycardia Induced by Lower Concentrations of Carbachol. I further
examined the effect of tertiapin in the presence of lower
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Langendorff Preparation of Rabbit Hearts. Male JapaneseWhite rabbits weighing 1.5 to 1.7 kg were injected with heparin
sodium (200 U/kg b.wt.) through an ear vein. Approximately 20 min
later, the rabbits were anesthetized with pentobarbital sodium or
thiopental sodium (30 mg/kg b.wt.) injected through a vein in another ear. As soon as the rabbits lost a nociceptive response, the
heart was quickly removed and immediately reperfused with oxygenated modified Tyrode’s solution (for composition, see below) at
37°C in the Langendorff apparatus in a retrograde manner.
Recording Electrocardiogram from Isolated Rabbit Hearts.
The isolated heart was hung over a glass funnel ⬃5 cm in diameter
in such a way that its apex lightly touched the inner surface of the
lowest and narrowest part of the funnel. A few sheets of thin paper
(Kimwipes; Kimberly-Clark Co., Tokyo, Japan) filled the space between the ventricular wall and the inner surface of the funnel. After
the paper was wetted with the cardiac effluent (i.e., the modified
Tyrode’s solution), the standard bipolar lead ECG was recorded from
the electrodes connected to the paper with a conventional ECG
recorder (Cardiofax; Nihon Kohden Co. Ltd., Tokyo, Japan).
After the heart exhibited a stable sinus rhythm, various drugs
were applied to the heart through coronary arteries by continuously
monitoring ECG. ECG was recorded on paper once every 1 to 5 min,
at which the PP, PQ, QRS, and QT intervals were measured. The
sinus rate was calculated as follows: sinus rate (min⫺1) ⫽ 60n/x,
where x is the duration of n PP intervals in seconds, and n is an
integer between 3 and 10. The QT intervals were corrected with
Bazett’s formula (QTc ⫽ QT/RR1/2).
Isolation of Atrial Myocytes and Patch-Clamp Study. The
methods of isolation of atrial myocytes and patch-clamp experiments
were precisely described in a previous paper (Kitamura et al., 2000).
Briefly, the heart in the Langendorff apparatus was perfused with
150 ml of the nominally calcium-free Tyrode’s solution (for composition, see below) and then that with 0.04% (w/v) collagenase (Yakult
Pharmaceutical, Tokyo, Japan) for 10 to 15 min. The digested heart
was stored in the KB solution (for composition, see below) at 4°C.
Atrial myocytes were isolated from the digested atrial tissue in the
modified Tyrode’s solution in a recording chamber set on an inverted
microscope (Axiovert 135; Carl Zeiss, Jana, Germany).
The ion channel currents were measured from the myocytes in the
whole-cell configuration of the patch-clamp method (Hamill et al.,
1981) with the patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA). The patch pipette was filled with the pipette
solution (for composition, see below). The cells were continuously
perfused with the modified Tyrode’s solution at 37°C. Throughout
experiments, ion channel currents were recorded for off-line analyses
on videocassette tapes through a pulse code modulation recorder
(VR10B; InstruTECH Corp., Port Washington, NY). The data were
subsequently reproduced by the same pulse code modulation recorder, filtered at 2 kHz (⫺3 dB) by a Bessel filter (Multifunction
Filter 3611; NF Electronic Instruments, Yokohama, Japan), digitized at 5 kHz with an AD converter (ITC16I; InstruTECH Corp.),
and analyzed with a computer and commercially available software
(Patch Analyst Pro; MT Corp., Hyogo, Japan).
Solutions and Chemicals. The modified Tyrode’s solution contained 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2,
5.5 mM glucose, and 5.5 mM HEPES (pH adjusted to 7.4 with
NaOH). For the nominally calcium-free Tyrode’s solution, CaCl2 was
simply omitted from the modified Tyrode’s solution. The KB solution
contained 10 mM taurine, 10 mM oxalic acid, 70 mM glutamic acid,
25 mM KCl, 10 mM KH2PO4, 11 mM glucose, 0.5 mM EGTA, and 10
mM HEPES (pH adjusted to 7.3 with KOH). The pipette solution
contained 140 mM KCl, 5 mM MgCl2, 5 mM EGTA, 3 mM ATP, 0.1
mM GTP, and 5 mM HEPES (pH adjusted to 7.4 with KOH). The
calculated free Mg2⫹ concentration in this solution was 1.6 mM. In
the ECG experiments, 5 mM ascorbic acid was added to the modified
Tyrode’s solution (pH readjusted to 7.4 with NaOH) to prevent oxygenation of tertiapin. Ascorbic acid (5 mM) alone did not affect the
ECG. Tertiapin was purchased from Peptide Institute Inc. (Osaka,
Japan). Tertiapin was dissolved in distilled water at 100 ␮M immediately before use, further diluted to desired concentrations with the
oxygenated modified Tyrode’s solution with ascorbic acid, and immediately applied to the hearts. CCh and propranolol were purchased
from Sigma Chemical Co. (St. Louis, MO). Atropine was from Wako
Pure Chemical (Osaka, Japan).
