© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 1745-1751 doi:10.1242/jeb.098509
RESEARCH ARTICLE
A new potassium ion current induced by stimulation of M2
cholinoreceptors in fish atrial myocytes
Denis V. Abramochkin1,2,*, Svetlana V. Tapilina1 and Matti Vornanen3
KEY WORDS: Acetylcholine, Muscarinic receptors, Fish heart,
Atrial myocytes, Ionic currents, IKACh.
INTRODUCTION
Cardiac contractility is adjusted to circulatory demands on a beatto-beat basis by the interaction of sympathetic and parasympathetic
nervous systems via β-adrenergic receptors and muscarinic (M2)
cholinergic receptors, respectively (Hartzell, 1988). Effects of
sympathetic activation are rapidly reversed by parasympathetic
stimulation, despite the continued sympathetic tone via the
‘accentuated antagonism’ of the autonomic nerves (Levy, 1971). It
is widely recognized that the main parasympathetic neurotransmitter,
acetylcholine (ACh), induces negative chronotropic and inotropic
effects mainly via the activation of the second subtype (M2) of
muscarinic cholinoreceptors, which prevails in the mammalian
1
Department of Human and Animal Physiology, Moscow State University,
Leninskiye Gory, 1, 12, Moscow 119991, Russia. 2Department of Fundamental
and Applied Physiology, Russian National Research Medical University, Moscow
117997, Russia. 3Department of Biology, University of Eastern Finland, FI-80101
Joensuu, Finland.
*Author for correspondence (abram340@mail.ru)
Received 16 October 2013; Accepted 4 February 2014
myocardium (Dhein et al., 2001), even though the involvement of
M3 cholinoreceptors in mediation of ACh effects has been recently
confirmed (Abramochkin et al., 2012; Wang et al., 2007).
The crucial cardiotropic effects of M2 cholinoreceptors are
mediated by activation of the ACh-gated inwardly rectifying K+
current (IKACh) via Gi proteins. Binding of ACh to M2 receptors leads
to dissociation of Gi proteins and subsequent direct activation of KACh
channels by their βγ-subunits (Hibino et al., 2010). IKACh belongs to
the family of cardiac inward rectifiers (IKir), which includes two other
K+ current systems: the background inward rectifier current (IK1) and
the ATP-sensitive current. Consistent with its physiological function
in maintaining a stable negative resting membrane potential, the IK1
is significant in ventricular myocytes, less prominent in atrial
cardiomyocytes and practically absent in the pacemaker cells of the
sinoatrial node. Opposite to the IK1, the density of IKACh is much
higher in supraventricular cardiac tissues than in ventricular
myocardium, which appears in electric activity as high sensitivity of
atrial and pacemaker action potentials (APs) to muscarinic
modulation. Induction of the IKACh by stimulation of M2 receptors
leads to hyperpolarisation, which is particularly distinct in the
pacemaker cells, and AP shortening, which is more pronounced in the
atrial myocardium than in the pacemakers (Boyett et al., 1995).
In the present study, we demonstrate the presence of another K+
current associated with muscarinic stimulation in atrial
cardiomyocytes of fish. It can be clearly distinguished from the
conventional IKACh by its low sensitivity to Ba2+. The outward
component of the novel current (IKACh2) has a current–voltage
dependence very similar to that of the IKACh, but in contrast to the
IKACh the inward component of IKACh2 is very small. Activation of
this novel carbamylcholine chloride (CCh)-sensitive current seems
to be mediated by a pertussis toxin (PTX)-dependent pathway
involving M2 receptors.
RESULTS
Current induced by CCh in fish atrial myocytes and its ionic
nature
When studying the Na–Ca exchange current (INCX) in fish cardiac
myocytes, we noticed that the recorded current was strongly and
quickly increased by CCh, a muscarinic agonist. Conditions for INCX
recording require Cs+-based pipette and external solutions (for
composition, see Materials and methods) and various ion channel
blockers (tetrodotoxin, nifedipine, E-4032 and BaCl2 for sodium,
calcium, delayed rectifier and inward rectifier currents, respectively)
applied into the external saline solution. Because of the experimental
setting for INCX recording, it was initially thought that activation of
the current represented parasympathetic modulation of the INCX.
