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INTRODUCTION |
In the heart, predominantly in supraventricular tissue, in various
neurons and endocrine cells, stimulation of receptors coupled to
pertussis toxin-sensitive G proteins (Gi/o) activates G
protein-gated inward rectifying K+
(GIRK)1 channels, resulting
in reduction of excitability (1-3). Upon exposure of a myocyte to an
appropriate receptor agonist, the current is activated within less than
1 s. Activation is followed by desensitization, i.e. a
decay of current, made up of several components with different kinetics
of onset and recovery (4). Slow components seem to represent receptor
desensitization, presumably mediated by phosphorylation via receptor
kinase(s) and subsequent events such as receptor internalization
and down-regulation (5, 6), but a fast component, developing with a
time constant in the range of seconds, is localized downstream of the
activating receptor. Several mechanisms have been proposed for fast
desensitization. Shui et al. (7) suggested channel
dephosphorylation as the underlying mechanism, whereas Hong et
al. (8) reported the contribution of a nonidentified cytosolic
protein. The major properties of fast desensitization, such as the rate
of current decay, its membrane-delimited nature, and its dependence on
receptor density, can be accounted for by a model relating fast
desensitization, analogous to activation, to the nucleotide exchange
and hydrolysis cycle of the G protein (9). More recently, an attractive
novel mechanism has been proposed (10). These authors suggested that activation and fast desensitization of atrial IK(ACh) result from costimulation of two muscarinic receptors: the
M2 subtype (M2AChR) which causes activation via
Gi/o, whereas stimulation of the M3 subtype
(M3AChR) via activation of Gq/11 stimulates phospholipase C (PLC) and subsequent depletion of phosphatidylinositol bisphosphate (PtIns(4,5)P2) in the inner leaflet of the
membrane. Such a mechanism would be in line with the finding that
PtIns(4,5)P2 is an important cofactor for activation of
GIRK channels (11-13) and other members of the Kir channel family
(14-16) and supports the notion that PtIns(4,5)P2 meets
the criteria of a receptor-controlled second messenger (17). However,
this hypothesis is inconsistent with one major property of acute
desensitization, namely its heterologous nature. In atrial cells
IK(ACh) can be activated not only by M2AChR but
also, e.g. by A1 adenosine receptors (18, 19)
and a sphingolipid receptor of the EDG family (20, 21). These
studies have shown that ACh rapidly desensitizes the response to Ado
and vice versa; correspondingly, sphingosine 1-phosphate,
presumably via a receptor of the EDG family (22), desensitizes
the response to ACh and vice versa. In rat atrial myocytes
overexpression of A1-receptors, a classic Gi/o-coupled species, not only increased density of
Ado-induced IK(ACh) but also substantially enhanced fast
desensitization of current activated via stimulation of this receptor
(19).
In the present study we investigated the effect of stimulating two
different intrinsic Gq/11-coupled receptors on
IK(ACh) in atrial myocytes. The results support the notion
that stimulation of the PLC pathway via depletion of
PtIns(4,5)P2 causes inhibition of IK(ACh).
However, this inhibition is slower than fast desensitization by a
factor of 10. Moreover there is no evidence of significant expression
of functional expression of a Gq/11-coupled muscarinic
receptor, such as M3AchR, in rat atrial myocytes.
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EXPERIMENTAL PROCEDURES |
Isolation and Culture of Atrial Myocytes--
Experiments were
performed with local ethics committee approval. Wistar Kyoto rats of
either sex (around 200 g) were anesthetized by intravenous
injection of urethane (1 g/kg). The chest was opened, and the heart was
removed and mounted on the cannula of a sterile Langendorff apparatus
for coronary perfusion at constant flow. The method of enzymatic
isolation of atrial myocytes has been described elsewhere (see Ref. 4).
The culture medium was fetal calf serum-free bicarbonate-buffered M199
(Life Technologies, Inc.) containing 25 µg/ml gentamycin (Sigma) and
25 µg/ml kanamycin (Sigma). Cells were plated at a low density
(several thousand cells/dish) on 36-mm culture dishes. Medium was
changed 24 h after plating and then every 2nd day. Myocytes were
used experimentally from day 0 until day 4 after isolation. No effects
of time in culture were found for the key experiments.