Statistical Analysis. All statistical values are indicated as
mean ⫾ S.E. The statistical difference was evaluated by Student’s t
test. For multiple comparison between pairs, difference was assessed
with analysis of variance and the Bonferroni method. A value of p ⬍
0.05 was considered statistically significant.
The Role of Muscarinic Kⴙ Channel in Sinus Bradycardia
683
TABLE 1
Parameters in electrocardiogram of rabbit hearts
The numbers indicate the mean ⫾ S.E.
Sinus Rate
PQ
QRS
min⫺1
Control
Propranol
Propranol ⫹ CsCl
193.4 ⫾ 4.0
164.8 ⫾ 3.9*
135.8 ⫾ 9.0†
QT
QTc
n
183.8 ⫾ 3.4
200.7 ⫾ 3.9*
220.9 ⫾ 8.6†
328.1 ⫾ 5.1
330.1 ⫾ 4.2
327.9 ⫾ 9.8
37
37
12
ms
49.0 ⫾ 1.0
52.3 ⫾ 0.9*
52.4 ⫾ 1.2*
52.3 ⫾ 1.1
52.6 ⫾ 1.0
49.2 ⫾ 1.8
Propranol, in the presence of propranolol (100 nM); Propranol ⫹ CsCl, in the presence of propranolol (100 nM) and CsCl (2 mM); n, the number of observations.
* p ⬍ 0.025 vs. control; † p ⬍ 0.025 vs. propranol.
concentrations of CCh (Fig. 2). Tertiapin (100 nM) reversed
the negative chronotropic effect of 300 nM CCh by ⬃41%
(Fig. 2A). The same concentration of tertiapin, however, antagonized the effect of 100 nM CCh only by ⬃16% (Fig. 2B)
and barely antagonized that of 30 nM CCh (Fig. 2C). In the
absence of CCh, 100 or 300 nM tertiapin did not significantly
alter the sinus rate, although it reduced the effect of subsequently applied 1 ␮M CCh by 73% (Fig. 2D and data not
shown). Therefore, the effect of tertiapin was less as the
concentration of CCh decreased.
Figure 3A summarizes the concentration-response relationship of the CCh-induced sinus bradycardia in the absence
and presence of 100 or 300 nM of tertiapin. Under control
conditions, CCh decreased the sinus rate in a concentrationdependent manner in the range of concentrations between 1
nM and 10 ␮M with IC50 of ⬃300 nM. Tertiapin (100 nM)
significantly increased the sinus rate only in the presence of
300 nM and 1 ␮M CCh. Tertiapin (300 nM), which inhibits
ACh (1 ␮M) induced KACh channel currents by ⬃95% (Kitamura et al., 2000), further increased the sinus rate in the
presence of 1 ␮M CCh. Tertiapin (300 nM) also significantly
increased the sinus rate in the presence of 10 ␮M CCh.
The inset of Fig. 3A depicts the negative chronotropic effect
of CCh, and its tertiapin-sensitive and -insensitive components as a percentage of the maximum effect of CCh. The
tertiapin-sensitive component exhibited a steep concentration-response curve in the range of concentrations between
100 nM and 1 ␮M and accounted for ⬃75% of the maximum
effect of CCh. In contrast, the tertiapin-insensitive component showed a less steep concentration-response curve in the
range of concentrations between 1 and 300 nM and accounted
for ⬃25% of the maximum effect of CCh.
Concentration-Dependent Effect of Carbachol on
the KACh Channel. I next examined the effect of CCh on
KACh channel currents in atrial myocytes (Fig. 3B). In the
whole-cell configuration of the patch-clamp method, CCh
(⬎10 nM) induced KACh channel currents with characteristic
slow relaxation (Fig. 3B, a). The current flowed inward at
⫺100 mV and outward at ⫺40 and ⫹10 mV. CCh induced
KACh channel currents in a concentration-dependent manner
in the range of concentrations between 1 nM and 10 ␮M (Fig.