Indeed, both atrial and ventricular myocytes of crucian carp and
rainbow trout hearts demonstrate a definite background INCX before
CCh addition (Fig. 1). During the experimental protocol, both
inward and outward components of the INCX always increased
during the first 2–3 min after gaining access to the whole-cell
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The Journal of Experimental Biology
ABSTRACT
A novel potassium ion current induced by muscarinic stimulation
(IKACh2) is characterized in atrial cardiomyocytes of teleost fishes
(crucian carp, Carassius carassius; rainbow trout, Oncorhynchus
mykiss) by means of the whole-cell patch-clamp technique. The
current is elicited in atrial, but not ventricular, cells by application of
carbamylcholine (CCh) in moderate to high concentrations
(10−7–10−4 mol l−1). It can be distinguished from the classic IKACh,
activated by the βγ-subunit of the Gi-protein, because of its low
sensitivity to Ba2+ ions and distinct current–voltage relationship with
a very small inward current component. Ni2+ ions (5 mmol l−1) and KBR7943 (10−5 mol l−1), non-selective blockers of the sodium–calcium
exchange current (INCX), strongly reduced and completely abolished,
respectively, the IKACh2. Therefore, IKACh2 was initially regarded as a
CCh-induced outward component of the INCX. However, the current is
not affected by either exclusion of intracellular Na+ or extracellular
Ca2+, but is completely abolished by intracellular perfusion with K+free solution. Atropine (10−6 mol l−1), a non-selective muscarinic
blocker, completely eliminated the IKACh2. A selective antagonist of M2
cholinoreceptors, AF-DX 116 (2×10−7 mol l−1) and an M3 antagonist,
4-DAMP (10−9 mol l−1), decreased IKACh2 by 84.4% and 16.6%,
respectively. Pertussis toxin, which irreversibly inhibits Gi-protein
coupled to M2 receptors, reduced the current by 95%, when applied
into the pipette solution. It is concluded that IKACh2, induced by
stimulation of M2 cholinoceptors and subsequent Gi-protein activation,
represents a new molecular target for the cardiac parasympathetic
innervation.
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509
List of symbols and abbreviations
acetylcholine
action potential
carbamylcholine chloride
background inward rectifier K+ current
acetylcholine-activated inward rectifier current
NCX current
muscarinic receptor
sodium–calcium exchanger
pertussis toxin
small conductance Ca2+-activated K+ channel
recording, and then remained relatively stable for up to 30 min or
more. This background current could be completely blocked by an
organic blocker KB-R7943 (10−5 mol l−1) or suppressed by 60–80%
with 5 mmol l−1 Ni2+ (data not shown).
CCh at concentrations of 10−6–10−4 mol l−1 induced a marked
increase in the recorded current in atrial myocytes of both crucian
carp and rainbow trout (Fig. 1). We define the CCh-induced current
as the difference between the current in the presence of CCh and the
current recorded just before CCh application (see current–voltage
curves in Fig. 1C,D). In contrast to the basal KB-R7943-sensitive
current, which had both inward and outward current components,
the CCh-induced current was exclusively in the outward direction.
CCh-induced current had the same shape in atrial myocytes of both
fish species (Fig. 1C,D). However, the density of the current
produced by 10−4 mol l−1 CCh was ~42% larger in crucian carp
(10.48±1.89 pA pF−1) than in rainbow trout (7.39±1.62 pA pF−1)
myocytes (P<0.05, Mann–Whitney test). In ventricular myocytes of
both species, CCh up to the concentration of 10−4 mol l−1 failed to
produce any distinguishable effects. This is not surprising in light of
the well-known insensitivity of fish ventricular myocardium to
muscarinic agonists (Vornanen and Tuomennoro, 1999).
A
200
Control
150
CCh 10–5 mol l–1
0
–50
–90 mV
–100
200
Time (ms)
400
0
–5
–100
mol l–1+
–5
CCh 10
KB-R7943 10 mol l–1
100
200
300
Time (ms)
0
–200
C
100
–46 mV
–46 mV
100
Background current (INCX)
CCh 10–6 mol l–1
CCh 10–5 mol l–1
CCh 10–4 mol l–1
CCh 10–5 mol l–1+
Ni2+ 5 mmol l–1
50
0
0
100
200
300
Time (ms)
–50 Control
400
400
500
500
–100
14
I (pA pF–1)
Current (pA)
300
B
60 mV
50
D
12
Background current (INCX)
CCh 10–6 mol l–1
10
CCh 10–5 mol l–1
CCh 10–4 mol l–1
8
10
8
6
6
4
4
2
2
–90
–70
–50
–30
0
–10
10
30
50
–2 Membrane potential (mV)
–90
–70
–50
–30
0
–10
–2
10
30
50
Membrane potential (mV)
Fig. 1. Basal current and carbamylcholine (CCh)-induced current in fish atrial myocytes. (A,B) Original current–voltage recordings from two
representative experiments. Black line shows the background sodium–calcium exchanger current (INCX or Control) after 5 min access to the whole-cell
configuration in the absence of CCh; red and green lines indicate the current in the presence of 10−5 mol l−1 CCh and 10−5 mol l−1 CCh + 10−5 mol l−1 KB-R7943
(A) or 10−5 mol l−1 CCh + 5 mmol l−1 Ni2+ (B), respectively. The inset shows the voltage ramp used to elicit the current. Experiments were conducted using the
external Cs+-based Tyrode solution and pipette solution 1 (for composition, see Materials and methods). (C,D) Mean current–voltage curves of the background
sodium–calcium exchange current (INCX) (C, n=25; D, n=10) and CCh-induced current recorded in the presence of 10−6 mol l−1 (C, n=25; D, n=14), 10−5 mol l−1
(C, n=40; D, n=16) and 10−4 mol l−1 (C, n=9; D, n=16) CCh in atrial cardiomyocytes from crucian carp (C) and rainbow trout (D). Current–voltage curves of the
CCh-induced current were obtained after subtraction of the basal INCX current. Cells were obtained from five crucian carp and four rainbow trout.
