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INTRODUCTION |
Phosphatidylinositol 4,5-bisphosphate
(PIP2)1 is well
known as a central molecule in the phosphoinositide cycle, by serving as the precursor of important signaling molecules such as inositol trisphosphate (IP3), diacylglycerol, or
phosphatidylinositol 3,4,5-trisphosphate. Recently, it was shown
that PIP2 is not just a precursor, but also exerts a direct
role in the regulation of various ion transporters, such as
Na+/Ca2+ exchanger (1), IP3
receptor Ca2+ channel (2), Na+-activated
nonselective cation channel (3), and several inwardly rectifying
K+ channels including ROMK1 (4, 5), IRK1 (4, 6),
KATP channels (1, 4, 7-9), and G protein-gated inward
rectifying K+ (GIRK) channels (4, 10). The underlying
mechanism of PIP2 action was investigated in detail for
GIRK, and it was shown that the activation of GIRK channels by G
depends on the presence of PIP2 (4, 6, 10-12). This result
may imply that PIP2 is a final regulator molecule for GIRK
channel activity and that G
exerts its effect by strengthening
the interaction of the channel with PIP2.
The KACh channel in cardiac myocytes is believed to be
heterotetrameric complex formed by GIRK1 and GIRK4. Slowing of heart rate by vagal stimulation is known to be mediated by the activation of
KACh channels, which leads to hyperpolarization of membrane potentials in pacemaking cells and in atrial myocytes (13-17). It is
also well known that the effect of vagal stimulation fades gradually
(vagal escape), due to desensitization of KACh currents (18-22). Molecular mechanisms of KACh activation and
desensitization have been subjects of intense researches for more than
a decade. However, it is not yet clear whether the recent view on the
mechanism of GIRK channel regulation is valid for native
KACh channels. It has been generally believed that direct
binding of G
to the channel results in activation (23, 24), but
the role of PIP2 in this process is still controversial. In
rat atrial myocytes, exogenously applied PIP2 and other
phospholipid were reported to block agonist-mediated KACh
channel activation (25).
The aim of the present study is to investigate the role of
PIP2 in normal signaling pathway for the regulation of
native KACh channels. Considering that PIP2
content in the membrane can be controlled by the PLC-linked receptor
(26, 27), we used mouse atrial myocytes that possess both PLC-linked
receptors, such as
1-adrenergic receptor and
KACh channels. The results of the present study show that
the
1-adrenergic agonist accelerates desensitization of
the KACh channel, possibly through the depletion of the
PIP2 pool in plasma membrane, supporting the hypothesis
that PIP2 is acting as a signal molecule in the regulation
of KACh channels through
1-adrenergic pathway.
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EXPERIMNTAL PROCEDURES |
Cell Isolation--
The isolation of single atrial myocytes from
mice was performed as described by Harrison et al. (28) with
minor modifications. Mice were killed by cervical dislocation, and the
heart was quickly removed. The heart was cannulated by a 24-gauge
needle and then retrogradely perfused via the aorta on a Langendorff
apparatus. During coronary perfusion all perfusates were maintained at
37 °C and equilibrated with 100% O2. Initially the
heart was perfused with normal Tyrode solution for 2-3 min to
clear the blood. The heart was then perfused with Ca2+-free
solution for 2 min. Finally the heart was perfused with enzyme solution
for 14-16 min. Enzyme solution contains 0.14 mg ml
1 collagenase (Sigma Type 5) in
Ca2+-free solution. After perfusion with enzyme solution,
the atria were separated from the ventricles and chopped into
small pieces. Single cells were dissociated in high K+, low
Cl
storage medium from these small pieces using blunt-tip
glass pipette. Cells were stored at 4 °C until use.
Materials and Solutions--
Normal Tyrode solution
contained (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 10 glucose, 5 HEPES, titrated to pH 7.4 with NaOH.
Ca2+-free solution contained (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 10 glucose, 5 HEPES, titrated to pH 7.4 with NaOH.