3B, b). The line indicates the fit of the results with the Hill
equation (see figure legend), which provided an estimate of
the half-maximum effective concentration of 435 nM and the
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Fig. 1. Carbachol-induced sinus bradycardia and effect of tertiapin in an isolated rabbit heart. A, in the Langendorff preparation of a rabbit heart,
drugs indicated above the graph were applied through coronary arteries in a retrograde manner. Throughout the experiment, ECG was monitored.
The measured sinus rate (ordinate) is plotted against the experimental time in minutes (abscissa). The numbers in parentheses correspond to those
in B. B, representative ECG recordings obtained at the timings indicated by numbers in A. Note a single ventricular escaped beat in (6).
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Yamada
Hill coefficient of 1.52. This curve is replotted in the inset of
Fig. 3A. This curve was very similar to that of the tertiapinsensitive component in a range of CCh concentrations of ⱕ1
␮M. However, in the presence of ⬎1 ␮M CCh, the KACh
current increased in a concentration-dependent manner,
whereas that for the tertiapin-sensitive component reached a
plateau because ⬎1 ␮M CCh caused sinus arrest.
Effect of CsCl on the Sinus Rate and Carbachol-Induced Bradycardia. The tertiapin-insensitive component
may be mediated by suppression of the basal cAMP level. It
is reported that ACh inhibits If currents at lower concentrations than activating KACh channels by reducing the basal
cAMP level (DiFrancesco and Tromba, 1988; DiFrancesco et
al., 1989). Thus, I examined the effect of CsCl on the heart
rate because CsCl is reported to selectively inhibit If currents
at 1 to 2 mM in rabbit SA node cells (Denyer and Brown,
1990). As shown in Fig. 4A, 2 mM CsCl reduced the sinus rate
to ⬃66% of the control (from recordings 1 and 2), indicating
that If currents contribute to but are not essential for sinus
automaticity (Irisawa et al., 1993). CsCl (2 mM) did not
significantly change the QRS or QTc intervals (Table 1),
indicating that it did not block the voltage-dependent or
inwardly rectifying K⫹ currents in ventricular and Purkinje
myocytes at least effectively in the physiological range of
membrane potentials. CCh (1 ␮M) applied after washout of
CsCl caused sinus arrest (recording 3), and 300 nM tertiapin
applied under this condition increased the sinus rate to the
level similar to that in the presence of 2 mM CsCl alone
(recordings 2– 4). On average, the sinus rate was 78.5 ⫾ 4.2%
of controls in the presence of 2 mM CsCl alone (n ⫽ 12) and
72.1 ⫾ 4.3% in the presence of 1 ␮M CCh and 300 nM
tertiapin (n ⫽ 5) (Fig. 3A). These values were not significantly different (p ⫽ 0.382). In Fig. 4B, 1 ␮M CCh was added
in the presence of 2 mM CsCl. CCh decreased the sinus rate
more slowly than in the absence of CsCl probably because
KACh channels were blocked by CsCl more potently as the
membrane potential was hyperpolarized (Argibay et al.,
1983). In the presence of 2 mM CsCl, 300 nM tertiapin
completely antagonized the effect of 1 ␮M CCh (recordings
2– 4). Thus, it is likely that the tertiapin-insensitive component of the CCh-induced sinus bradycardia results from inhibition of the If current.
Discussion
By using tertiapin as a selective KACh channel blocker in
the heart (Kitamura et al., 2000), I divided the negative
chronotropic effect of CCh into the tertiapin-sensitive (i.e.,
KACh current-dependent) and -insensitive (i.e., KACh currentindependent) components. The KACh current-dependent component exhibited a steep concentration-response curve in the
range of CCh concentrations between 100 nM and 1 ␮M, and
accounted for ⬃75% of the maximum effect of CCh. On the
other hand, the KACh current-independent component was
induced by 1 to 300 nM CCh and accounted for ⬃25% of the
maximum effect of CCh. Thus, the KACh current-dependent
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Fig. 2. Effect of tertiapin in the presence and absence of 300, 100, and 30 nM carbachol. Tertiapin (100 nM) was applied in the presence of 300 nM
(A), 100 nM (B), and 30 nM (C) of CCh or in the absence of CCh (D).