Solutions and Chemicals--
For the patch clamp measurements an
extracellular solution of the following composition was used (in
mM): 120 NaCl, 20 KCl, 2.0 CaCl2, 1.0 MgCl2, 10.0 Hepes/NaOH, pH 7.4. The solution for filling
the patch clamp pipettes for whole cell voltage clamp experiments
contained (in mM) 110 potassium aspartate, 20 KCl, 10 NaCl,
1.0 MgCl2, 2.0 MgATP, 2.0 EGTA, 0.01 GTP or 0.5 GTP
S, 10.0 Hepes/KOH, pH 7.4. Standard chemicals for electrophysiological experiments were from Merck (Darmstadt, Germany). EGTA, Hepes, MgATP,
adenosine, GTP, acetylcholine chloride, phenylephrine, methoxamine, and
endotheline-1 were from Sigma; PtIns(4,5)P2, Pasteurella multocida toxin and U23187 were from Calbiochem. 4-Diphenylacetoxy-N-methypiperidine (4-DAMP) was from
Tocris. Drugs were prepared as concentrated stock solutions either in distilled water or dimethyl sulfoxide. PtIns(4,5)P2 was
dissolved in pipette solution at a nominal concentration of 500 µM. The solution was sonicated intermittently on ice for
30 min. Sonification was repeated each time before filling a new
pipette. PtIns(4,5)P2 solutions were used for 1 day only.
Current Measurement--
Membrane currents were measured using
whole cell patch clamp. Pipettes were fabricated from borosilicate
glass and were filled with the solution listed above (DC resistance
4-6 megohms). Currents were measured by means of a patch clamp
amplifier (LM/EPC 7, Darmstadt, Germany). Signals were analog filtered
(corner frequency of 1-3 kHz), digitally sampled at 5 kHz, and stored
on a computer, equipped with a hardware/software package (ISO2 by MFK,
Frankfurt/Main, Germany) for voltage control and data acquisition.
Experiments were performed at ambient temperature (22-24 °C). Cells
were voltage clamped at
90 mV, i.e. negative to
EK (
50 mV), resulting in inward K+ currents.
Current-voltage relations were determined by means of voltage ramps
from
120 mV to +60 mV. Rapid superfusion of the cells for application
and withdrawal of different solutions was performed by means of a
custom made solenoid-operated flow system that permitted switching
between up to six different solutions (t1/2
100 ms). Performance of this system was dependent on the positioning of
the outlet tube in relation to the cell studied. This was routinely
optimized by measuring the time course of the blocking action of
Ba2+ on IK(ACh). As shown previously, activation kinetics of IK(ACh) was not limited by the rate
of agonist application (19).
RT-PCR--
RNA from cell samples was isolated using
Trizol reagent (Life Technologies, Inc.). RNA in 20 µl of RNase-free
H2O (Qiagen, Hilden, Germany) was treated with 7.5 units of
DNase I (Amersham Pharmacia Biotech) for 15 min at 37 °C. DNase was
inactivated by adding 2.5 µl of 25 mM EDTA and heating at
65 °C for 10 min.
The culture medium was replaced by rinsing the cells for 30 min with a
solution containing (in mM): 120 NaCl, 20 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes/NaOH, pH 7.4. 0.1%
diethyl pyrocarbonate was added. The solution was autoclaved before
use. 10 myocytes were aspirated into glass capillaries with a
~30-µm tip diameter using a water hydraulic micromanipulator
attached to an inverted microscope. Alternatively, the cells were
scratched from the dish, and a corresponding volume of the cell
suspension was aspirated, which should contain also nonmyocyte cells.
Capillaries were rinsed with hexamethyldisilazane (Sigma) and baked for
7 h at 240 °C before use. The content of the capillary (<0.5
µl) was expelled into a tube filled with 5 µl of lysis buffer
containing: 0.8% (w/v) Nonidet P-40 substitute (Fluka, Buchs,
Switzerland), 100 µg/ml yeast tRNA, 10 mM dithiothreitol (both from Life Technologies, Inc.), and 8 units/µl RNAguard
(Amersham Pharmacia Biotech).
In the experiments on selected myocytes 2.5 µl of RNase-free
H2O (Qiagen) and 0.5 µl of oligo(dT)15 primer
(500 µg/ml; Promega, Madison, WI) were added to the lysis solution
and incubated at 42 °C for 10 min. 11 µl of RT mixture was added,
containing 2.4 µl of RNase-free H2O, 4 µl of first
strand buffer (5-fold), 2 µl of dithiothreitol (100 mM),
1 µl of SuperScript II RT (200 units/µl; all from Life
Technologies, Inc.) and 1.6 µl of dNTP mixture (6.25 mM
each; Epicentre Technologies, Madison, WI). The RNA was transcribed for
1 h at 42 °C. The reaction was stopped by heating to 95 °C
for 10 min.
