Correspondence to: Joel Bard, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472. Fax:(617) 972-1761
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Abstract |
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Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (IKACh) by G proteincoupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of Gß subunits to the NH2- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate IKACh. Because of the very low basal activity of native IKACh, it has been difficult to determine the single channel effect of Gß
subunit binding on IKACh activity. Through analysis of a novel G proteinactivated chimeric inward rectifier channel that displays increased basal activity relative to IKACh, we find that single channel activation can be explained by a G proteindependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.
Key Words: ion channel, patch-clamp, G protein, gating, acetylcholine
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INTRODUCTION |
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Secretion of acetylcholine (ACh)1 by the vagus nerve slows the heartbeat rate by activating an inward rectifier potassium current (IKACh) in the pacemaker cells of the sinoatrial node (
The biochemical events underlying the stimulation of IKACh have been well studied. ACh activates the m2 muscarinic acetylcholine receptor (mAChR) which, in turn, catalyzes the exchange of GDP for GTP on the subunit of the heterotrimeric G protein Gi. The dimer of the G protein ß and
subunits becomes dissociated from the GTP-bound
subunit and activates IKACh by a direct, membrane-delimited mechanism (
The molecular cloning of genes encoding the proteins that form inward rectifier potassium channels has allowed a more detailed understanding of how Gß activates IKACh (
In a previous study ( activation. We constructed a chimeric molecule (termed GR7.1) in which the NH2 terminus and the distal portion of the COOH-terminal cytoplasmic domains of GIRK1 were fused to the pore region and proximal COOH-terminal cytoplasmic domain of RB-IRK2 (see Fig 1). The homomeric channels formed by GR7.1 have a high basal activity similar to that of RB-IRK2, but are further activated by stimulation of m2 mAChRs. Furthermore, G protein ß
subunits bind directly to bacterially expressed glutathioneS-transferase fusion proteins of the GIRK1 domains included in the chimera, suggesting that activation occurs through a direct interaction between the G protein and the channel.
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The link between binding events involving the intracellular domain of the channel and gating of the channel pore remains unclear. One approach to this problem is to measure the properties of single channels before and after activation to see which parameters are affected. This has not been possible for GIRK1/GIRK4 channels because of their very low basal activity. In this study, we take advantage of the relatively high basal activity of GR7.1 to determine the single channel effect of G protein binding. We find that activation of single channels by mAChR stimulation leads to an increase in the mean duration of bursts of channel openings. These observations suggest a simple molecular model of G protein activation of inward rectifier potassium channel activity.
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MATERIALS AND METHODS |
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Xenopus oocytes were injected with cRNA for the m2 mAChR (10 ng) and either a mixture of GIRK1 (
10 ng) and GIRK4 (
1 ng), GR7.1 (
10 ng), or RB-IRK2 (
0.5 ng;
.
Recordings of Patches Containing Multiple Channels
Cell-attached patches having many channels were subjected to a voltage protocol consisting of a pulse to -60 mV for 800 ms followed by a pulse to +60 mV for 800 ms; both were repeated every 5 s. 3 µl of 10 mM carbachol was added to a 3-ml bath volume after 100 s and allowed to mix by diffusion. In control whole cell experiments, this generally led to activation within 2 min. After 100 episodes, the perfusion tube was transferred to a reservoir containing 20 µM carbachol and the pipet was perfused by suction until the volume of the pipet solution had doubled. The observed delay in activation reflects the time required for the drug to diffuse to the tip of the pipet and varied considerably with pipet geometry.
Recordings of Patches Containing Single Channels
Patches that appeared to contain only one channel were subjected to voltage ramp protocols to determine rectification properties, and were recorded continuously for 500 s at -60 mV. The data were filtered at 1 kHz, and the sampling frequency was 10 kHz. After the initial recording, the pipet was perfused as before. After several minutes, during which the drug was allowed to diffuse to the tip of the pipet, another 500 s of data were acquired. Single channel data were idealized using Fetchan (Axon Instruments) with a filter frequency of 150 Hz for GR7.1 and RB-IRK2; no filter was applied for the GIRK1/GIRK4 analysis. Analysis of the dwell-time histograms was performed using Pstat (Axon Instruments). The burst delimiter for burst analysis was determined by finding the minimum of the fit between the second and third exponential terms. It was generally between 175 and 300 ms.
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RESULTS |
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To use GR7.1 as a model channel for G protein activation, we first needed to demonstrate that this chimeric channel is activated through a similar mechanism to that of IKACh. Membrane-delimited activation by G proteincoupled receptors is a hallmark property of IKACh. In cell-attached patches from rabbit atrial cells, the addition of a mAChR agonist to the recording pipet activates the current, whereas the addition of the same agonist to the bath does not (
To test whether activation of GIRK1/GIRK4 and chimera GR7.1 occurs by a similar membrane-delimited mechanism, we recorded from patches containing multiple channels while adding the mAChR agonist carbachol first to the bath and then to the pipet. Oocytes expressing GIRK1/GIRK4 and m2 mAChRs showed very little activity in the absence of carbachol or when carbachol was added to the bath. However, when carbachol was added to the pipet, there was a large increase in activity (Fig 2 A). Patches from cells expressing RB-IRK2 or GR7.1 showed significant activity in the absence of carbachol. This activity did not increase upon addition of the agonist to the bath. Importantly, the addition of carbachol to the pipet had no effect on RB-IRK2, but significantly increased the activity of GR7.1 (Fig 2, BD). These results show that activation of GR7.1 and GIRK1/GIRK4 share the feature of membrane-delimited regulation as originally defined for IKACh.
