Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes.

G protein-gated inwardly rectifier K+ current in atrial myocytes (I(K(ACh))) upon stimulation with acetylcholine (ACh) shows a fast desensitizing component (t(1/2) approximately 5 s). After washout of ACh, I(K(ACh)) recovers from fast desensitization within < 30 s. A recent hypothesis suggests that fast desensitization is caused by depletion of phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)), resulting from costimulation of phospholipase C (PLC)-coupled M3 receptors (M3AChR). The effects of stimulating two established PLC-coupled receptors, alpha-adrenergic and endothelin (ET(A)), on I(K(ACh)) were studied in rat atrial myocytes. Stimulation of these receptors caused activation of I(K(ACh)) and inhibition of the M2AChR-activated current. In myocytes loaded with GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), causing stable activation of I(K(ACh)), inhibition via alpha-agonists and ET-1 was studied in isolation. Stimulation of either type of receptor under this condition, via G(q/11), caused a slow inhibition (t(1/2) approximately 50 s) by about 70%. No comparable effect on GTPgammaS-activated I(K(ACh)) was induced by ACh, suggesting that PLC-coupled M3AChRs are not functionally expressed in rat myocytes, which was supported by the finding that M3AChR transcripts were not detected by reverse transcriptase-polymerase chain reaction in identified atrial myocytes. Supplementing the pipette solution with PtIns(4,5)P(2) significantly reduced inhibition of I(K(ACh)) but had no effect on fast desensitization. From these data it is concluded that stimulation of PLC-coupled receptors causes slow inhibition of I(K(ACh)) by depletion of PtIns(4,5)P(2), whereas fast desensitization of I(K(ACh)) is not related to PtIns(4,5)P(2) depletion. As muscarinic stimulation by ACh does not exert inhibition of I(K(ACh)) comparable to stimulation of alpha(1)- and ET(A) receptors, expression of functional PLC-coupled muscarinic receptors in rat atrial myocytes is unlikely.

In the heart, predominantly in supraventricular tissue, in various neurons and endocrine cells, stimulation of receptors coupled to pertussis toxin-sensitive G proteins (G i/o ) activates G protein-gated inward rectifying K ϩ (GIRK) 1 channels, result-ing in reduction of excitability (1)(2)(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 I K(ACh) result from costimulation of two muscarinic receptors: the M 2 subtype (M 2 AChR) which causes activation via G i/o , whereas stimulation of the M 3 subtype (M 3 AChR) via activation of G q/11 stimulates phospholipase C (PLC) and subsequent depletion of phosphatidylinositol bisphosphate (PtIns(4,5)P 2 ) in the inner leaflet of the membrane. Such a mechanism would be in line with the finding that PtIns(4,5)P 2 is an important cofactor for activation of GIRK channels (11)(12)(13) and other members of the Kir channel family (14 -16) and supports the notion that PtIns(4,5)P 2 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 I K(ACh) can be activated not only by M 2 AChR but also, e.g. by A 1 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 A 1receptors, a classic G i/o -coupled species, not only increased density of Ado-induced I K(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 G q/11 -coupled receptors on I K(ACh) in atrial myocytes. The results support the notion that stimulation of the PLC pathway via depletion of PtIns(4,5)P 2 causes inhibition of I K(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 G q/11 -coupled muscarinic receptor, such as M 3 AchR, in rat atrial myocytes.

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 g/ml gentamycin (Sigma) and 25 g/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.
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 Ϫ90 mV, i.e. negative to E K (Ϫ50 mV), resulting in inward K ϩ currents. Current-voltage relations were determined by means of voltage ramps from Ϫ120 mV to ϩ60 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 (t 1/2 Յ 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 Ba 2ϩ on I K(ACh) . As shown previously, activation kinetics of I K(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 l of RNase-free H 2 O (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 l 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 CaCl 2 , 1 MgCl 2 , 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 ϳ30-m 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 (Ͻ0.5 l) was expelled into a tube filled with 5 l of lysis buffer containing: 0.8% (w/v) Nonidet P-40 substitute (Fluka, Buchs, Switzerland), 100 g/ml yeast tRNA, 10 mM dithiothreitol (both from Life Technologies, Inc.), and 8 units/l RNAguard (Amersham Pharmacia Biotech).