The extracted RNA from suspended atrial cultures (see above) was
transcribed by adding 10 µl of first strand buffer, 0.5 µl of
SuperScript II RT, 5 µl of dithiothreitol, 1.6 µl of dNTP mixture, 1.6 µl of oligo(dT)15 primer, 0.5 µl of RNAguard, and
7.2 µl of RNase-free H2O (same reagents as above). The
sample was incubated at 42 °C for 1 h before heat inactivation
at 95 °C for 10 min.
cDNA from the myocyte RT was amplified by dividing the content of
the RT tube into two aliquots of 9.5 µl and adding 40.5 µl of
PCR mixture to each tube. The PCR mixture contained 34 µl of
distilled water (Life Technologies, Inc.), 0.5 µl of Taq
DNA polymerase (5,000 units/ml), 5 µl of PCR buffer (10-fold; both Amersham Pharmacia Biotech), 0.5 µl of sense primer, 0.5 µl of antisense primer (both 100 pmol/µl; Life Technologies, Inc.), either
for M3AChR and GAPDH or M2AChR and GAPDH.
PCR from cDNA of atrial cell suspension RT was performed by mixing
1 µl of RT product, 16 µl of distilled water, 2 µl of PCR buffer,
0.4 µl of dNTP mixture, 0.2 µl of M3 primer (sense), 0.2 µl of M3
primer (antisense), and 0.2 µl of Taq DNA polymerase (same
reagents as above).
The sequences of primers were as follows: M2AChR,
5'-TGGCTTGGCTATTACCAGTC-3' (sense) and 5'- ACGATGAACTGCCAGAAGAG-3'
(antisense); M3AChR, 5'-GTCGCTGTCACTTCTGGTTC-3' (sense) and
5'-CGGCAGACTCTAACTGGATG-3' (antisense); GAPDH, 5'-TCCGCCCCTTCCGCTGAT-3'
(sense) and 5'-CACGGAAGGCCATGCCAGTGA-3' (antisense). The primers
amplify a 340-base pair fragment of GAPDH, a 468-base pair fragment of
M2AChR, and a 378-base pair fragment of M3AChR.
Samples were overlaid with mineral oil and incubated in a thermal
cycler (PTC-150, MJ Research, Watertown, MA), first for 5 min at
94 °C, followed by 50 cycles for 45 s at 94 °C, 1 min at
52 °C, 2 min at 72 °C, and a final extension at 72 °C at 10 min. 10 µl of RT-PCR products was subjected to electrophoresis on a
2% agarose gel, stained with ethidium bromide, and photographed under
UV transillumination.
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RESULTS |
Definition of Fast Desensitization of Atrial
IK(ACh)--
As shown previously, atrial
IK(ACh) in the presence of an agonist shows different
components of desensitization, depending on multiple factors such as
agonist concentration, receptor density, speed, and duration of agonist exposure (4, 19, 23). The slower components, occurring upon agonist
exposure on a time scale of minutes to hours, are supposed to reflect
receptor desensitization, analogous to other receptor-G
protein-effector pathways (4, 23-25). The fastest component, which
occurs without a measurable delay upon exposure to ACh, represents a
specific property of the pathway under study.
The key properties of fast desensitization are defined in Fig.
1. 1A shows a representative
response of a native myocyte to a saturating concentration of ACh (10 µM) superfused for 50 s. After rapid activation of
inward IK(ACh), the current decayed to a quasi steady-state
level of 54% of the peak value. The decay could be approximated by a
single exponential with a half-time of 5.28 ± 0.43 s
(n = 30). A second slower phase of desensitization
within the short period of time contributed to the total decay in
current with less than 5% and thus could be ignored when exposure to
ACh did not exceed 1 min or so.

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Fig. 1.
Properties of fast desensitization of atrial
IK(ACh). Panel A, original trace showing
desensitizing inward current induced by rapid superfusion with 10 µM ACh-containing solution and complete recovery from
desensitization 30 s after washout. Panel B,
heterologous nature of fast desensitization. Concentrations of ACh and
adenosine were 10 and 100 µM, respectively. The
arrow indicates the amplitude of adenosine-induced
IK(ACh). Rapid vertical deflections represent changes in
membrane current induced by voltage ramps from 120 to + 60 mV, which
were routinely superimposed and served to identify IK(ACh)
by its strongly inward rectifying I/V relation and to check for
stability of the recording conditions. In most figures these have been
cut off for graphical reasons. Panel C, representative
response of a cell to two consecutive exposures to a high concentration
of ACh (100 µM) for ~2 min; a, low speed
recording of membrane current; b, superimposed rising phases
on an expanded time base. Numbers correspond to the labeling
in a. The trace labeled 2 has been scaled up
vertically to match the amplitude of 1.
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We have shown previously that this second component is likely to
reflect desensitization of the M2AChR (4). A characteristic feature of fast desensitization of atrial IK(ACh) is its
rapid reversibility. 30 s after starting washout of ACh-containing solution, a second challenge by the agonist resulted in a current of
identical amplitude, i.e. rapid desensitization was reversed as soon as IK(ACh) had decayed to its basal level. Longer
periods of exposure to ACh and/or substantially higher concentrations
could result in a significant reduction of the second response, most
likely because of receptor desensitization.