1746
The Journal of Experimental Biology
mol l
–1
I (pA pF–1)
CCh 10
–5
Membrane potential
(mV)
400
Current (pA)
ACh
AP
CCh
IK1
IKACh
INCX
M-receptor
NCX
PTX
SKCa
KB-R7943 (10−5 mol l−1), a non-selective blocker of NCX,
abolished both the CCh-sensitive and the background current (INCX),
leaving only a tiny leakage current (Fig. 1A). A concentration of
5 mmol l−1 Ni2+, a non-specific inorganic blocker of the NCX, strongly
reduced or abolished (70–100%) the overall CCh-sensitive and
background current (Fig. 1B). However, Ni2+ turned out to be toxic to
fish cardiac myocytes (most of the studied cells died 20–30 s after the
start of Ni2+ application), which prevented its routine use as an NCX
blocker. The noticed sensitivity of the CCh-induced current to
standard blockers of INCX suggested that the recorded current
represented parasympathetic stimulation of the outward INCX. Ion
substitution experiments involving exclusion of Na+ from the pipette
solution or omission of Ca2+ from the extracellular saline solution did
not, however, support this assumption. In the first series of
experiments, 5 mmol l−1 Na2ATP in the pipette solution was
substituted by 5 mmol l−1 MgATP. The CCh-induced current was still
present and not less than in the controls (Fig. 2A,B). In the second
series of experiments, we used a Ca2+-free external solution
supplemented with 5 mmol l−1 EGTA to ensure practical absence of
intracellular free Ca2+. The application of this solution led to an
expected decrease in the outward component of the background INCX,
but subsequent addition of 10−5 mol l−1 CCh produced a large outward
current (Fig. 2A,C). Substitution of Na+ with Li+ in the composition
of the Ca2+-free Tyrode, a well-known way to suppress INCX (Sanders
et al., 2006), also failed to affect the CCh-induced current, although
the basal INCX was reduced in a manner similar to the experiments
with normal Ca2+-free Tyrode (Fig. 2A,D). Therefore, the CChinduced current is evidently not a component of the INCX.
In all experiments described above we used Cs+-based pipette
solution 1 (for composition, see Materials and methods). However,
this solution contained 20 mmol l−1 BAPTA K+ salt for intracellular
Ca2+ buffering. Therefore, in the next series of experiments we used
a modified pipette solution 1, where K4BAPTA was substituted with
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509
7
6
5
4
3
2
1
B
K+-free pipette
solution
C
60
40
Control
20
0
0
100
200
300
400
–40
Current (pA)
n=5
140
120
Control
100
80
2+
60 Ca -free
Tyrode
40
20
0
500
100
–20 0
–40
–60
–80
Time (ms)
Ca2+-free Tyrode
+CCh 10–5 mol l–1
CCh 10–5 mol l–1
80
–60
D
200
300
400
500
200
300
400
500
E
300
CCh 10–5 mol l–1
250
Ca2+-free Tyrode + Li+
+CCh 10–5 mol l–1
Ca2+-free
Tyrode + Li+
200
150
80
60
40
20
CCh 10–5 mol l–1
0
Control
100
–20
50
0
100
–40
0
0
–50
*
n=7
Ca2+-free Tyrode,
Na+ changed to Li+
n=5
n=6
Ca2+-free Tyrode
n=40
Na+-free pipette
solution
0
100
–20
Fig. 2. Dependence of the CCh-induced current on
intracellular Na+ and K+ and extracellular Ca2+ in
crucian carp atrial myocytes. (A) Comparison of the
CCh-induced current density at +20 mV in different
experimental conditions. The results are means ±
s.e.m. of five to 40 myocytes as indicated above the
bars. An asterisk indicates a statistically significant
difference (P<0.05, Mann–Whitney test) between the
K+-free conditions and all other experimental conditions.
(B–E) Original traces of the basal INCX and the total
current after CCh application recorded in different
experimental conditions. (B) Na+-free pipette solution 1
was used (Na2ATP was substituted with MgATP).
(C,D) Normal pipette solution 1 was used, but modified
external Cs+-based Tyrode solutions were applied
during the recordings. (E) Pipette solution 1 with
K4BAPTA replaced with EGTA. In all experiments, the
same voltage ramp pulse as shown in the inset in Fig. 1
was applied to elicit the current.
8
Control
conditions
A
CCh-induced current (pA pF–1)
RESEARCH ARTICLE
100
200
300
400
500
–60
Control
–80
EGTA (free acid). Under these conditions, 10−5 mol l−1 CCh failed
to induce any outward or inward current (Fig. 2A,E), clearly
indicating that the studied current was carried by K+ ions. Thus,
although the CCh-induced current can be elicited using an NCX
protocol (voltage ramp) and is blocked by Ni2+ and KB-R7943, it is
independent of the NCX and is carried by K+.