In the experiments on selected myocytes 2.5 l of RNase-free H 2 O (Qiagen) and 0.5 l of oligo(dT) 15 primer (500 g/ml; Promega, Madison, WI) were added to the lysis solution and incubated at 42°C for 10 min. Rapid vertical deflections represent changes in membrane current induced by voltage ramps from Ϫ120 to ϩ 60 mV, which were routinely superimposed and served to identify I K(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 M) for ϳ2 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.
l 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 l of first strand buffer, 0.5 l of SuperScript II RT, 5 l of dithiothreitol, 1.6 l of dNTP mixture, 1.6 l of oligo(dT) 15 primer, 0.5 l of RNAguard, and 7.2 l of RNase-free H 2 O (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 l and adding 40.5 l of PCR mixture to each tube. The PCR mixture contained 34 l of distilled water (Life Technologies, Inc.), 0.5 l of Taq DNA polymerase (5,000 units/ml), 5 l of PCR buffer (10-fold; both Amersham Pharmacia Biotech), 0.5 l of sense primer, 0.5 l of antisense primer (both 100 pmol/l; Life Technologies, Inc.), either for M 3 AChR and GAPDH or M 2 AChR and GAPDH.
PCR from cDNA of atrial cell suspension RT was performed by mixing 1 l of RT product, 16 l of distilled water, 2 l of PCR buffer, 0.4 l of dNTP mixture, 0.2 l of M3 primer (sense), 0.2 l of M3 primer (antisense), and 0.2 l of Taq DNA polymerase (same reagents as above).

RESULTS
Definition of Fast Desensitization of Atrial I K(ACh) -As shown previously, atrial I K(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)(24)(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 M) superfused for 50 s. After rapid activation of inward I K(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 Ϯ 0.43 s (n ϭ 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.
We have shown previously that this second component is likely to reflect desensitization of the M 2 AChR (4). A characteristic feature of fast desensitization of atrial I K(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 I K(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. Inhibition of Atrial GIRK Current by PtIns(4,5)P 2 Depletion 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 I K(ACh) by 10 M Ado, a concentration that is saturating for this agonist, via stimulation of A 1 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 A 1 receptors (19). Superfusion of 10 M ACh in the presence of Ado resulted in a peak inward current of only 70% of the current induced by 2 M 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 M 2 AChR/sphingolipid, or M 2 AChR/␤-adrenergic (21,28). These properties of fast desensitization are contradictory to the hypothesis that it is mediated by stimulation of a muscarinic M 3 receptor, unless one would assume that stimulation of each receptor causing activation of I K(ACh) is paralleled by costimulation of a G q/11coupled subtype causing desensitization.

Inhibition of I K(ACh) by ␣ 1 -Adrenergic Receptors and ET A
Receptors-On principle, information on how activation of PLC interferes with I K(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 I K(ACh) and, concomitantly, an inhibition of I K(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).
To study the inhibitory effect of the ␣ 1 -adrenergic agonist or ET-1 in isolation and to obtain information on its kinetic properties, I K(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 M phenylephrine (Phe) and 10 nM ET-1 both caused an inhibition of I K(ACh) . A comparable effect was also found if 100 M methoxamine was used as ␣-agonist (not shown).The summarized data in Fig. 4A demonstrate that both, ␣ 1 -adrenergic stimulation as well as stimulation of ET A receptors caused inhibition of I K(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 Ϯ 7.9 s (Phe, n ϭ 10) and 57.3 Ϯ 8.8 s (ET-1, n ϭ 10), respectively. On average, the GTP␥S-activated current in the absence of Phe or ET-1, respectively, decayed spontaneously by ϳ20% 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 I K(ACh) by activation of ␣ 1 -adrenergic or ET A 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 M 2 AChR-G i/o pathway, using activation of I K(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.
