Correspondence to: Diomedes E. Logothetis, Department of Physiology and Biophysics, Mount Sinai School of Medicine of the New York University, New York, NY 10029. Fax: 212-860-3369; E-mail:logothetis{at}msvax.mssm.edu.
Released online: 11 October 1999
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Abstract |
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Native and recombinant G proteingated inwardly rectifying potassium (GIRK) channels are directly activated by the ß subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP2) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP2 or application of exogenous PIP2 increases the mean open time of GIRK channels and sensitizes them to gating by internal Na+ ions. In the present study, we show that the activity of ATP- or PIP2-modified channels could also be stimulated by intracellular Mg2+ ions. In addition, Mg2+ ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na+ and Mg2+ ions exert their gating effects independently of each other or of the activation by the Gß
subunits. At high levels of PIP2, synergistic interactions among Na+, Mg2+, and Gß
subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K+ channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.
Key Words:
G proteingated inwardly rectifying potassium channels, phosphatidylinositol-bis-phosphate, Gß gating, Mg2+ gating, Na+ gating
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INTRODUCTION |
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In atrial tissue, acetylcholine released by the vagus nerve binds to muscarinic type 2 receptors, activates KACh channels via pertussis toxinsensitive G proteins, and slows the heart rate. Upon activation, the heterotrimeric G protein dissociates, allowing the Gß subunits to directly activate the KACh channel (
subunits (
subunits (
Here, using both native and recombinant GIRK channels, we show that Na+ as well as Mg2+ ions gate the ATP- or PIP2-modified channels. While the two ions seem to exert their effects at distinct sites on the channel protein, they showed synergistic effects on gating. In the presence of exogenous PIP2, Gß and Na+ and Mg2+ ions showed great synergism in activating the channel. However, in the absence of exogenous PIP2, preactivation by G protein ß
subunits sensitized the channel to gating by Na+ but not Mg2+ ions. These data suggest that the synergism between Mg2+ and Gß
subunits in gating GIRK channels shows a much greater dependence on PIP2 levels than the synergism between Na+ and Gß
. The synergism among ions and Gß
proteins in the gating of GIRK channels implies that variations of the concentrations of these molecules in the local environment of these channels could play an important role in the "fine tuning" of their activity.
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MATERIALS AND METHODS |
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Expression of Recombinant Channels in Xenopus Oocytes
Recombinant channel subunits (GIRK1, GenBank accession No.
U39196; GIRK4, GenBank accession No.
U39195) were expressed in Xenopus oocytes as described previously (
Preparation of Chicken Atrial Myocytes
The procedure used for isolating cardiac myocytes from chicken embryos has been described previously (
Reagents
General chemical reagents, including GTP and ATP, were purchased from Sigma Chemical Co. PIP2 (Boehringer Mannheim) was sonicated on ice for 30 min before application. Purified recombinant G protein subunits dimer ß17 was kindly provided by Dr. J. Garrison (University of Virginia, Charlottesville, VA). The stock of ß1
7 (0.86 µg/µl) was dissolved in 20 mM HEPES, 1 mM EDTA, 200 mM NaCl, 0.6% CHAPS, 50 mM MgCl2, 10 mM NaF, 30 µM AlCL3, 3 mM dithiothreitol (DTT), 3 µM GDP, pH 8.0. The final concentration was 20 nM in a solution containing 0.012% CHAPS, and 20 µM DTT. QEHA peptide (
Single-Channel Recording and Analysis
Single-channel activity was recorded in the cell-attached or inside-out patch configurations (. All experiments were conducted at room temperature (2022°C). Single-channel recordings were performed at a membrane potential of -80 mV with acetylcholine (ACh, 5 µM) in the pipette, unless otherwise indicated. Single-channel currents were filtered at 12 kHz, sampled at 510 MHz, and stored directly into the computer's hard disk through the DIGIDATA 1200 interface (Axon Instruments). PCLAMP (v. 6.03; Axon Instruments) was used for data acquisition.