Effect of CCh is mainly mediated via M2 muscarinic
receptors
Using pharmacological blockers, we tried to find out which
subtype(s) of cholinoreceptors might mediate the activation of the
CCh-induced current. In atrial myocytes from both species, atropine
(10−6 mol l−1), which blocks muscarinic cholinoreceptors without
subtype discrimination, completely abolished the outward current
induced by 10−5 mol l−1 CCh (Fig. 3A,B). Atropine blocked only the
CCh-induced current, without effect on the basal INCX. These
findings strongly suggest that the induction of the outward current
by CCh is mediated by muscarinic cholinoreceptors.
The major role of M2 muscarinic receptors in the mediation of
cardiac cholinergic response is widely recognized. However, the
presence of physiologically relevant M3 cholinoreceptors was
confirmed during the last decade for mammalian myocardium
(Hellgren et al., 2000; Kitazawa et al., 2009), and therefore cannot
be neglected as a possible signaling pathway in fish cardiac
myocytes. To this end, we have investigated effects of the selective
M3 blocker 4-DAMP (10−9 mol l−1) and selective M2 antagonist AFDX 116 (2×10−7 mol l−1) on the CCh (10−5 mol l−1)-induced current.
The selected agonist concentrations have been shown in a previous
patch-clamp study (Wang et al., 1999) to exert subtype-specific
effects on muscarinic signaling, while higher concentrations may
lead to non-selective binding of the blockers.
Application of AF-DX 116 suppressed the CCh-induced current
by 84.3% (Fig. 3A,C). In contrast to the marked blocking effect of
AF-DX 116, 4-DAMP produced only a slight reduction of the
current by 16.6%, although this decrease was significant (P<0.05;
Fig. 3A,D). These results suggest a predominant role of M2 receptors
in the mediation of the CCh effect. To confirm this assumption, we
conducted a series of experiments with PTX.
Inhibition of the CCh-induced current by PTX
PTX is widely used as a selective inhibitor of Gi-protein-mediated
signaling pathways. It irreversibly blocks the activity of αi-subunit by
ADP-ribosylation, which prevents the αi-subunit from interacting with
the receptor molecule. The routine way of PTX application is
1747
The Journal of Experimental Biology
Time (ms)
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509
A
8
CCh 10–5 mol l–1
300
7
*
6
CCh–5 mol l–1+
Atr 10–6 mol l–1
200
100
0
5
–100
4
0
200
400
C
400
3
CCh 10–5 mol l–1
300
Current (pA)
2
*
0
*
0
+4-DAMP 10–9 mol l–1
+AF-DX116 2×10–7 mol l–1
+Atr 10–6 mol l–1
CCh 10–5 mol l–1
n=40 n=13 n=15 n=12
–1
100
–100
0
400
200
D
CCh 10–5 mol l–1
400
CCh 10–5 mol l–1+
4-DAMP 10–6 mol l–1
300
200
100
0
–100
0
200
400
Time (ms)
incubation of isolated myocytes in a medium containing the toxin.
Several pilot experiments using the protocols of PTX application to
mammalian and frog cardiac myocytes (2–4 h of incubation at room
temperature in a medium containing up to 6 μg ml−1 PTX) failed to
induce any decrease of the CCh-induced current in fish atrial
myocytes. This negative result could be due to the absence of a
membrane receptor for PTX or any other downstream step responsible
for endocytosis of the toxin and its intracellular activation in fish
cardiomyocytes. Therefore, we decided to add PTX directly to pipette
solution 1 (1 μg ml−1) before patching and wait for diffusion of PTX
inside the cell. A similar method of PTX administration was
previously successfully used in outer cells of guinea pig cochlea
(Kakehata et al., 1993), although to our knowledge we have applied
it in cardiomyocytes for the first time. To examine PTX action, we
recorded the effect of 10−5 mol l−1 CCh on membrane current every
10 min during the 30 min internal perfusion of the cell with pipette
solution containing PTX. Similar long-term control recordings were
performed using a normal PTX-free pipette solution.
After the first minute of recording with pipette solution containing
PTX, the cells demonstrated a normal strong increase of the outward
current (6.03±1.95 pA pF−1) in response to 10−5 mol l−1 CCh (Fig. 4).
However, later on, the CCh-induced current started to gradually
fade, so that after 30 min of internal perfusion it was only
0.3±0.33 pA pF−1 (P<0.05), i.e. 5% of the initial value. In control
experiments without PTX in the pipette solution, the amplitude of
CCh-induced current was not significantly changed during the
30 min internal perfusion (5.54±0.81 versus 6.2±1.83 pA pF−1,
respectively; P>0.05). Thus, PTX applied to the pipette solution
effectively inhibits the response to CCh. Taken together with the
data obtained using subtype-selective muscarinic blockers, these
findings strongly suggest a crucial role of M2 cholinoreceptors in
mediation of the outward current induction by CCh.