The high K+, low Cl
storage medium contained
(mM): 70 KOH, 40 KCl, 50 L-glutamic acid, 20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose, 10 HEPES, 0.5 EGTA. Pipette solution contained (mM): 140 KCl, 10 HEPES, 1 MgCl2, 5 EGTA, titrated to pH 7.2 with KOH. ACh chloride
(Sigma), phenylephrine (Sigma), and neomycin (Biomol) were dissolved in
deionized water to make a stock solution and stored at
20 °C. On
the day of experiments one aliquot was thawed and used. Calphostin C
(Biomol) and wortmannin (WMN; Biomol) were first dissolved in dimethyl
sulfoxide (Me2SO) as a stock solution and then used
at the final concentration in the solution. All experiments were
conducted at 35 ± 1 °C. In the presence of ACh, 10 µM glibenclamide was applied to inhibit the ATP-sensitive
K+ channel. Cells were superfused with solution by gravity
at ~5 ml/min. Approximately 30 s were required to change
completely the bath contents.
Voltage Clamp Recording and Analysis--
Membrane currents were
recorded in nystatin-perforated patch configuration using an
Axopatch-1C amplifier (Axon Instruments). Nystatin forms
voltage-insensitive ion pores in the membrane patch that are somewhat
selective for cations over anions but are impermeant to
Ca2+ and other multivalent ions or molecules >0.8 nm in
diameter (29). This method, therefore, minimizes dialysis of
intracellular constituents with the internal pipette solution. Nystatin
was dissolved in Me2SO at a concentration of 50 mg/ml and
then added to the internal pipette solution to yield a final nystatin
concentration of 200 µg/ml. The patch pipettes were pulled from
borosilicate capillaries (Clark Electromedical Instruments, Pangbourne,
United Kingdom) using a Narishige puller (PP-83; Narishige, Tokyo,
Japan). We used patch pipettes with a resistance of 2-3
megaohms when filled with the above pipette solutions. The
electrical signals were displayed during the experiments on an
oscilloscope (Tektronix, TDS 210) and a chart recorder (Gould). Data
were digitized with pClamp software 5.7.1 (Axon Instruments) at a
sampling rate of 1-2 kHz and filtered at 5 kHz. Voltage clamp and data
acquisitions were performed by a digital interface (Digidata 1200, Axon
Instruments) coupled to an IBM-compatible computer using pClamp
software 5.7.1 (Axon Instruments) at a sampling rate of 1-2 kHz and
filtered at 5 kHz.
Statistics and Presentation of Data--
The results in the text
and in the figures are presented as means ± S.E.,
n = number of cells tested. Statistical analyses were
performed using the Student's t test. The difference
between two groups was considered to be significant when
p < 0.01 and not significant when p > 0.05.
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RESULTS |
Activation and Desensitization of
IKACh--
Acetylcholine-activated K+ current
(IKACh) was activated by adding 10 µM acetylcholine (ACh) to the bath solution, while the cell was voltage-clamped at the holding potential of
40 mV (Fig. 1A). Upon the application
of ACh, a rapid increase in outward current was observed. Despite the
continuous presence of ACh, the activation of
IKACh was not sustained at its peak, but the amplitude of IKACh decreased slowly. We regarded
this decrease as a result of ACh-induced desensitization of
IKACh. The ACh-induced desensitization was
recovered after washout of ACh, so that the amplitude of
IKACh at the second exposure to ACh in a 6-min
interval was similar to that at the first exposure. In subsequent
experiments, such a paired application of ACh was used for
investigating the effect of various signal molecules on regulation of
KACh channel, regarding the IKACh at
the first response as the control.

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Fig. 1.
Characteristics of ACh-activated
K+ current recorded with nystatin-perforated whole cell
clamp technique. A, chart recordings of the whole-cell
current at the holding potential of 40 mV. The dotted line
indicates zero current level. The application of 10 µM
ACh is indicated by the horizontal bar above the trace. ACh
was applied for 4 min, two times in a 6-min interval. The vertical
deflections of current trace are the responses to voltage ramps.