To remove the vitelline membrane, Xenopus oocytes were placed in a hypertonic solution (
The pipette solution contained 96 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.35. The bath solution contained 96 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.35. When high concentrations of Mg2+ ions (>5 mM) were used in the bath solution, the KCl concentration was reduced accordingly to maintain osmolarity. Gadolinium chloride at 100 µM was routinely added to the pipette solution to suppress native stretch channel activity in the oocyte membrane. For chick atrial cells, the experimental solutions were the same as those used with oocyte recordings, except that the KCl concentration was 140 mM without gadolinium chloride.
Free Mg2+ and ATP concentrations were estimated as described previously (
In experiments shown in Figure 7, where exogenous PIP2 was applied throughout the experiment (i.e., Figure 7A and Figure C), occasional applications of the same ion as a function of time in the experiment were used as control to ascertain that the synergistic effects described were not due to a time-dependent accumulation of PIP2 in the membrane patch. Similar precautions were taken in the experiments shown in Figure 4. Experiments used to generate the data shown in these two figures were never longer than 14 min (usually 1013 min). Na+ and/or Mg2+ ions were applied for 30 s.
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RESULTS |
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MgATP/Na Activation of the KACh Channel Can Proceed Independently of the Involvement of G Proteins
It has been shown previously that Na+ ions can stimulate KACh activity in an ATP-dependent manner in the absence of agonist and internal GTP (
To further test for a dependence of the MgATP/Na+ activation on G protein gating of KAch, we designed experiments where G proteindependent activation of the channel was impaired. As shown in Figure 1 A, the KACh channel in an inside-out atrial myocyte patch was activated persistently by 10 µM GTPS, a nonhydrolyzable analogue of GTP. Activation of the channel by GTP
S was blocked upon perfusion of the QEHA peptide. QEHA is a 27 amino acid long peptide derived from the COOH terminus of the Gß
-sensitive adenylate cyclase 2 isoform. It has been shown to block Gß
activation of several different effectors, including the KACh channel (
S activation of KACh in <2 min (n = 3). After washout, the channel activity remained very low, suggesting the persistence of the QEHA-blocking effect. However, under these conditions, the KACh channel could be activated by MgATP/Na+ (5/20 mM). QEHA coapplication with MgATP/Na+ failed to block channel activation, whereas QEHA did block GTP
S-induced activation in the same oocyte patches (n = 3) (data not shown).
Another way we impaired the G protein regulation of the GIRK channels was by coexpressing them in oocytes with a ß-binding protein. We used the PH domain of ßARK (ßARK-PH), which specifically binds the ß
subunits of G proteins, and thus acts as a "ß
sink" (
S did not induce channel activity. This suggests that the ßARK-PH protein bound the oocyte endogenous G proteins, such that no ß
subunits were available for channel activation (Figure 1 B). However, in the same patches, MgATP/Na+ (5/20 mM) caused a >30-fold increase in channel activity. Summary data revealed that channel activities (NPo) before, during, and after GTP
S application were similar, 0.0070 ± 0.0039, 0.0077 ± 0.0037, and 0.0097 ± 0.0045, respectively (mean ± SEM, n = 4). During application of MgATP/Na+, NPo was 0.313 ± 0.221 (n = 4).
In control experiments using inside-out patches from oocytes of the same batch that coexpressed the recombinant channels GIRK1/GIRK4 alone, GTPS caused great channel activation (n = 3, data not shown). Similar results were obtained in experiments in which we applied Na+ ions with PIP2 rather than MgATP (n = 4, data not shown).
These results suggest that even when G protein regulation is impaired, Na+ ions are still able to activate the channel. Thus, Na+ gating of the channel can indeed proceed independently of Gß gating.
Gß Subunits Sensitize GIRK Channels to Gating by Na+ Ions
Na+ ions can gate GIRK channels when membrane PIP2 levels are maintained (i.e., via hydrolysis of ATP). We next tested under conditions that did not maintain PIP2 at a constant high level whether Na+ ions could gate these channels after Gß activation.