A typical experiment, qualitatively representative of 20 myocytes
studied using such a protocol, is illustrated in Fig. 1C. The cell was challenged by a high concentration of ACh (100 µM) for about 2 min. A second exposure after an ACh-free
period of 140 s resulted in a current of 68% of the initial peak
amplitude (C, a); the fast desensitizing
component was almost completely abolished. The half time of activation,
which, at saturating agonist concentrations, is closely related to
functional density of the receptor (4, 19, 23, 26), was increased from
120 to 210 ms (C, b). A slowing in activation
rate, however, per se results in a diminution of the fast
desensitizing component (19). Therefore, comparing peak currents of two
responses elicited in succession provides little reliable information
on fast desensitization because receptor desensitization,
i.e. a reduction in density of functional receptors, might
contribute to reduction of the amplitude of the second response (10).
Another property defining fast desensitization, in contrast to receptor
desensitization, is represented by its heterologous nature. Fig.
1B shows that activation of IK(ACh) by 10 µM Ado, a concentration that is saturating for this
agonist, via stimulation of A1 receptors, induced a current
of about 35% of the peak current that could be induced by ACh. As
shown previously, sensitivity to Ado in this system is limited by low
density of functional A1 receptors (19). Superfusion of 10 µM ACh in the presence of Ado resulted in a peak inward
current of only 70% of the current induced by 2 µM ACh
alone. Thus, although the Ado-induced current did not show
desensitization in terms of a visible kinetic component, the system was
desensitized by exposure to Ado within few seconds. This subadditive
behavior, first described by Kurachi et al. (27), has been
found for various combinations of receptor agonists, such as
M2AChR/sphingolipid, or M2AChR/
-adrenergic (21, 28). These properties of fast desensitization are contradictory to
the hypothesis that it is mediated by stimulation of a muscarinic M3 receptor, unless one would assume that stimulation of
each receptor causing activation of IK(ACh) is paralleled
by costimulation of a Gq/11-coupled subtype causing desensitization.
Inhibition of IK(ACh) by
1-Adrenergic
Receptors and ETA Receptors--
On principle, information
on how activation of PLC interferes with IK(ACh) can be
obtained by stimulation of any type of putative PLC-linked receptor
expressed in the system under study. In line with a previous
publication (29), stimulation of
1-adrenergic receptors
resulted in a small activation of IK(ACh) and,
concomitantly, an inhibition of IK(ACh) activated by brief
pulses of ACh. A representative example is illustrated in Fig.
2. Whereas there was distinct inhibition
in all experiments of this type, the activation could be very small or
absent. Effects comparable to those observed upon
1-adrenergic stimulation were evoked by endothelin-1
(ET-1) (not shown, compare Fig. 3) in
line with a previous publication (30).

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Fig. 2.
Simultaneous activation and inhibition of
IK(ACh) by Phe. 2 µM ACh and 100 µM Phe were superfused as indicated. The
arrowhead denotes a gap of 30 s in duration in the
recording. The identities of currents activated by ACh and Phe were
verified by their identical current-voltage relations (not
shown).
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Fig. 3.
Inhibition of IK(ACh) in
GTP S-loaded myocytes by Phe and ET-1.
Pipette-filling solutions contained 500 µM GTP S.
Panel A, representative recording showing stable,
irreversible activation of IK(ACh) by repetitive exposure
of a myocyte to 2 µM ACh. Panels B and
C, representative traces showing inhibition of
IK(ACh) by 100 µM Phe and by 10 nM ET-1.
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To study the inhibitory effect of the
1-adrenergic
agonist or ET-1 in isolation and to obtain information on its kinetic properties, IK(ACh) was routinely activated by loading the
cells with the hydrolysis-resistant GTP analogue GTP
S. To accelerate
activation, the cells were repetitively exposed to ACh until a steady
current level was reached. Fig. 3A shows a control trace.
Once activated, the current remained fairly constant for several
minutes of recording. The sample traces in Fig. 3, B and
C, illustrate that 100 µM phenylephrine (Phe) and 10 nM ET-1 both caused an inhibition of
IK(ACh). A comparable effect was also found if 100 µM methoxamine was used as
-agonist (not shown).The
summarized data in Fig. 4A
demonstrate that both,
1-adrenergic stimulation as well
as stimulation of ETA receptors caused inhibition of
IK(ACh) in GTP
S-loaded myocytes to about the same degree
and with identical time course. The mean half-times of inhibition were
53.4 ± 7.9 s (Phe, n = 10) and 57.3 ± 8.8 s (ET-1, n = 10), respectively. On average,
the GTP
S-activated current in the absence of Phe or ET-1,
respectively, decayed spontaneously by ~20% within 3 min (Fig.