B, I-V relationships obtained at the points
indicated by a-d in A. C, the
I-V curves for net IKACh at peak and
at 4 min in ACh. Data were calculated from data in B. The
reversal potential was 83 mV.
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Characteristics of IKACh activation and
desensitization were further investigated from the current-voltage
(I-V) curves. I-V curves were obtained from the
current response induced by voltage ramps between +60 and
120 mV (at
a speed of ± 0.6 V s
1) from the holding
potential of
40 mV. The ramps were applied before ACh application
(a), at peak (b), 4 min in ACh (c),
and washout of ACh (d), as indicated in Fig. 1A.
Corresponding I-V curves were plotted in Fig. 1B:
the reversal potentials were shifted slightly to negative potentials
toward K+ equilibrium potential, and the
shape of I-V curves was changed by ACh. The degree of inward
rectification was less strong in the presence of ACh (b and
c) compared with that in control (a). A strong
inward rectification in "a" is considered to be typical for inward rectifying K+ currents, IRK (30). The
I-V curves for net IKACh were
obtained by subtracting the control curve (a) from the
I-V curves in the presence of ACh, as shown in Fig.
1C: "b-a" represents
IKACh at peak (IKACh,
peak), and "c-a" represents IKACh
at 4 min in ACh (IKACh, 4 min). The shape of
inward rectification and the reversal potential of two curves were not
different, indicating that the decrease in current amplitude during
exposure to ACh occurs uniformly over the voltage range tested.
Desensitization of IKACh Is Accelerated by
1-Adrenergic Agonist--
In Fig.
2A, 100 µM
phenylephrine (PE) was applied together at the second exposure to ACh.
From the continuous recording of current trace at
40 mV, it was
noticed that the process of desensitization was markedly accelerated by
PE, resulting in a greater reduction of IKACh
after the same period (Fig. 2A). But, the amplitude of IKACh at peak was not significantly affected by
PE. We regarded this increased desensitization by PE as PE-induced
desensitization. The effect of PE on IKACh
desensitization was well illustrated in I-V curves: the
difference between IKACh, peak
(b'-a') and IKACh, 4 min
(c'-a') in the presence of PE was significantly
greater compared with the control (Fig. 2, B and
C). But PE did not affect the degree of inward rectification
and the reversal potential, indicating that PE modulates
IKACh itself, rather than modulating other
current systems. PE-induced desensitization was also reversible after a
6-min recovery period, and the third exposure to ACh elicited an
outward current with a similar peak amplitude and desensitization (Fig.
2A).

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Fig. 2.
Effect of PE on IKACh.
A, the IKACh was activated by the
perfusion of 10 µM ACh. The applications of ACh and 100 µM PE are indicated by the horizontal bars
above the trace. B, the I-V curves for net
IKACh at peak and at 4 min in ACh during control
ACh exposure. C, I-V curves of net
IKACh during PE exposure. D, summary
of the amplitude of IKACh at peak and 4 min in
control and PE-treated cell. IKACh was measured
at 40 mV. The numbers in parentheses indicate numbers of
cells.
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The data were summarized in Fig. 2D. The amplitude of
IKACh was measured at
40 mV to minimize the
possible contamination of IRK and voltage-activated K+
currents. The amplitude of IKACh at peak was not
significantly different between control (775.90 ± 90.29 pA,
n = 12) and PE (644.96 ± 94.21 pA,
n = 9). However, the amplitude of
IKACh at 4 min in ACh was significantly smaller
in PE, indicating that desensitization was increased by PE. When the
extent of desensitization was determined as a proportion of the current
decrease during 4 min, it was 44.27 ± 2.38% (n = 12) in control and increased significantly to 69.34 ± 2.22%
(n = 9) by PE.
PLC, but Not PKC, Is Involved in the Increased Desensitization by
PE--
To elucidate the mechanisms for PE-induced
desensitization, we blocked each step of the signal transduction
pathway related with PE. When PKC inhibitor, calphostin C (2.5 µM), was pretreated before the application of PE at the
second exposure to ACh, acceleration of IKACh
desensitization by PE was still observed (Fig.