The Role of Muscarinic Kⴙ Channel in Sinus Bradycardia
685
component was ⬃10 times less sensitive to CCh but ⬃3 times
more effective than the KACh current-independent component in decreasing the sinus rate.
The concentration-response curve for the CCh-induced
KACh current closely resembled that for the tertiapin-sensitive component in a range of CCh concentrations of ⱕ1 ␮M
with a similar steepness (Figs. 3A, inset; the Hill coefficient
ⱖ1). This positive cooperativity may arise from the interaction between the G protein ␤␥ subunits and KACh channels
(Hosoya et al., 1996). In the presence of ⬎1 ␮M CCh, the
CCh-induced KACh current increased in a concentration-dependent manner, whereas that for the tertiapin-sensitive
component reached a plateau. This is because ⬎1 ␮M CCh
caused sinus arrest.
Recently, Drici et al. (2000) identified the role of KACh
channels in the parasympathetic regulation of AV conduction
by showing that tertiapin attenuated the ACh-induced advanced AV block in guinea pig hearts. They also briefly
mentioned the effect of tertiapin on the negative chronotropic
effect of ACh. In guinea pig hearts, they found that tertiapin
almost completely reversed the effect of 0.5 ␮M ACh. In
rabbit hearts, however, they found that 5 ␮M ACh reduced
the sinus rate by only 20%, and that this response was
insensitive to tertiapin. This is very different from my observation. I found in preliminary experiments that ⬎1 ␮M ACh
usually caused sinus arrest as is the case for CCh. At lower
concentrations, however, ACh caused more variable effect
than CCh (data not shown), indicating that cholinesterase
significantly affects the effect of ACh in whole heart preparation (Löffelholz and Pappano, 1985). Thus, it is possible
that the maximum effective concentration of ACh in their
rabbit heart preparation might have been much higher than
5 ␮M.
It is widely accepted that the negative chronotropic effect
of vagal nerve activity or parasympathomimetics results
from hyperpolarization of sinus node cells due to activation of
KACh channels (Gaskell, 1887; Burgen and Terroux, 1953; del
Castillo and Katz, 1955; Hutter and Trautwein, 1955 and
1956; Harris and Hutter, 1956; Hutter, 1957; Trautwein an
Dudel, 1958; Noma and Trautwein, 1978; Sakmann et al.,
1983). On the other hand, weak vagal stimulation or low
concentrations of ACh is known to cause the negative chro-
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Fig. 3. Carbachol-induced negative chronotropic effect in the presence and absence of tertiapin, and carbachol-induced muscarinic K⫹ channel
currents in rabbit atrial myocytes. A, relationship between the sinus rate and concentrations of CCh in the absence (circles) and the presence of 100
nM (triangles) or 300 nM (squares) of tertiapin. The sinus rate is indicated as a percentage of the control. 多, p ⬍ 0.05 versus control; 多多, p ⬍ 0.025
versus control; and †, p ⬍ 0.025 versus 100 nM tertiapin. Symbols and bars indicate the mean and S.E., respectively. The number of observations at
each point was 4 to 10. Inset, the negative chronotropic effect of CCh in the absence of tertiapin (open circles), and its tertiapin-sensitive (diamonds)
and -insensitive (squares) components are depicted as a percentage of the maximum effect of CCh. The tertiapin-sensitive component was calculated
by subtracting the percentage of the sinus rate in the presence of tertiapin from that in the absence of tertiapin. The tertiapin-insensitive component
was calculated by subtracting the percentage of the sinus rate in the presence of tertiapin from 100%. The data in the presence of 1 ␮M CCh and 100
nM tertiapin were omitted from the calculation. The broken line is the curve shown in panel B, b (see below). Panel B: a, in the whole-cell configuration
of the patch-clamp method, CCh-induced KACh channel currents (bottom panel) were recorded from a rabbit atrial myocyte. From the holding potential
of ⫺40 mV, a set of three 300 ms voltage pulses to ⫺100, ⫺40, and ⫹10 mV were applied every 3 s (top panel). Arrowheads and dotted lines indicate
the zero potential (top panel) and the zero current (bottom panel) levels. b, relationship between concentrations of CCh and the steady-state
CCh-induced KACh channel currents at ⫺40 mV expressed as a percentage of the maximum (I). Symbols and bars indicate the mean and S.E.,
respectively. The solid line is the fit of the data with the following Hill equation: I ⫽ 100/(1 ⫹ (4.35 ⫻ 10⫺7/[CCh])1.52).