Figure 2A and Figure B, show representative and summary data from experiments where Na+ ions gated GIRK1/GIRK4 channels after activation by G proteins. Inside-out patches from oocytes expressing these channels showed no channel activity upon application of 20 mM Na+. This result suggested a low presence of PIP2 in the membrane. However, this PIP2 concentration was sufficient to allow persistent channel activation by a brief exposure to 10 µM GTPS. Reapplication of Na+ ions produced a more than fourfold increase in the channel activity above the level obtained with GTP
S. It should be noted that the effect of Na+ ions on the basal channel activity was variable from patch to patch, presumably reflecting different levels of endogenous PIP2 at the time of Na+ application.
Na+ ions also gated GIRK channels after stimulation of activity by purified Gß subunits. In Figure 2C and Figure D, Na+ ions (20 mM) applied on an inside-out patch did not affect significantly the basal activity of the channel. After washout of the Na+ ions, recombinant Gß1
7 was applied on the patch at a concentration of 20 nM, causing a slow channel activation. After washout of Gß1
7, and as activity stabilized, a second application of Na+ ions produced a more than threefold increase in channel activity, above the level obtained with ß1
7. Combined together, these data suggested that the G protein ß
subunits sensitized GIRK channels to gating by Na+ ions. It has been shown that the mean open time (MTo) increased in the presence of PIP2 that is generated by hydrolysis of ATP or exogenous application (
-dependent gating effects of Na+ ions.
It has been shown that Li+ ions stimulate GIRK channels modified by ATP to ~10% the activity level achieved by comparable Na+ ion concentrations (S. In three patches, the mean NPo of the GIRK channel was 0.028 ± 0.013 in control conditions, 0.127 ± 0.035 after application of 10 µM of GTP
S, 0.578 ± 0.15 in the presence of 20 mM Na+ ions, and 0.099 ± 0.045 in the presence of 20 mM Li+ ions (data not shown). When applied together, Li+ ions were also unable to affect the gating of the GIRK channel by Na+ ions. This suggests that the gating effect of Na+ ions on the GIRK channel activated by G protein ß
subunits is specific to Na+ ions.
Mg2+ Ions Gate GIRK Channels After Channel Modification by ATP or PIP2
In certain experiments, 5 mM MgATP increased the activity of the GIRK channels in the absence of Na+ ions (e.g.,
Using PIP2, we could test the ability of different Mg2+ concentrations to activate the GIRK channels. Figure 4 represents normalized activity of GIRK channels for different concentrations of Mg2+ ions. The NPo for each concentration was calculated in reference to the NPo measured at 1 mM Mg2+. Mg2+ ions could activate the GIRK channels at concentrations as low as 100300 µM. Maximal activity could be obtained at a concentration of ~7 mM Mg2+. At higher concentrations (e.g., 20 mM), Mg2+ ions resulted in a decrease of channel activity relative to lower concentrations (e.g., 7 mM). It has been shown that, at high concentrations, divalent cations can trigger aggregation of PIP2 molecules (
In another set of experiments, we showed that Mg2+, like Na+ gating, can occur independently of G proteins. Patches excised from oocytes coexpressing the ßARK-PH domain and GIRK channels were exposed to PIP2 (2.5 µM) and subsequently to Mg2+ ions. In these patches, GTPS (10 µM) was unable to activate the GIRK channels, giving a NPo of 0.08 ± 0.03, identical to the NPo measured in PIP2 (0.078 ± 0.02). Mg2+ ions (1 mM) could increase the channel activity approximately sixfold (n = 4, data not shown) above the activity measured in PIP2, showing that Mg2+ gating could proceed independently of Gß
gating.
These results suggest that when modified by ATP or PIP2, GIRK channels become sensitive to either Na+ or Mg2+ ions.