The slow decay of GTP␥S-activated I K(ACh) by about 20% within 3 min in the control group could reflect an inhibitory effect caused by costimulation of putative intrinsic G q/11 -coupled muscarinic receptors. However, exposure of a GTP␥Sloaded cell to a high concentration of ACh (100 M), following activation of I K(ACh) either by ACh at a low concentration (2 M) or by adenosine via A 1 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 M ACh, the decrease in current after 3 min was 16.2 Ϯ 4.8% (n ϭ 6), which was not significantly different from the decrease without exposure to ACh (18.1 Ϯ 4.2%, n ϭ 12). We propose that the slight "spontaneous" decay of I K(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 M 3 AChRs are involved in regulating I K(ACh) in adult atrial myocytes.
Whereas the M 2 AChR 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 M 3 AChR expression in the heart, without identification of the cell type, are functionally meaningless. In rat ventricular myocytes no M 3 AChR transcripts were detected using single cell RT-PCR, which was confirmed by Southern blot analysis and immunocytochemistry, whereas these cells were positive for both M 1 AChR and M 2 AChR (31). Corresponding data are not available for atrial myocytes.
In atrial myocytes collected under optical control no M 3 AChR 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 M 3 AChR transcripts in identified atrial myocytes, whereas transcripts for GAPDH and M 2 AChR could be detected. On the other hand, using the same primers, M 3 AChR 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 G q/11 -coupled muscarinic receptor subtype.
The hypothesis that fast desensitization in atrial cells is mediated by M 3 AChR (10) was primarily based on pharmacological evidence. 4-DAMP, a compound described as a selective M 3 AChR antagonist, in that study resulted in a reduction of desensitization or a desensitizing component of ACh-induced I K(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 M 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 M 2 AChR antagonist. This is in line with the reported inhibition constants ranging from 4 ϫ 10 Ϫ9 M to 1.6 ϫ 10 Ϫ8 M for the M 2 AChR and from 1.2 ϫ 10 Ϫ9 M to 5 ϫ 10 Ϫ10 for the M 3 AChR (32). Thus, despite its frequent use as pharmacological tool, this compound is not sufficiently selective to be suitable for discriminating between M 3 AChR and M 2 AChR. when the concentration of GTP␥S should be low, and activation of G i/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.

Inhibition of I K(ACh) Is Mediated by the PLC-linked Signaling
Pathway-␣ 1 -Adrenergic receptors and ET A receptors are supposed to couple preferentially but not exclusively to G proteins of the G q/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 (InsP 3 ) and diacylglycerol from hydrolysis of PtIns(4,5)P 2 . To verify the contribution of this class of G proteins to inhibition of I K(ACh) , myocytes were pretreated with P. multocida toxin, a protein toxin that has been described to uncouple G q 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 g/ml for ϳ 20 h), activation of I K(ACh) by M 2 AChR and/or intracellular GTP␥S was not affected (Fig. 8). However, inhibition of I K(ACh) by 100 M Phe was completely abolished. This was also confirmed in four experiments using 10 nM ET-1 (not shown). Contribution of G q/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 M 3 AChR and subsequent depletion of PtIns(4,5)P 2 represent the mechanism of fast desensitization. As shown in Fig. 9, however, U 73122 at 1 M caused a reduction in GTP␥S-activated I K(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.
Receptor-induced Inhibition of I K(ACh) Is Reduced by PtIns(4,5)P 2 -We next tried to obtain further information on the signaling mechanism subsequent to underlying slow inhibition of I K(ACh) via G q/11 -coupled receptors. Whereas InsP 3 is the ubiquitous Ca 2ϩ -mobilizing second messenger, diacylglycerol causes activation of protein kinase C. 100 M 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 ki- nase C is not causally involved. As in the present study a moderate pipette Ca 2ϩ -buffering capacity (2 mM EGTA) was used, Ca 2ϩ released from InsP 3 -sensitive stores as mediator cannot be excluded. Supplementing the pipette solution with a 10 mM concentration of the fast Ca 2ϩ chelator BAPTA did not affect inhibition of I K(ACh) by Phe (10 cells, not shown), suggesting that Ca 2ϩ -dependent processes are not involved.