4A). The identical kinetics of inhibition by the two receptor agonists suggest that it is not determined by the receptor species but by downstream reactions of a common signaling pathway. Compared with fast desensitization (compare Fig. 1), inhibition of
IK(ACh) by activation of
1-adrenergic or
ETA receptors was slower by about 1 order of magnitude. The slow time course of inhibition is unlikely to reflect slower activation of the G protein coupling to these receptors when GTP is exchanged by
GTP
S. At least for the M2AChR-Gi/o pathway,
using activation of IK(ACh) as an assay, activation rates
were identical few seconds after breaking the patch under the pipette,
i.e. before equilibration of the cytosol with GTP
S, and
about 30 s later, when the response to ACh was completely irreversible, as shown in Fig. 4B,which is qualitatively
representative of 10 experiments using such a protocol.

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Fig. 4.
Panel A, time course of
IK(ACh) inhibition by Phe and ET-1, summarized from
experiments as shown in Fig. 3. As a routine, Phe or ET-1 was applied
~60 s after getting access to the cytoplasm by rupturing the patch
under the tip of the patch clamp pipette. This corresponds to time zero
in plots of fractional current versus time also for the
control group. Data represent mean values ± S.D.
(n = 10 for each group). Panel B, activation
of IK(ACh), i.e. receptor-controlled activation
of Gi/o, is fast in myocytes loaded with GTP S.
a, low speed recording of membrane current. The first
challenge by 10 µM ACh started 4 s after rupturing
the patch under the recording pipette, i.e. when the
concentration of GTP S should be low, and activation of
Gi/o should predominantly reflect GTP binding. The rate of
activation upon a second exposure, 23 s after washout, was
identical, as illustrated by the expanded traces (b). The
response labeled 2 has been expanded vertically and
superimposed on response 1. A third exposure to ACh
(3) demonstrates that the current was activated maximally by
the second exposure.
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The slow decay of GTP
S-activated IK(ACh) by about 20%
within 3 min in the control group could reflect an inhibitory effect
caused by costimulation of putative intrinsic Gq/11-coupled
muscarinic receptors. However, exposure of a GTP
S-loaded cell to a
high concentration of ACh (100 µM), following activation
of IK(ACh) either by ACh at a low concentration (2 µM) or by adenosine via A1 receptors, i.e. without prior muscarinic stimulation, did not cause any
inhibition, as shown in Fig. 5. After
activation by adenosine and subsequent exposure to 100 µM
ACh, the decrease in current after 3 min was 16.2 ± 4.8%
(n = 6), which was not significantly different from the
decrease without exposure to ACh (18.1 ± 4.2%, n = 12). We propose that the slight "spontaneous" decay of
IK(ACh) resulted from slow receptor-independent activation
by GTP
S of the G protein involved in the inhibitory pathway. In
contrast to a recent report (10), from the data presented so far there is no evidence that intrinsic M3AChRs are involved in
regulating IK(ACh) in adult atrial myocytes.

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Fig. 5.
ACh does not cause inhibition of
IK(ACh). Panel A, in a GTP S-loaded
myocyte IK(ACh) was activated by a brief (4-s) pulse of 2 µM ACh. Activation was maximal because before application
of ACh the cell had been dialyzed with GTP S for 15 s, which
caused some "spontaneous" inward current. The dashed
line indicates the current level immediately after rupturing the
membrane patch. 100 µM ACh applied for 2 min did not
cause inhibition, but a subsequent exposure to 100 µM Phe
resulted in a decrease in current by about 2 nA. Panel B,
IK(ACh) in a GTP S-dialyzed myocyte was activated
maximally by three exposures to 100 µM Ado. 100 µM ACh failed to cause inhibition. 2 mM
BaCl2 was applied briefly in this experiment to measure the
background current level.
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Whereas the M2AChR represents the classic cardiac ACh
receptor, the issue of expression of other subtypes in cardiac myocytes is controversial. Identification of subtypes on pharmacological grounds
only is problematic, as selectivity of the ligands presently available
is limited (see Fig. 7). Moreover, positive data on global
M3AChR expression in the heart, without identification of
the cell type, are functionally meaningless. In rat ventricular myocytes no M3AChR transcripts were detected using single
cell RT-PCR, which was confirmed by Southern blot analysis and
immunocytochemistry, whereas these cells were positive for both
M1AChR and M2AChR (31). Corresponding data are
not available for atrial myocytes.
In atrial myocytes collected under optical control no
M3AChR transcripts could be detected in the RT-PCR
products, as shown in Fig. 6A.