Mg2+ Ion Gating Occurs at a Site Distinct from that of Na+ Action
Recent work has identified an aspartate amino acid residue as the site of action of Na+ ions on GIRK channels, GIRK2 (D228) and GIRK4 (D223) (
Mg2+ Ions Reduce the Conductance of the GIRK Channels
We observed, particularly at high concentrations (>5 mM), that internal Mg2+ ions reduced the amplitude of single GIRK channel currents. In Figure 6 A, the activity of the coexpressed channel subunits GIRK1/GIRK4 from an inside-out patch was recorded at -120 mV. After activation by 10 µM GTPS, channel activity was recorded in a solution containing 1 mM Mg2+ ions, showing an approximate amplitude of -3.2 pA. When the solution applied to the patch was switched to one containing 20 mM Mg2+ ions, the amplitude of the single openings was rapidly reduced to a lower value, approximately -2.5 pA (n = 5). In Figure 6 B, the activity of native KACh channels in an inside-out patch from an atrial cell was recorded at -90 mV. After exposure to 5 µM PIP2, the patch was perfused with a solution containing 20 mM Mg2+ ions, giving an amplitude of approximately -2.2 pA. When the solution applied to the patch was switched to one containing 20 mM Na+ and 1 mM Mg2+ ions, the channel amplitude immediately increased to a value of approximately -3.5 pA. This amplitude was also obtained in control conditions, where 1 mM Mg2+ ions were present (n = 5). The reduction in the single-channel amplitude was observed at various voltages. Since it was present at negative potentials (i.e., -80, -90, and -120 mV) where no rectification occurs, it is likely to proceed by a mechanism distinct from that of the rectification phenomenon. Mg2+ ions at high concentrations also decreased the amplitude of GIRK single channels when applied together with Na+ ions (data not shown). Thus, regardless of their ability to gate GIRK channels (see Figure 3 and Figure 7), Mg2+ ions at high concentrations (>5 mM) show a clear inhibition on single-channel current amplitudes. These data suggest that the inhibitory effect of Mg2+ ions on the single-channel amplitude was not dependent on their ability to gate the channel.
Synergistic Interactions among Ions and G Protein Subunits in Gating GIRK Channels
ATP modification of GIRK channels (native or recombinant) is likely to proceed through changes in the level of membrane PIP2 in the local environment of the channel (
We next tested whether Mg2+ ions, like Na+ ions (Figure 2), can further gate GIRK channels after channel activation by GTPS, under conditions that do not maintain PIP2 at constant high levels. Figure 7 B shows that the basal activity of GIRK1/GIRK4 recombinant channels was not affected by Mg2+ ions. 10 µM GTP
S increased the activity of the channel approximately fivefold above basal levels. After GTP
S washout, the channel activity was stable and, when applied to the patches, Mg2+ ions were unable to increase channel activity further. In contrast, Na+ ions (10 mM) increased activity by another twofold above the GTP
S effect. When Mg2+ ions were applied together with Na+ ions, no further increase in channel activity above the levels obtained with Na+ ions was seen. Thus, G protein activation sensitized the GIRK channels to gating by Na+ ions, but not Mg2+ ions.
In Figure 7 C, we show the effects of Mg2+ and Na+ ions after stimulation of the channel by GTPS under conditions that kept PIP2 at a constant high level. As shown earlier, in the absence of Mg2+ and Na+ ions, PIP2 was not able to increase the basal activity of the GIRK channels. When Mg2+ ions (10 mM) were applied to the patches in the presence of PIP2, a greater than eightfold increase over control or PIP2 activity levels occurred. Mg2+ and Na+ ions (each 10 mM) in combination could raise channel activity by 50-fold over control levels. We then applied GTP
S and studied the effects of ions on G proteinstimulated channel activity in the continuous presence of PIP2. GTP
S was able to activate the channel >14-fold above control basal levels. After washout of GTP
S, channel activity was stable. When Mg2+ ions (10 mM) were applied to the patches after the GTP
S treatment in the continuous presence of PIP2, they could enhance channel activity to levels >100-fold higher than those obtained under control conditions. Thus, in the continuous presence of PIP2, this high level of activity was greater than that obtained with Mg2+ or GTP
S alone or their sum, suggesting synergistic interactions among the three molecules. Finally, when Mg2+ and Na+ ions were applied together, the channel total activity was increased 400-fold compared with control.