Thus, the CCh-induced current is carried by K+ ions and can be
activated by stimulation of the cardiac muscarinic receptors.
Because of the similarity of the CCh-induced current to the IKACh,
1748
we decided to refer to it as IKACh2 and tested its possible identity with
the classic IKACh.
Distinguishing IKACh2 from other K+ currents
We attempted to determine whether IKACh2 is a novel and separate
current entity or just a CCh-sensitive component of the IKACh or
other known cardiac K+ currents. The presence of Cs+ in both
external and intracellular solution and the addition of E-4031 to Cs+based Tyrode allowed to exclude the involvement of the delayed
rectifier K+ currents, which also have almost linear current–voltage
dependence and are not coupled to muscarinic receptors.
PS with 1 µg ml–1 PTX
PS without PTX
*
10
*
8
6
4
2
0
–2
1
10
20
30
Time (min)
Fig. 4. Effect of intracellular perfusion with PTX on the density of CChinduced current in atrial myocytes of the crucian carp. PTX was included
in pipette solution (PS) 1 and the myocytes were perfused for 30 min with
continuous recording of the current. An asterisk indicates a statistically
significant difference (P<0.05, Mann–Whitney test) between two columns.
The same voltage ramp pulse as shown at the inset in Fig. 1 was used to
elicit the current. Experiments were conducted using the external Cs+-based
Tyrode solution. The number of experiments (n) was 8 and 6 cells for control
and PTX experiments, respectively.
The Journal of Experimental Biology
1
CCh 10–5 mol l–1+
AFDX-116
10–6 mol l–1
200
CCh-induced current (pA pF–1)
CCh-induced current (pA pF–1)
Fig. 3. Effect of cholinoreceptor antagonists on the CChinduced current in crucian carp atrial myocytes. The current
was first elicited with 10−5 mol l−1 CCh and then different blockers
were applied in the continuous presence of CCh. (A) Mean
(±s.e.m.) effects of atropine, AF-DX116 and 4-DAMP at +20 mV. An
asterisk indicates a statistically significant difference (P<0.05,
Mann–Whitney test) from the control (CCh alone). Original traces
from representative experiments are shown in B–D. The same
voltage ramp pulse as shown at the inset in Fig. 1 was used to elicit
the current. Experiments were conducted using the external Cs+based Tyrode solution and pipette solution 1.
B
400
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509
Normal pipette
solution
A
7
6
CCh 10–5 mol l–1+
KB-R7943 10–5 mol l–1
6
4
–5
–1
2+
–4
–1
2 CCh 10 mol l +Ba 10 mol l
5
CCh-induced
current (pA pF–1)
4
Ca2+-free pipette
solution
3
–120
–100
Control
–80
–60
–40
CCh 10–5 mol l–1
–20
I (pA pF–1) –6
–8
Membrane potential (mV)
2
1
0
–90
–70
–50
–30
–10
0
20
40
–2 0
Membrane potential (mV)
–4
10
30
50
–1
60 mV
50
–12
0
0
–50
200 400 600 800 1000 1200 1400
Time (ms)
–80 mV
–80 mV
–100
Membrane potential (mV)
–10
–120 mV
–16
–18
–20
–22
–150
Fig. 5. Representative current–voltage curves of IKACh2 recorded in
crucian carp atrial myocytes with the use of the standard pipette
solution 1 and the same solution without CaCl2. The intracellular calcium
concentration was 105 nmol l−1 in control conditions and less than 1 nmol l−1
when using Ca2+-free pipette solution. The same voltage ramp pulse as
shown at the inset in Fig. 1 was used to elicit the current. Experiments were
conducted using the external Cs+-based Tyrode solution.
–14
B
5
IKACh
IKACh2
–120
–100
–80
–60
–40
–20
0
0
20
40
Membrane potential (mV)
–5
I (pA pF–1)
–10
–15
–20
Fig. 6. Comparison of IKACh2 with the inward rectifiers in crucian carp
atrial myocytes. (A) Original current–voltage curves of basal IKir (IK1), total
current after CCh application (IK1 + IKACh + IKACh2), the current recorded in the
presence of CCh and Ba2+ (IKACh2) and the leakage current obtained after
addition of KB-R7943. The inset shows the voltage ramp pulse, which was
used to elicit the current. Experiments were conducted using the external K+based Tyrode solution and pipette solution 2 (for composition, see Materials
and methods). (B) Averaged current–voltage curves (n=11, 3 fish) of IKACh
and IKACh2. The first curve was obtained by subtraction of basal IK1 + IKACh2
from the total current recorded under CCh.
typical inward rectifier, very large at potentials below the EK, while
the latter has a tiny inward but a significant outward component.
Thus, two currents not only differ in sensitivity to BaCl2, but
demonstrate principally different current–voltage relationships.