This gel, which is representative of five experiments, is negative for
M3AChR transcripts in identified atrial myocytes, whereas
transcripts for GAPDH and M2AChR could be detected. On the
other hand, using the same primers, M3AChR transcripts
could be detected in samples obtained from suspended cultures (Fig. 6B), which might reflect expression of this subtype in
vascular smooth muscle and/or endothelial cells, which contaminate the cultures, though at a low level. Although negative RT-PCR data alone
cannot be taken as a proof that the corresponding protein is not
expressed, the lack of a signal supports our conclusion drawn from the
functional data that in adult rat atrial myocytes ACh does not
stimulate a Gq/11-coupled muscarinic receptor subtype.

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Fig. 6.
Panel A, gel electrophoresis of RT-PCR
products amplified from identified atrial myocytes. As described under
"Experimental Procedures," 10 myocytes had been picked under
optical control for the RT reaction. The solution containing the RT
products was split and used in two different PCRs with either primers
for the M2AChR and GAPDH (second and third
lanes from the left) or M3AChR and GAPDH
(fourth and fifth lanes). Panel B, the
message for the M3AChR is apparent in RT-PCR products from
samples of suspended atrial cultures (rightmost lane) but
not in atrial myocytes (second from left), which
are positive for GAPDH (third lane).
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The hypothesis that fast desensitization in atrial cells is mediated by
M3AChR (10) was primarily based on pharmacological evidence. 4-DAMP, a compound described as a selective
M3AChR antagonist, in that study resulted in a reduction of
desensitization or a desensitizing component of ACh-induced
IK(ACh) in atrial cells. As shown in Fig.
7, however, selectivity of 4-DAMP for
muscarinic receptor subtypes is poor. This compound, at 10 nM, caused a reversible reduction of the current induced by
2 µM ACh by about 40%. As with any other muscarinic
antagonist, such as atropine, inhibition of the current by 4-DAMP was
paralleled by slowing of activation and blunting of the rapidly
desensitizing component. Because the current in GTP
S-loaded myocytes
was not sensitive to 4-DAMP (not shown) the compound most likely also acts as an M2AChR antagonist. This is in line with the
reported inhibition constants ranging from 4 × 10
9 M to 1.6 × 10
8 M for the M2AChR
and from 1.2 × 10
9 M to
5 × 10
10 for the M3AChR
(32). Thus, despite its frequent use as pharmacological tool, this
compound is not sufficiently selective to be suitable for
discriminating between M3AChR and M2AChR.

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Fig. 7.
4-DAMP reversibly inhibits
IK(ACh) and blunts the fast desensitizing component.
Pipette-filling solution contained 50 µM GTP. 10 µM ACh and 10 nM 4-DAMP were applied as
indicated.
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Inhibition of IK(ACh) Is Mediated by the PLC-linked
Signaling Pathway--
1-Adrenergic receptors and
ETA receptors are supposed to couple preferentially but not
exclusively to G proteins of the Gq/11 family. The
subsequent step in the common pathway controlled by this class of G
proteins is activation of PLC
, yielding the second messengers
inositol (3,4,5)trisphosphate (InsP3) and
diacylglycerol from hydrolysis of PtIns(4,5)P2. To verify
the contribution of this class of G proteins to inhibition of
IK(ACh), myocytes were pretreated with P. multocida toxin, a protein toxin that has been described to
uncouple Gq proteins from their receptors, presumably by
first causing activation followed by a modification that prevents
reactivation after deactivation (33, 34). In cells pretreated with the
toxin (1 µg/ml for ~ 20 h), activation of
IK(ACh) by M2AChR and/or intracellular GTP
S
was not affected (Fig. 8). However,
inhibition of IK(ACh) by 100 µM Phe was
completely abolished. This was also confirmed in four experiments using
10 nM ET-1 (not shown). Contribution of Gq/11
to mediating the inhibition is consistent with the notion that
activation of PLC represents the subsequent signaling step, although
contributions by alternative mechanisms cannot be ruled out a
priori, such as direct inhibitory actions of
G
dimers containing the
5
subunit (35). A frequently used PLC inhibitor is the aminosteroid U
73122. Because many side effects not related to inhibition of PLC have
been described for this compound, its use as pharmacological tool is
limited and requires careful control experiments. This compound has
been used previously to support the hypothesis that activation of PLC
by M3AChR and subsequent depletion of
PtIns(4,5)P2 represent the mechanism of fast
desensitization. As shown in Fig. 9,
however, U 73122 at 1 µM caused a reduction in
GTP
S-activated IK(ACh) by about 50%. Although this was
not investigated further in detail, this compound is likely to act as a
blocker of Kir3.0 channels and thus is not suited as a tool to study
PLC-related effects in this system.