Similar data were obtained when the G protein ß17 subunits rather than GTP
S were used. In three cells, the total channel activity measured as the mean NPo was 0.027 ± 0.023 in control conditions, 0.022 ± 0.02 in the presence of 2.5 µM PIP2, 0.12 ± 0.09 in the presence of PIP2 and 10 mM Mg2+ ions, and 1 ± 0.55 in the presence of PIP2 and Mg2+ and Na+ ions. When 20 nM ß1
7 was applied in the presence of PIP2, it gave a steady state activity of the channel corresponding to a mean NPo of 0.25 ± 0.12. In the continuous presence of PIP2 and after stimulation of the channel by ß
subunits, the mean NPo was 1.23 ± 0.23 in the presence of Mg2+ ions and 2.61 ± 0.24 in the presence of Mg2+ and Na+ ions. It should be noted that the differences in channel activity (mean NPo) for the same condition applied to the patches (for example PIP2 + Mg2+ in Figure 7A and Figure C) may be related to differences in the level of channel expression between different batches of oocytes. Taken together, these data make four points. (a) Mg2+ ions can gate the channel after modification by PIP2. At a concentration of 10 mM, their gating potency is comparable with that of 10 mM Na+ ions. (b) When applied together, Mg2+ and Na+ ions show synergistic effects, resulting in levels of activity higher than those induced by each of the ions separately or their summed responses. (c) After activation by GTP
S (in the absence of exogenous PIP2 or MgATP), the GIRK channels are not further gated by Mg2+ ions, suggesting an important difference between Mg2+ and Na+ ions in gating these channels. (d) After channel modification by the combination of PIP2 and GTP
S, Mg2+ ions do stimulate the GIRK channel activity to higher levels than those obtained with PIP2 alone, suggesting that PIP2 renders the ß
-activated channels sensitive to gating by Mg2+ ions.
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DISCUSSION |
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In the present study, we have shown that Mg2+ ions at physiological concentrations are additional activators of G proteingated potassium channels. These K+ channels can be activated independently either by the ß subunits of GTP-binding proteins (
GIRK Channel Activation by Na+ Ions Can Be Independent of G Protein Subunit Involvement
Previous results from our laboratory showed that intracellular solution containing MgATP/Na+ was able to stimulate K+ channel activity in the absence of acetylcholine in the pipette, suggesting a G proteinindependent mechanism of activation ( from the channel with either QEHA perfusion or ßARK-PH coexpression) did not prevent the MgATP/Na+ stimulation of activity (Figure 1). Thus, we have provided further evidence that Na+ ion gating of the channel modified by ATP (or PIP2) can be independent of G protein subunit activation.
Mg2+ Gating of the G Protein-gated K+ Channel
Mg2+ ions have been shown to play an essential role in the rectification properties of inwardly rectifying K+ channels. Unitary currentvoltage relations for G proteinsensitive K+ channels become ohmic if the internal face of the patch is exposed to Mg2+-free solutions. Inward rectification is restored when Mg2+ is reintroduced in the bathing solutions (
Mg2+ ions are involved in many other reactions as essential cofactors. subunit (also see
Our present data show that Mg2+ ions, in addition to their involvement in the processes mentioned above, are able to activate the ATP- or PIP2-modified G proteinsensitive channel (Figure 3 and Figure 7). In the presence of PIP2, similar concentrations of Mg2+ and Na+ ions yielded comparable levels of channel activity, suggesting equivalent gating abilities for both ions. Since PIP2 mimics the MgATP effects on the channel, we have been able to study directly Mg2+ gating effects.
Mutation of the amino acid responsible for Na+-ion activation of GIRK channels did not interfere with Mg2+-ion activation. This result strongly suggests that Mg2+ and Na+ ions act on distinct sites to gate the channel.