DISCUSSION
Muscarinic receptor stimulation plays a central role in the
parasympathetic control of cardiac function by modulating heart rate
(chronotropic effect), contractility (inotropic effect) and conduction
velocity (dromotropic effect) (Brodde and Michel, 1999). These
effects are particularly strong in pacemaker and atrial tissues. The M2
muscarinic receptors prevail in the mammalian myocardium, although
some M3 receptors are also present and physiologically active
(Pönicke et al., 2003; Wang et al., 2007). The even-numbered
muscarinic receptors, including M2, are coupled to Gi proteins and act
via inhibition of adenylate cyclase and a subsequent decrease in
cellular cAMP content (Dhein et al., 2001). However, M2 and M3
receptors can activate different effectors without the second messenger
systems. The channels responsible for the inward rectifier IKACh are
directly stimulated by βγ-subunits of the Gi protein, while joint
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The Journal of Experimental Biology
In our experiments, we did not specifically block the putative
Ca2+-dependent K+ current, which is carried by the small
conductance Ca2+-activated K+ channels (SKCa) in mammalian atrial
and ventricular myocytes (Xu et al., 2003). However, in murine and
human cells, this current has a prominent inward component at the
potentials below potassium equilibrium potential, while the outward
current is substantial only at potentials more positive than −40 mV.
Although Ca2+-dependent K+ current has never been registered in
fish cardiomyocytes, in mammalian cells the current density
strongly depends on intracellular Ca2+ content and is negligible at
10−8 mol l−1 intracellular Ca2+ (Xu et al., 2003). Experiments with
Ca2+-free pipette solution (BAPTA substituted with EGTA, no
CaCl2, free [Ca2+]in <10−9 mol l−1) were conducted in crucian carp
atrial cells to prevent SKCa from contributing to the CCh-induced
current. The IKACh2 at +20 mV was significantly less than in the
control conditions (5.22±1.11 versus 6.16±0.83 pA pF−1,
respectively; P>0.05; Fig. 5), but still far from inhibited. Obviously,
IKACh2 is different from SKCa, which would be completely blocked
in the practical absence of intracellular free Ca2+.
Finally, there was a possibility of incomplete inhibition of inward
rectifiers (IKir) by 10−4 mol l−1 BaCl2. Then IKACh2 could be just a part
of the classic IKACh. To test this assumption, we recorded IKir in the
K+-based Tyrode solution without BaCl2. The density of the basal
IK1 was very low, like it usually is in fish atrial cardiomyocytes
(Fig. 6A). Application of 10−5 mol l−1 CCh produced a large increase
in the inwardly rectifying current and, especially, the outward IKir.
The density of recorded current was 4.87±0.42 pA pF−1 at +20 mV
and −19.23±0.51 pA pF−1 at −120 mV. Subsequent addition of
10−4 mol l−1 BaCl2 blocked the inward current by 97%
(−0.58±0.06 pA pF−1 at −120 mV), while the outward current
persisted (1.45±0.69 pA pF−1 at +20 mV). Increase in Ba2+
concentration up to 3×10−4 mol l−1 did not lead to further decrease
of the outward current (1.45±0.69 pA pF−1 at +20 mV). On the
contrary, addition of 10−5 mol l−1 KB-R7943 practically abolished
the outward current (0.27±0.07 pA pF−1 at +20 mV). It seems that
10−4 mol l−1 Ba2+ completely blocks the inward rectifiers (both IK1
and IKACh), while IKACh2 resists Ba2+, but not KB-R7943.
The current–voltage curves for IKACh and IKACh2 were obtained
from these data (Fig. 6B). It is clear that the former current is a
RESEARCH ARTICLE
1750
of the IKAch, i.e. shortening of atrial action potential duration with
consequent reduction of atrial contraction. However, activation of
the IKACh2 requires relatively high (micromolar) agonist
concentrations and therefore it is likely to appear only at a strong
parasympathetic tone. In contrast to mammals, in fish and
amphibians, strong cholinergic stimulation not only reduces AP
duration, but gradually decreases AP amplitude in atrial myocardium
until the full cessation of electrical activity (Abramochkin et al.,
2010). This effect is putatively attributed to induction of a strong
outward K+ current, which overwhelms all inward currents and
makes the depolarization impossible. The described IKACh2 may be
involved in mediation of this cholinergic effect. Further research,
including distribution of Kir3 channels in supraventricular tissues of
the fish heart, could reveal possible physiological implications of
this novel K+ current.
Conclusions
In fish atrial cardiomyocytes, stimulation of the M2-mediated second
messenger pathway leads to induction of a novel type of K+ current,
the IKACh2. Different from the IKACh, this current has a very small
inward component and is relatively insensitive to Ba2+ blocking.