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Fig. 8.
-Receptor-induced inhibition of
IK(ACh) is abolished by treatment with P. multocida toxin. Panel A, representative
current recording from a myocyte treated with the 1 µg/ml toxin ( 20
h). 10 µM ACh and 100 µM Phe were applied
as indicated. Panel B, summarized data. Untreated cells were
from the same batches as P. multocida toxin-treated cells.
The difference between the control group and the P. multocida toxin-treated group was not significant.
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Fig. 9.
Inhibition of
GTP S-activated IK(ACh) by U
73122. The current was activated by brief exposure to ACh as in
Fig. 2 (not shown). U 73122 was applied as indicated.
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Receptor-induced Inhibition of IK(ACh) Is Reduced by
PtIns(4,5)P2--
We next tried to obtain further
information on the signaling mechanism subsequent to underlying slow
inhibition of IK(ACh) via Gq/11-coupled
receptors. Whereas InsP3 is the ubiquitous
Ca2+-mobilizing second messenger, diacylglycerol causes
activation of protein kinase C. 100 µM
12-O-tetradecanoylphorbol-13-acetate, a potent activator of
PKC, in 10 cells studied failed to mimic the effects of
-adrenergic
agonists or ET-1, respectively (not shown), suggesting that protein
kinase C is not causally involved. As in the present study a moderate
pipette Ca2+-buffering capacity (2 mM EGTA) was
used, Ca2+ released from InsP3-sensitive stores
as mediator cannot be excluded. Supplementing the pipette solution with
a 10 mM concentration of the fast Ca2+ chelator
BAPTA did not affect inhibition of IK(ACh) by Phe (10 cells, not shown), suggesting that
Ca2+-dependent processes are not involved.
In a number of recent publications evidence has been provided that
PtIns(4,5)P2 represents a cofactor for activation of Kir3.0 channels by G
(11, 36) and is also
required for activity of other inward rectifying K+
channels (13, 16, 17, 37, 38). We therefore studied whether loading of
atrial myocytes with PtIns(4,5)P2 via the pipette filling
solution affects properties of macroscopic IK(ACh) and/or
1-adrenergic inhibition. Fig.
10A demonstrates that
inclusion of PtIns(4,5)P2 (nominally 500 µM)
in the pipette without GTP
S had no effect on amplitude and
desensitization properties of IK(ACh). This was found in
six out of six myocytes loaded with PtIns(4,5)P2. On the
other hand, supplementation of the pipette solution with PtIns(4,5)P2 caused a significant reduction of the
Phe-induced inhibition of GTP
S-activated IK(ACh). Representative traces from this series of experiments are shown in Fig.
10B. As shown in Figs. 2 and 4, 100 µM Phe
caused an inhibition of steady-state IK(ACh) by about 70% (C). These data suggest first that under normal conditions
the concentration of PtIns(4,5)P2 in the membrane is not a
limiting factor for activation of IK(ACh). Second,
inhibition of IK(ACh) by heptahelical receptors activating
PLC is caused by depletion of PtIns(4,5)P2. Moreover,
depletion of PtIns(4,5)P2 is not causally related to fast
desensitization.

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Fig. 10.
Loading of myocytes with
PtIns(4,5)P2. Panel A, receptor-activated
IK(ACh) in the absence of intracellular GTP S is not
affected by supplementing the pipette solution with
PtIns(4,5)P2. The first response to ACh was induced within
6 s after rupturing the patch under the recording pipette.
Panel B, inhibition of GTP S-activated IK(ACh)
is reduced by adding PtIns(4,5)P2 to the pipette solution
(representative recording). Panel C, control measurement
from a cell of the same batch as B without
PtIns(4,5)P2. Panel D, summarized data
(n = 6 for either group).
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DISCUSSION |
The present study has provided evidence that activation of
-adrenergic and ETA receptors causes inhibition of
atrial IK(ACh) by depletion of PtIns(4,5)P2.
The rate of inhibition with a half-time of around 50 s was
identical for the two receptors, suggesting a common signaling
mechanism. Interestingly, the rate of inhibition was similar to the
rate of inhibition of heterologously expressed K(ATP)
channels by stimulation of coexpressed M1AChR receptors, described recently as an example of a receptor-controlled signal that
is mediated via depletion of PtIns(4,5)P2 (17).
The GIRK (Kir3.0) channel complex represents the paradigmatic target of
G protein 
subunits. It is generally assumed that physiological
activation of Kir3.0 channels is specific to 
subunits of
pertussis toxin-sensitive G proteins (Gi/o),
although examples of activation under certain conditions of atrial
IK(ACh) via the Gs pathway have been presented
recently (28, 39).