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To examine the mechanism of channel activation by G protein binding, we analyzed recordings of individual GR7.1 channels before and after perfusion of the patch pipet with carbachol. The trace in Fig 3 A shows the activity of a single GR7.1 channel before addition of agonist to the pipet. Bursts of activity are separated by long inactive periods of up to several seconds. The trace in Fig 3 B shows the activity of the same channel after addition of carbachol to the pipet. For this channel, there was a 60% increase in open probability (0.20 before carbachol and 0.32 after carbachol), which is wholly accounted for by a similar increase in the burst duration (1.2 s before carbachol and 2.1 s after carbachol; see Fig 4E and Fig F). This enhanced open probability results both from an increase in the duration of openings within bursts (94 ms before carbachol and 126 ms after carbachol; Fig 4A and Fig B) and an increase in the number of openings per burst (13.2 before carbachol and 16.2 after carbachol). Since each opening within a burst is preceded and followed by a closing, an increase in the number of openings per burst is reflected in the closed time distribution as an increased population of the region of the distribution corresponding to short closings that occur during bursts (Fig 4C and Fig D, leftmost peaks). Importantly, the interburst interval remains unchanged after stimulation implying that bursts do not occur more frequently after stimulation (Fig 4G and Fig H). Similar activation was seen in five out of seven single channel experiments with GR7.1; it may be that there were no mAChRs in those patches where no activation was observed. Except for a small change in open duration, no significant changes in single channel properties were observed in control experiments with RB-IRK2 (Fig 4 I).
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To determine whether wild-type channels display similar changes in single channel activity, we recorded from oocytes injected with cRNAs encoding GIRK1, GIRK4, and the m2 mAChR. Because of the low basal open probability of GIRK1/GIRK4 channels, we could not conclusively determine if our unstimulated patches contained only a single channel and, therefore, could not perform the sort of quantitative analysis applied to GR7.1. However, there is a clear qualitative similarity between the activation of GIRK1/GIRK4 and that of GR7.1. When carbachol is not present in the pipet, recordings from cells expressing GIRK1/GIRK4 and m2 mAChRs are characterized by brief, solitary openings. This can be seen in Fig 5 A, which shows the current from a patch containing multiple GIRK1/GIRK4 channels in the absence of carbachol. In contrast, when carbachol is present in the pipet, GIRK1/GIRK4 single channel activity is similar to that of GR7.1: bursts of openings are separated by long closings (Fig 5 B). As with GR7.1, this enhanced tendency to remain in the bursting state appears as a large peak representing short closings within bursts in the closed time distribution observed with carbachol in the pipet (Fig 5 D). The corresponding region of the closed time distribution for patches containing an unknown number of channels in the absence of carbachol contains very few events (Fig 5 C).
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DISCUSSION |
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We have used patch-clamp methodology to study the mechanism of activation of inward rectifier potassium channels by mAChRs. Our results confirm that GIRK1/GIRK4 and chimera GR7.1 channels are both activated by a similar membrane delimited mechanism. Furthermore, at the single channel level, activation appears as an increase in the duration of bursts of channel openings.
There have been a number of studies in which the single channel properties of GIRKs were examined in the presence of various concentrations of neurotransmitters or exogenously applied Gß (
The simpler single channel behavior and robust unstimulated activity of GR7.1 enable us to investigate the role of the G protein signal in the regulation of single channel kinetics of this model system. If one assumes a simplified model of a bursting channel in which a single open state (O) can be exited either to a short-lived intraburst closing (CS) or a long-lived interburst closing (CL), then both the increased duration of bursts and the increased o of activated GR7.1 can be explained if the relative probability of exiting an opening to CL rather than CS is decreased upon binding to Gß
.
This explanation of activation by Gß can be extended to account for our qualitative observations of the single channel behavior of GIRK1/GIRK4 channels. In this case, the rare solitary openings observed in the absence of stimulation can be considered as the limiting case of bursts with only a single opening, i.e., the probability of leaving O for CL is much larger than for CS. A decrease in the probability of exiting O for CL might allow the bursts that are observed in the presence of stimulation to occur. Although such an explanation can account for the large-scale differences between GIRK channels in the presence and absence of Gß
, it cannot account for the observation of Ivanova-Nikolova and Breitwieser that the Po within a burst increases when the concentration of Gß
is increased (
Our recordings suggest that the only difference between GR7.1 and IRK2 is in the G protein sensitivity of the transition from the bursting state to the long closed state. Because the sequences of these two channels differ only in their intracellular domains, it is likely that this transition is mediated by conformational changes in this intracellular domain. This would be the case if, as several groups have suggested ( is thought to function by increasing the affinity of its targets for the membrane (
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Footnotes |
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Dr. Bard's current address is Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472. Dr. Kunkel's current address Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, MO 63110
1 Abbreviations used in this paper: ACh, acetylcholine; GIRK, G proteinactivated inward rectifier potassium channel; IRK, inward rectifier potassium channel; mAChR, muscarinic acetylcholine receptor.
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Acknowledgements |
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This work is dedicated to the memory of Dr. Ernest G. Peralta (19591999).
The authors would like to thank Markus Meister, Guido Guidotti, Gary Yellen, and Jennifer Hill for helpful discussions and comments on the manuscript, and Karl Magleby, Anthony Morielli, and Reed Carroll for invaluable technical advice.
This work was supported by a National Institutes of Health grant GM42843-09 and by a Howard Hughes Medical Institute predoctoral fellowship to J. Bard.
Submitted: 26 July 2000
Revised: 13 September 2000
Accepted: 14 September 2000
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