Together with IKACh, IKACh2 may provide shortening of APs in fish
atrial myocardium and particularly under a strong cholinergic
influence. The similarity of results obtained from crucian carp
(family Cyprinidae) and rainbow trout (family Salmonidae) atrial
myocytes suggests that the described CCh-dependent activation of
IKACh2 might be a common mechanism for different fish groups, and
raises the question of whether this mechanism is also relevant for
other vertebrates.
MATERIALS AND METHODS
Animals
Experiments were conducted on atrial myocytes of two fish species, crucian
carp (Carassius carassius L., n=31) and rainbow trout (Oncorhynchus
mykiss W., n=7). Fish were held separately in 500 l stainless steel aquaria
with continuous flow of aerated (11 mg O2 l−1) groundwater at constant
temperature of either 18°C (crucian carp) or 14°C (rainbow trout) and under
a 12 h:12 h light:dark photoperiod. During the laboratory maintenance
(>4 weeks), the fish were fed aquarium fish food five times a week. The
investigation conforms with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication
no. 85–23, revised 1996) and the experimental protocols were approved by
the Animal Experiment Board in Finland (permission no. STH252A).
Isolated myocyte preparation
Fish were stunned with a blow to the head, the spine was cut immediately
behind the head and the heart was rapidly excised. Atrial myocytes were
isolated by retrograde perfusion of the heart with proteolytic enzymes
(collagenase type 1A, 0.75 mg ml−1; trypsin type IX, 0.5 mg ml−1; fatty-acidfree BSA, 0.75 mg ml−1) as described previously in detail (Vornanen, 1997).
Isolated cells were stored for up to 8 h at 5°C in low-Na+ solution containing
(in mmol l−1): NaCl 100, KCl 10, KH2PO4·2H2O 1.2, MgSO4·7H2O 4,
taurine 50, glucose 10 and Hepes 10 at a pH of 6.9.
Measurement of sarcolemmal ionic currents using the wholecell patch-clamp method
Atrial myocytes were placed in the experimental chamber (RCP-10T,
Dagan, Maryland, MI, USA, volume 150 μl) and superfused with an external
saline solution. In most of the experiments, we used Cs+-based Tyrode
solution containing (in mmol l−1): NaCl 150, CsCl 5.4, NaH2PO4 0.4,
MgSO4 1.5, CaCl2 1.8, glucose 10 and Hepes 10 at pH 7.6 (adjusted with
CsOH). In other experiments, we used a K+-based Tyrode solution of the
same content with the exception of CsCl and CsOH substitution for KCl and
KOH, respectively. Tetrodotoxin (5×10−7 mol l−1), nifedipine (10−5 mol l−1),
The Journal of Experimental Biology
activity of βγ- and αq-subunits is believed to be involved in the
stimulation of the delayed rectifier type K+ current, IKM3 (Wang et al.,
1999). The molecular basis of the IKM3 current is still unresolved, but
it was recently proposed to be carried by the same channels as the
IKACh (Navarro-Polanco et al., 2013). The delayed rectifier-like
properties of the IKM3 could be explained by the increasing affinity of
muscarinic receptors to their agonists at positive membrane potentials.
In the present study, we describe a novel K+ current, induced by
muscarinic stimulation, with a distinct current–voltage relationship
and pharmacological properties from the known ACh-activated
currents, IKACh and IKM3. Clear dependence of the current on
intracellular and extracellular K+ concentration and reversal of the
current close to the Nernst potential of K+ ions indicate that the
current is carried by K+ ions. Furthermore, the current is
independent from intracellular Na+ and external Ca2+. Unlike the
cardiac inward rectifiers, the new current cannot be blocked by
10−4 mol l−1 Ba2+, which, unlike mammalian cells, is sufficient to
abolish both IK1 and IKACh in crucian carp myocytes (Hassinen et al.,
2008). Different from IKACh and IK1, it is mainly an outward current
with only a tiny inward component. Because of its inwardly
rectifying properties at positive voltages and its activation by the
intracellular signaling pathway of the M2 receptors (similar to the
IKACh), we refer to it as IKACh2.
Experiments using the subtype-selective muscarinic antagonists
and PTX strongly suggest a dominant role of M2 cholinoreceptors
in CСh activation of IKACh2, a property shared with the IKACh. AFDX 116, which at the concentration of 2×10−7 mol l−1 is considered
to be relatively specific against M2 cholinoreceptors (Doods et al.,
1987; Giachetti et al., 1986), almost completely abolished the IKACh2.
In contrast, an M3 cholinoreceptor blocker 4-DAMP (Doods et al.,
1987), had only a weak inhibitory effect on the CСh-induced
current. M2 cholinoreceptors are coupled to the Gi proteins and
convey their physiological effects via the αi-subunit by decreasing
cellular cAMP content or via direct coupling of the βγ-subunit to
their target (channels of inward rectifier IKACh) (Brodde and Michel,
1999). Our experiments on CCh were conducted in the absence of
β-adrenergic stimulation, i.e. without activation of the cAMPdependent pathway. Therefore, it could be argued that the αi-subunit
of the G protein and cAMP might not be involved in IKACh2
induction. It is, however, possible that there is a basal activation of
adenylate cyclase in the absence of β-adrenergic stimulation, which
is antagonized by a CCh-dependent mechanism. Then the increase
in cAMP intracellular content caused by noradrenaline or other
adenylate cyclase-stimulating compounds should suppress the
IKACh2. So, further research should shed light on the mechanism of
IKACh2 activation.