3A). To focus on the change in
desensitization, I-V curves for desensitized current were
plotted in Fig. 3B. It was noticed that desensitized current
in the presence of PE and calphostin C (b'-c')
was almost completely overlapped by that in the presence of PE only
(b-c). The extent of desensitization in the presence of PE
and calphostin C was 79.20 ± 4.46% (n = 4),
indicating no significant difference from that in the presence of PE
only (69.34 ± 2.22%). Furthermore phorbol 12-myristate
13-acetate (100 nM), a specific PKC activator, did
not mimic the effect of PE on the desensitization of
IKACh (n = 5, data not shown).
The amplitude of IKACh at peak was affected
neither by calphostin C nor by phorbol 12-myristate
13-acetate.

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Fig. 3.
Effect of calphostin C and neomycin on PE
effect. PE (100 µM) was applied together with ACh
(10 µM), resulting in acceleration of desensitization of
IKACh. Application of drugs are indicated by
horizontal bars. A, calphostin C (100 µM) was pretreated 2 min before the second application of
ACh and PE. B, difference I-V curve from data in
A, corresponding to the decrease in current amplitude during
4-min exposure to PE and ACh in the absence (b-c) and in the
presence of calphostin C (b'-c'). C,
neomycin (500 µM) was pretreated before the second
application of ACh and PE. D, difference I-V
relation from data in C, corresponding to the decrease in
current during 4-min exposure to PE and ACh in the absence
(b-c) or presence of neomycin (b'-c').
E, summary of the extent of desensitization in various
conditions. The numbers in parentheses indicate
numbers of cells. * indicates p < 0.01.
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We then tested the effect of PLC inhibitor, neomycin. When neomycin
(500 µM) was pretreated before the application of PE at the second exposure to ACh, the increase in desensitization by PE was
no longer observed (Fig. 3C). This finding suggests that PE-induced desensitization of IKACh
is antagonized by neomycin. The I-V curve for desensitized
current in the presence of neomycin and PE
(b'-c') was significantly smaller than that in
the presence of PE (b-a). The extent of desensitization in
the presence of PE and neomycin was 48.79 ± 3.95%
(n = 9), showing a significant difference from the
extent of desensitization in the presence of PE (69.34 ± 2.22%),
but not different from the control (44.27 ± 2.38%). Neomycin
itself did not significantly affect the activation of
IKACh and ACh-induced desensitization
(IKACh, peak: 759.20 ± 93.43 pA;
desensitization: 48.65 ± 3.76%, n = 7).
The extent of desensitization obtained in various conditions was
summarized in Fig. 3E. Based on these results, it is
suggested that PE-induced desensitization of
IKACh is the result of the activation of PLC,
but not through the activation of PKC.
Effect of the Depletion of PIP2 Pool by Wortmannin on
IKACh--
We postulated that PLC involvement is related
with PIP2, and this possibility was tested by using WMN (an
inhibitor of PI 3-kinase and PI 4-kinase). It was reported that WMN
inhibits the replenishment of PIP2 after the depletion of
PIP2 by the receptor-mediated activation of PLC (27).
Therefore, we examined whether the inhibition of PIP2
replenishment by WMN affects PE-induced desensitization of
KACh current and its recovery. In Fig.
4A, we first applied ACh and
PE simultaneously to induce the increased desensitization of
IKACh and confirmed the full recovery of
IKACh from desensitization after a 6-min washout
with normal Tyrode solution. Then the same series of experiment
was performed in the presence of 100 µM WMN (Fig.
4B). PE-induced desensitization of
IKACh was greatly accelerated by WMN, and
IKACh, 4 min became very smaller. The extent of
desensitization in the presence of WMN was 97.25 ± 7.63%
(n = 6), and this value was significantly greater than
64.22 ± 5.70% (n = 7) in the absence of WMN. In
contrast, WMN alone without PE did not significantly affect the
activation and desensitization of IKACh
(IKACh, peak: 603.14 ± 80.63 pA;
desensitization: 45.84 ± 4.07%, n = 6, data not
shown). These results suggest that blockade of PIP2
synthesis by WMN facilitated PE-induced desensitization, but not
ACh-induced desensitization.