Our data also show that Mg2+ ions reduced the conductance of the G proteingated channels in a manner independent of their stimulatory effect on gating. Since this inhibitory effect of Mg2+ ions on conductance was present at negative potentials (-120, -90, and -80 mV), where no rectification is occurring (
Synergism Among G Proteins and Ions in the Gating of GIRK Channels
At higher PIP2 concentrations, the combination of Na+ and Mg2+ ions resulted in a stimulation of channel activity that was greater than the sum of their individual effects, suggesting synergistic interactions of these ions on channel gating (Figure 7 A).
Na+ ions gate the K+ channel in the presence of hydrolyzable ATP or PIP2 ( subunits, but not of Na+ or Mg2+ ions, results in stimulation of channel activity (Figure 2 and Figure 7 B). Our previous study (
or Na+) could activate the channel. In the present experiment under conditions that we do not expect to have depleted PIP2, Gß
subunits caused a much greater stimulation of activity than Na+ or Mg2+ ions. This result suggests that the dependence on PIP2 for channel gating is greater for Mg2+ and Na+ than for Gß
. Under these conditions, we find that Na+ ions do stimulate channel activity after preactivation by GTP
S or by purified Gß
subunits (Figure 2 and Figure 7 B). This result suggests that Gß
activation sensitizes the K+ channel gating to Na+ ions. Moreover, in such experiments, Gß
subunits and Na+ ions act synergistically in gating the channel. Interestingly, Mg2+ ions were unable to gate the channel after channel preactivation by GTP
S. These data underscore an interesting difference in the gating of this channel by ions, namely at low PIP2 levels Gß
subunits synergize with Na+ but not Mg2+ ions to gate the channel.
This difference of the two ions on channel gating is lost at higher PIP2 concentrations (Figure 7 C). In such experiments, not only were the synergistic effects of the ions shown in Figure 7 A reproduced, but also Mg2+ as well as Na+ ions cooperated with Gß. When applied in combination, all three gating particles showed synergistic effects (Figure 7 C).
We have previously shown that block of the Na+/K+ pump activates KACh in atrial myocytes with kinetics similar to those seen for Na+ accumulation resulting from the block of the pump ( subunits implies that variations in the local levels of these molecules could have a profound impact on the dynamic range of GIRK channel activity under normal or pathophysiologic states.
A Gating Model for GIRK Channels
Channel binding sites for PIP2, Gß, and Na2+ ions have been identified (
, Na+, or Mg2+ are unable to activate the channel (closed state C2). However, in the presence of PIP2, any of the gating molecules can cause channel activation.
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We envision two possible mechanisms for the synergistic action of gating molecules to activate the channel. Ions and Gß subunits maybe exerting their combined effects by synergistic interactions of channel sites with PIP2. Published reports have already suggested a stronger interaction of channel with PIP2 in the presence of either Gß
subunits or Na+ ions (
, unlike Na+ or Mg2+ ions, can still gate the channels. This result suggests a stronger influence of Gß
than of Na+ or Mg2+ ions on channel gating, possibly proceeding in a PIP2-independent manner. Further work will be required to distinguish between these possibilities.
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Footnotes |
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Dr. Sui's present address is Cambridge Neuroscience, Cambridge, MA 02139.
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Acknowledgements |
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We are grateful to Lidiya Lontsman and Xixin Yan for technical support and preparation of the oocytes. We thank Drs. James Garrison, Eitan Reuveny, and Ravi Iyengar for their kind gifts of Gß17 subunits, the ßARK-PH construct and QEHA peptide, respectively. We also thank Drs. Vladimir Brezina, Sherman Kupfer, Tooraj Mirshahi, Tibor Rohacs, and Hailin Zhang for critical comments on the manuscript.
This work was supported by grants from the Aaron Diamond Foundation (J.L. Sui), the National Institutes of Health (HL54185), and the American Heart Association, National Center (96011620) (D.E. Logothetis).
Submitted: 16 July 1999
Revised: 25 August 1999
Accepted: 26 August 1999
1used in this paper: ACh, acetylcholine; ßARK-PH, ß-adrenergic receptor kinase plekstrin homology domain; GIRK channel, G proteingated inwardly rectifying potassium channel; PIP2, phosphatidylinositol-bis-phosphate
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