The present study did not try to clarify the molecular basis of the
IKACh2. However, the M2-dependent activation pathway and the
inwardly rectifying properties of the current strongly suggest that it
is closely related to the IKACh, which in the mammalian heart is
carried by Kir3.1 and Kir3.4 channels (Hibino et al., 2010). The
molecular basis of ACh-activated inward rectifiers of the fish heart
is not yet known, but because of the whole genome duplication in
the teleost fishes (Jaillon et al., 2004), the diversity of this current
system may be greater in fishes than mammals. For example, the
rapid component of the cardiac delayed rectifier current is
represented by at least two different ERG channels in the zebrafish
heart (Langheinrich et al., 2003; Milan et al., 2003). Molecular
cloning of the fish Kir3 channels and their expression in
heterologous system is needed to resolve this issue.
Considering the biophysical similarities of IKAch2 and IKAch, the
physiological role of the IKAch2 is assumed to be comparable to that
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509
RESEARCH ARTICLE
Drugs
Tetrodotoxin, E-4031, KB-R7943, AF-DX 116 {11-([2-[(diethylamino)methyl]l-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6on, a selective M2 cholinoreceptor blocker}, 4-DAMP (4-diphenylacetoxyN-methylpiperidine methiodide, a selective M3 receptor antagonist) and
PTX, which irreversibly inhibits Gi signaling protein, were all purchased
from Tocris (Bristol, UK). Collagenase, trypsin, nifedipine (a blocker of Ltype Ca2+ channels) and carbamylcholine chloride (a muscarinic agonist)
were purchased from Sigma (St Louis, MO, USA).
Statistics
All data in the text and figures except the original recordings are presented
as means ± s.e.m. for n experiments. Effects of CCh on sarcolemmal ionic
current relative to the respective basal value of the current were compared
using the Wilcoxon test. The density of CCh-induced current in normal
conditions and in the presence of muscarinic blockers, PTX, KB-R7943 and
other agents was compared using the Mann–Whitney test. P<0.05 was
adopted as the level of statistical significance.
Acknowledgements
The authors thank Anita Kervinen for skillful technical assistance.
Competing interests
The authors declare no competing financial interests.
Author contributions
M.V. and D.A. designed the research and experimental design. All authors
performed experiments and participated in writing and editing of the paper.
Funding
This work was supported by the Academy of Finland [project No. 14795] to M.V.
and the Russian Foundation for Basic Research [grant numbers 12-04-31737 and
14-04-01564] to D.A.
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The Journal of Experimental Biology
E-4031 (10−6 mol l−1) and BaCl2 (10−4 mol l−1) were added to external
solution to block Na+ channels, L-type Ca2+ channels, K+ channels of the
fast delayed rectifier K+ current (IKr) and K+ channels of the inward rectifier
K+ current (IK1), respectively. In experiments with IK1 and IKACh, BaCl2 was
not added in advance to the K+-based Tyrode. A constant flow of external
solution (1.5–2 ml min−1) in the experimental chamber was maintained
throughout the experiment. Temperature of the saline solutions was
regulated at 18°C using a Peltier device (TC-100, Dagan). Pipette solution
1, which was used in the majority of experiments, contained (in mmol l−1):
CsCl 140, MgCl2 1, CaCl2 9, BAPTA 20, Na2ATP 5, Na2GTP 0.03 and
Hepes 10, adjusted to pH 7.2 with CsOH at 20°C. Under these conditions,
free intracellular Ca2+ concentration is buffered close to the diastolic level
(105 nmol l−1; calculated using MaxChelator). Pipette solution 2 was used
for recording of the inward rectifiers (IKir) and contained (in mmol l−1): KCl
140, MgCl2 1, EGTA 5, MgATP 4, Na2GTP 0.03 and Hepes 10 with pH
adjusted to 7.2 with KOH.
The whole-cell voltage clamp recording of ionic currents was performed
using an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA, USA)
and the pClamp 8.2 software package. Resistance of patch electrodes was
2–4 MΩ when filled with the pipette solution. Pipette capacitance, wholecell capacitance and access resistance were routinely compensated. In most
experiments, the current was elicited at 15 s intervals from the holding
potential of −46.5 mV (the calculated reversal potential of NCX) by 1 s
voltage ramp pulses (see Fig. 1A, inset). The current was measured during
the hyperpolarizing phase of the ramp. For recording of the IKir, the common
hyperpolarizing ramp (from +60 to −120 mV) protocol with 10 s intervals
was used. The holding potential was −80 mV.
The Journal of Experimental Biology (2014) doi:10.1242/jeb.098509