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Fig. 4.
The effect of wortmannin on PE effect.
The applications of ACh, PE, and WMN are indicated by the
bars above the recording. A, recovery of the PE
effect on desensitization of IKACh.
B, in the presence of WMN, IKACh was
not fully recovered at the second application of ACh 6 min after
washout of PE and ACh. C, activation of
IKACh when the cell was pretreated with WMN and
PE for 3 min prior to ACh. D, summary data of the relative
amplitudes (percent) of IKACh, 4 min and
IKACh, peak after recovery in respect to
IKACh, peak obtained from the experiments A and
B. IKACh was measured at 40 mV. The
numbers in parentheses indicate numbers of
cells.
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IKACh, peak was not different, but
IKACh, peak was significantly smaller in the
presence of WMN, indicating that PE-induced desensitization of
IKACh was further accelerated by WMN. The extent
of desensitization in the presence of WMN was 97.25 ± 7.63%
(n = 6), and it was significantly greater than
64.22 ± 5.70% (n = 7) in the absence of WMN. In
contrast, WMN alone without PE did not significantly affect the
activation and desensitization of IKACh
(IKACh, peak: 603.14 ± 80.63 pA;
desensitization: 45.84 ± 4.07%, n = 6, data
not shown). These results suggest that blockade of PIP2
synthesis by WMN facilitated PE-induced desensitization, but not
ACh-induced desensitization.
The recovery from PE-induced desensitization was also affected by WMN
(Fig. 4B). In the continuous presence of WMN, the amplitude of IKACh, peak measured at the second exposure to ACh was only 19.65 ± 2.61% (n = 6) of the preceding
level (Fig. 4D), indicating that the recovery from
desensitization was significantly inhibited by WMN. WMN also inhibited
the recovery from ACh-induced desensitization, but to a lesser extent:
IKACh, peak measured at the second exposure to ACh in the
absence of PE was 70.92 ± 9.18% of preceding level
(n = 6, data not shown). The degree of inhibition is
comparable with the magnitude reduction of basal PIP2
levels in unstimulated cells by WMN in this period time (27),
suggesting that the incomplete recovery of ACh-induced desensitization
was the result of basal reduction of PIP2 level by WMN.
Facilitation of PE effect by WMN was further confirmed in Fig.
4C, where PIP2 pool in the membrane was depleted
before the first exposure to ACh by pretreating WMN and PE for 3 min.
The amplitude of IKACh, peak was only 178.33 ± 85.36 pA (n = 3), and this value was significantly smaller
than IKACh, peak in control (775.90 ± 90.29 pA,
n = 12). Above results imply that depletion of
PIP2 pool by PE causes a decrease in
IKACh and that the increase in desensitization
by PE can be explained by the same mechanism.
Effect of PE on IRK--
As well as KACh channel, IRK
is known to be regulated by PIP2 (4, 6). We examined
whether IRK is affected by the various substances used in the present
study. IRK was determined from the I-V curve, as described
previously in Fig. 1B. The amplitude was measured at
120
mV, where IRK is large. IRK was not affected either by PE (100 µM) or by WMN (100 µM). However, the
addition of PE in the presence of WMN decreased IRK by 31.41 ± 1.93%. These results suggest that the change of PIP2 level
induced by normal signal molecule such as PE may not contribute to IRK
regulation, although IRK is inhibited when PIP2 level is
reduced further down by inhibiting its replenishment.
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DISCUSSION |
The main question addressed in the present study is whether
PIP2 is acting as a signal molecule for the regulation of
native KACh channels. The results obtained can be
summarized as follows: 1) PE,
1-adrenergic agonist,
accelerates desensitization of the IKACh; 2)
PE-induced desensitization was inhibited by PLC inhibitor, neomycin,
but not by PKC inhibitor, calphostin C; 3) when wortmannin, an
inhibitor PI 3-kinase and PI 4-kinase was applied with PE, desensitization of IKACh was further
accelerated, and the recovery from desensitization was inhibited. From
these results it was suggested that KACh channels are
regulated by
1-adrenergic agonist through the depletion
of the PIP2 pool in plasma membrane.