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Yamada
notropic effect by decreasing the slope of diastolic depolarization without causing hyperpolarization of the maximum
diastolic potential (Hutter and Trautwein, 1955 and 1956;
West, 1955; Bouman et al., 1963; Shibata et al., 1985; Campbell et al., 1989; DiFrancesco, 1993). This phenomenon may
be at least in part explainable in terms of the slow relaxation
of KACh channels (Yamada et al., 1998). ACh-induced inhibition of the L-type calcium channel current and/or the sustained inward current may also be responsible for this phenomenon (Irisawa et al., 1993). On the other hand,
DiFrancesco et al. ascribed the phenomenon to inhibition of
the If current resulting from the ACh-induced suppression of
the basal cAMP level (DiFrancesco and Tromba, 1988; DiFrancesco et al., 1989; DiFrancesco, 1993). They showed that
ACh inhibited If currents and slowed the spontaneous firing
rate of isolated rabbit SA node cells at ⬃20 times lower
concentrations than activating KACh channels. Although
there is some controversy on their hypothesis and contribution of If currents to the basal sinus automaticity (Irisawa et
al., 1993), 2 mM CsCl indeed reduced the sinus rate by ⬃20%
(Fig. 4) without causing a significant change in QRS or QTc
intervals (Table 1). Thus, the If current seems to contribute
to the basal cardiac pacemaking probably by counteracting
the hyperpolarizing influence of the atrial myocytes connected to SA node cells (DiFrancesco, 1993; Irisawa et al.,
1993). Furthermore, the sinus rate was similar in the presence of 2 mM CsCl alone and in the presence of 1 ␮M CCh
plus 300 nM tertiapin (Fig. 4A), and there was no tertiapininsensitive component in the presence of 2 mM CsCl (Fig.
4B). Therefore, the tertiapin-insensitive component seems to
result from suppression of If currents.
It was recently demonstrated that GIRK4 knock-out mice
deficient of KACh channels exhibited a normal mean resting
heart rate with impaired baroreflex (Wickman et al., 1998).
In view of the present study, the mean resting heart rate may
be regulated by relatively low concentrations of ACh through
the suppression of If currents (Fig. 3A). The study also indicates that the vagal activity under baroreflex provides sufficiently high concentrations of ACh to activate KACh channels
in the SA node cells (Fig. 3A). In the baroreflex, the efferent
cardiac vagal activity occurs more or less fixed to the cardiac
cycle (Jewett, 1964; Katona et al., 1970), and the effectiveness of vagal activity on the sinus automaticity is strongly
dependent on the relationship between the pacemaker cycle
and the duration, amplitude, and timing of the hyperpolarizing effect of vagal impulses (Jalife and Moe, 1979). KACh
channels possess a sufficiently fast response time to ACh to
mediate such a phasic effect of vagal activity (Breitwieser
and Szabo, 1988; Inomata et al., 1989), at least in part due to
the direct coupling of the channels with G proteins and the
regulator of G protein signaling proteins (Doupnik et al.,
1997; Yamada et al., 1998). The positive cooperativity found
in activation of KACh channels (Fig. 4B) will also help the
channels to promptly open when ACh concentration rises and
suddenly close when it decreases even slightly. It is, therefore, plausible that KACh channels play an essential role in
the baroreflex.
To summarize, I quantitatively assessed the contribution
of KACh channels to the negative chronotropic effect of CCh in
rabbit hearts by using tertiapin. I found that KACh currents
mediate the negative chronotropic effect of ⱖ300 nM CCh
and account for up to ⬃75% of the maximum effect of CCh.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 11, 2017
Fig. 4. Effect of tertiapin on carbachol-induced sinus bradycardia in the presence and absence of CsCl. A, CsCl (2 mM) was first applied alone. After
washout of CsCl, CCh and tertiapin were applied. B, CCh and tertiapin were applied in the presence of 2 mM CsCl.
The Role of Muscarinic Kⴙ Channel in Sinus Bradycardia
However, suppression of If currents might play a major role
for the effect of 1 to 100 nM CCh and account for ⬃25% of the
maximum effect of CCh. The KACh current-dependent mechanism may play an important role in the beat-to-beat regulation of the sinus rate under baroreflex, whereas suppression of If currents may participate in regulation of the mean
resting heart rate. Tertiapin is a useful pharmacological tool
to identify the physiological and pathophysiological roles of
KACh channels in parasympathetic regulation of the heart
rate of various animals under ex vivo and in vivo conditions.
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