In a number of recent publications evidence has been provided that PtIns(4,5)P 2 represents a cofactor for activation of Kir3.0 channels by G ␤␥ (11,36) and is also required for activity of other inward rectifying K ϩ channels (13,16,17,37,38). We therefore studied whether loading of atrial myocytes with PtIns(4,5)P 2 via the pipette filling solution affects properties of macroscopic I K(ACh) and/or ␣ 1 -adrenergic inhibition. Fig. 10A demonstrates that inclusion of PtIns(4,5)P 2 (nominally 500 M) in the pipette without GTP␥S had no effect on amplitude and desensitization properties of I K(ACh) . This was found in six out of six myocytes loaded with PtIns(4,5)P 2 . On the other hand, supplementation of the pipette solution with PtIns(4,5)P 2 caused a significant reduction of the Phe-induced inhibition of GTP␥S-activated I K(ACh) . Representative traces from this series of experiments are shown in Fig. 10B. As shown in Figs. 2 and 4, 100 M Phe caused an inhibition of steady-state I K(ACh) by about 70% (C). These data suggest first that under normal conditions the concentration of PtIns(4,5)P 2 in the membrane is not a limiting factor for activation of I K(ACh) . Second, inhibition of I K(ACh) by heptahelical receptors activating PLC is caused by depletion of PtIns(4,5)P 2 . Moreover, depletion of PtIns(4,5)P 2 is not causally related to fast desensitization. DISCUSSION The present study has provided evidence that activation of ␣-adrenergic and ET A receptors causes inhibition of atrial I K(ACh) by depletion of PtIns(4,5)P 2 . 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 heter-ologously expressed K (ATP) channels by stimulation of coexpressed M 1 AChR receptors, described recently as an example of a receptor-controlled signal that is mediated via depletion of PtIns(4,5)P 2 (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 (G i/o ), although examples of activation under certain conditions of atrial I K(ACh) via the G s pathway have been presented recently (28,39).
Apart from G ␤␥ regulation, channel activity has been shown to be modulated by PtIns(4,5)P 2 and intracellular [Na ϩ ]. Sensitivity to PtIns(4,5)P 2 has also been demonstrated for other members of the Kir channel family (12,15,16,37). It remained unclear, however, whether PtIns(4,5)P 2 is a necessary cofactor for Kir channels or if PtIns(4,5)P 2 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)P 2 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)P 2 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)P 2 and other phosphoinositides.
The notion of a second messenger role of PtIns(4,5)P 2 was supported by Kobrinsky et al. (10), who reported that activation of PLC-coupled receptors caused fast desensitization of native I K(ACh) in rat neonatal myocytes and Kir3.1/Kir3.4 currents expressed in a cell line. According to that study, fast desensitization of M 2 AChR-activated I K(ACh) in atrial myocytes results from PLC activation via costimulation of a PLC-linked muscarinic receptor subtype (M 3 AChR). The present findings confirm that activation of PLC-coupled receptors causes inhibition of atrial I K(ACh) in a PtIns(4,5)P 2 -sensitive manner. However, they disprove the hypotheses that intrinsic M 3 AChRs are involved in regulation of atrial I K(ACh) and that PtIns(4,5)P 2depletion causes fast desensitization.
First, fast desensitization of atrial I K(ACh) at room temperature has a half-time of in the order of 5 s, whereas inhibition of I K(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 G i/o was not affected by this condition.
Second, ACh did not induce a measurable inhibition of I K(ACh) .
Third, we have shown previously that atrial I K(ACh) , activated by A 1 receptors, displays fast desensitization, which was increased in myocytes overexpressing the A 1 receptor (19). Panel A, representative current recording from a myocyte treated with the 1 g/ml toxin (Ն20 h). 10 M ACh and 100 M 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.
FIG. 9. Inhibition of GTP␥S-activated I K(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.
FIG. 10. Loading of myocytes with PtIns(4,5)P 2 . Panel A, receptor-activated I K(ACh) in the absence of intracellular GTP␥S is not affected by supplementing the pipette solution with PtIns(4,5)P 2 . 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 I K(ACh) is reduced by adding PtIns(4,5)P 2 to the pipette solution (representative recording). Panel C, control measurement from a cell of the same batch as B without PtIns(4,5)P 2 . Panel D, summarized data (n ϭ 6 for either group).