Classical signal molecules produced by the activation of PLC-linked
receptors are IP3 and diacylglycerol. The role of
IP3 on ion channels has not been reported in cardiac
myocytes, but diacylglycerol may possibly contribute to the regulation
of ion channels via PKC. Desensitization of KACh channel
was known to be caused by phosphorylation of m2 muscarinic receptor
(31-34), so it is possible that PKC is involved in the increased
desensitization of KACh channel by PE. We tested this
possibility, but calphostin C, a PKC-specific inhibitor did not inhibit
PE action (desensitization of 79.20 ± 4.46%, Fig. 3,
A and E). Furthermore, the effect of PE was not
mimicked by direct pharmacological activation of PKC with phorbol
12-myristate 13-acetate. These results support the idea that the
mechanism for PE-induced desensitization was the depletion of
PIP2 rather than the production of PIP2
metabolites that may inhibit the KACh channel. Although we
did not carry out the biochemical measurements of PIP2
concentration in the present experiment, the decrease in
PIP2 concentration in the plasma membrane by PLC-linked
receptor has been reported previously in Chinese hamster ovary cells
and human neuroblastoma cells (26, 27). The involvement of
PIP2 in the PE effect was further supported by the finding
that the PE-induced desensitization became irreversible when the
replenishment of PIP2 was blocked by WMN (Fig.
4B). Furthermore when the PIP2 pool was depleted
by preincubation of PE and WMN, activation of the KACh
current was reduced (Fig. 4C).
IRK channels, on the other hand, showed a different response. In the
case of IRK, PE did not affect the channel activity, although PE with
WMN affected the channel activity. These data suggest that interaction
of IRK with PIP2 is stronger than that of KACh
channel, as suggested previously (4, 6, 35), and that the effect of
PIP2 depletion on IRK occurs at much lower concentration.
Another possibility that should be tested in future studies is a
co-localization of a specific PLC-linked receptor and a specific ion
channel. In this view, the PIP2 pool, which is regulated by
PE, may not be uniformly distributed over the whole membrane, but
localized closely with KACh channels.
It has previously been reported by other studies that several
PLC-linked receptor can inhibit KACh channel. Braun
et al. (36) reported that the selective
1-adrenergic agonist methoxamine reduced both the
IK1 and KACh current in rabbit
atrial myocytes. Yamaguchi et al. (37) reported that
endothelin-1 and endothelin-3 inhibited KACh current in
guinea pig atrial myocytes. Their observation is similar to the effect
of PE presented in this paper, but they failed to identify the
mechanism of the
1-adrenergic agonist or endothelin
induced inhibition. They only demonstrated that these effects were not
mediated by PKC or IP3. But it now seems to be very likely
that PIP2 is involved in those effects. Recently, channel
expression studies have demonstrated that PLC-linked receptors inhibit
GIRK1/GIRK4 channels (38) or KATP channels (39), and these
effects were mediated by depletion of the PIP2 pool in membrane.
The functional consequence of accelerated desensitization of
IKACh by
1-adrenergic receptor
may be an early cessation of parasympathetic effect in the continuous
presence of ACh. This discovery may be of particular importance, since
it provides a novel pathway for sympathetic-parasympathetic
interaction. Interaction between sympathetic and parasympathetic system
was recognized early in various experimental conditions, and this
interaction is also considered to be of clinical importance. However,
the precise signal transduction pathways involved in this interaction are not fully understood, except the inhibition of adenylate cyclase by
acetylcholine as a mechanism of parasympathetic antagonizism to
sympathetic stimulation. To our knowledge, the pathway presented in the
present study seems to be the first report of a reciprocal pathway
through which sympathetic stimulation can antagonize parasympathetic activity.
In conclusion,
1-adrenergic agonist accelerates the
desensitization of KACh channel through the regulation of
PIP2 pool, suggesting that the receptor mediated regulation
of the PIP2 pool may play an important role in the control
of cellular function through the modulation of ion channels.