Apart from G
regulation, channel activity
has been shown to be modulated by PtIns(4,5)P2 and
intracellular [Na+]. Sensitivity to
PtIns(4,5)P2 has also been demonstrated for other members
of the Kir channel family (12, 15, 16, 37). It remained unclear,
however, whether PtIns(4,5)P2 is a necessary cofactor for
Kir channels or if PtIns(4,5)P2 acts as a signaling molecule in terms of a second messenger. More recently, Xie et al. (17) demonstrated that PLC-coupled receptors can regulate K(ATP) channels composed of Kir6.2 and SUR2A subunits via
depletion of PtIns(4,5)P2 in an heterologous expression
system, providing direct evidence that phosphoinositides act as
messengers in a receptor-controlled signaling pathway. The
physiological relevance of modulation of Kir channels by
PtIns(4,5)P2 was also demonstrated in an elegant study on
expressed K(ATP) channels (37). These authors found a
decrease in ATP sensitivity of K(ATP) in cells coexpressing
a phosphoinositide-4-phosphate 5-kinase, which contributes to
up-regulating the levels of PtIns(4,5)P2 and other phosphoinositides.
The notion of a second messenger role of PtIns(4,5)P2 was
supported by Kobrinsky et al. (10), who reported that
activation of PLC-coupled receptors caused fast desensitization of
native IK(ACh) in rat neonatal myocytes and Kir3.1/Kir3.4
currents expressed in a cell line. According to that study, fast
desensitization of M2AChR-activated IK(ACh) in
atrial myocytes results from PLC activation via costimulation of a
PLC-linked muscarinic receptor subtype (M3AChR). The
present findings confirm that activation of PLC-coupled receptors
causes inhibition of atrial IK(ACh) in a PtIns(4,5)P2-sensitive manner. However, they disprove the
hypotheses that intrinsic M3AChRs are involved in
regulation of atrial IK(ACh) and that
PtIns(4,5)P2-depletion causes fast desensitization.
First, fast desensitization of atrial IK(ACh) at room
temperature has a half-time of in the order of 5 s, whereas
inhibition of IK(ACh) by stimulation of PLC-linked
receptors was slower by a factor of 10. The slow rate of inhibition is
an intrinsic property of the signaling pathway and does not result from
the experimental condition (loading with GTP
S) because the rate of
signaling via Gi/o was not affected by this condition.
Second, ACh did not induce a measurable inhibition of
IK(ACh).
Third, we have shown previously that atrial IK(ACh),
activated by A1 receptors, displays fast desensitization,
which was increased in myocytes overexpressing the A1
receptor (19).
With a background of these results any contribution of intrinsic
PLC-coupled muscarinic receptors to fast desensitization can be safely
excluded. Moreover, the lack of any inhibition of atrial
IK(ACh) by ACh, in contrast to
-agonists or ET-1, and
the negative RT-PCR for M3AChR transcripts argue against
expression of this receptor in rat atrial myocytes.
GIRK currents generate inhibitory postsynaptic potentials, which are
shaped by different phases of desensitization in terms of an adaptation
to synaptic inputs. The term desensitization is a
phenomenological one. It comprises ultrarapid conformational changes on
the microsecond time scale of ionotropic receptor/channel proteins,
such as the nicotinic ACh receptor, to phosphorylation-initiated reductions in sensitivity of a cell to a stimulus on the time scale of
hours as, e.g. in case of the paradigmatic
-adrenergic receptors, but also in the pathway that is subject to the present investigation.
Both the instantaneous onset of acute desensitization and its fast
recovery argue against phosphorylation/dephosphorylation of the channel
complex, as suggested previously (7, 40). The most convincing concept
to date suggests that fast desensitization is caused by the kinetics of
the nucleotide exchange and hydrolysis cycle of the G protein (9). The
model proposed by these authors takes into account that fast
desensitization is closely linked to activation of the current.
Although attractive, this model does not describe all of the properties
of IK(ACh) in a native myocyte. According to that model,
GTP
S-induced IK(ACh) should be larger, and in the
presence of the GTP analog, fast desensitization should be abolished.
Our data (e.g. Figs. 5 and 10) suggest that steady-state
currents in GTP
S-loaded myocytes are smaller than peak currents
activated by ACh. Moreover, desensitization is not abolished by
GTP
S. Therefore further experimental work is required to clarify the
mechanism of fast desensitization.
The present study confirms a second messenger role of
PtIns(4,5)P2. Dual regulation of Kir3.0 currents by
stimulatory Gi/o-coupled receptors via
G
and by inhibitory
Gq/11-coupled receptors via PtIns(4,5)P2
depletion represents a novel concept of synaptic integration at the
level of an ion channel.