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(Circulation Research. 1995;76:832-838.)
© 1995 American Heart Association, Inc.

Articles

Cardiac Muscarinic Potassium Channel Activity Is Attenuated by Inhibitors of G_ß

Lawrence A. Nair, James Inglese, Robert Stoffel, Walter J. Koch, Robert J. Lefkowitz, Madan M. Kwatra, Augustus O. Grant

From the Departments of Medicine, Biochemistry, and Anesthesiology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC.

Correspondence to Augustus O. Grant, MD, Box 3504, Duke University Medical Center, Durham, NC 27710.

	Abstract

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Abstract The cardiac muscarinic potassium channel (I_K.ACh) is activated by a G protein upon receptor stimulation with acetylcholine. The G protein subunit responsible for activation (G versus G_ß) has been disputed. We used G_ß inhibitors derived from the ß-adrenergic kinase 1 (ßARK1) to assess the relative importance of G_ß in I_K.ACh activation. In rabbit atrial myocytes, I_K.ACh had a conductance of 49±6.2 pS. In inside-out patches, the mean open time was 1.60±0.57 ms, mean time constant (_o) was 1.59±0.53 ms, and mean closed time was 3.02±1.35 ms (n=38). ßARK1 is a G_ß-sensitive enzyme that interacts with G_ß through a defined sequence near its carboxyl terminus. A 28-amino-acid peptide derived from the carboxyl terminus of ßARK1 (peptide G) increased the closed time to 10.04 ms (P<.001) and decreased opening probability (NP_o) by 71% (P<.001). Fusion proteins containing the entire carboxyl terminus of ßARK1, glutathione S-transferase ßARK1ct and hexahistidine ßARK1ct, decreased NP_o by 67% (P=.03) and 48% (P=.009), respectively. They also both significantly increased the closed time. None of the inhibitors affected mean open time or channel amplitude. A control peptide derived from a neighboring region of ßARK1 had no significant effect on I_K.ACh activity. These results provide further evidence for the role of G_ß in the activation of I_K.ACh.

Key Words: muscarinic receptor • potassium channel • G protein • ß-adrenergic receptor kinase

	Introduction

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Acetylcholine (ACh) activates a cardiac inwardly rectifying K⁺ channel (I_K.ACh) via M₂-muscarinic receptors, resulting in hyperpolarization of atrial tissue as well as sinoatrial and atrioventricular nodal cells. This effect can produce a negative chronotropic response, while the atrioventricular node and atrial tissue respond in a variable fashion. I_K.ACh is coupled to its receptor via a GTP-binding protein (G_i protein).^{1 2 3 4} Upon ligand binding to the receptor, the heterotrimeric G protein (G_K) dissociates into a GTP-bound -subunit (G_-GTP) and a ß-subunit (G_ß). Initial inquiry into the physiological roles of these subunits focused on G_-GTP as the active component in signaling.^{5 6 7 8} Subsequent investigation revealed G_ß (generally derived from oligomeric G_o/G_i preparations) to be an activator of I_K.ACh,^{9 10 11} as well as many other intracellular effectors, including phospholipase A₂, phospholipase C, types II and IV adenylyl cyclase, and the ß-adrenergic receptor kinase 1 (ßARK1).^{12 13 14 15 16 17 18} There has been some dispute regarding the relative importance of G versus G_ß in activating I_K.ACh.^{19 20 21} To further investigate this issue, we used specific G_ß inhibitors derived from ßARK1,²² a cytosolic enzyme known to phosphorylate ß-adrenergic²³ and muscarinic cholinergic receptors.²⁴ The translocation of ßARK1 to the plasma membrane is stimulated by G_ß, which is membrane-associated via the geranylgeranyl isoprenoid moiety of the -subunit.^{17 18} ßARK1, which lacks its own isoprenyl group, acquires its membrane affinity by binding G_ß.^{17 25} The G_ß binding site on ßARK1 has been localized to a 125-amino-acid residue region within its carboxyl terminus.^{17 18 22} Previous studies have shown that the carboxyl terminus of ßARK1 can inhibit the G_ß activation of phospholipase C-ß,²⁶ type II adenylyl cyclase,^{26 27} and a cloned muscarinic potassium channel (GIRK) expressed in Xenopus oocytes.²⁸ In this study, we used these specific G_ß inhibitors to examine the role of G_ß in I_K.ACh activation in native membrane patches.

	Materials and Methods

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Cell Isolation
Single atrial myocytes were isolated from adult male New Zealand White rabbits. Animals were anesthetized with ketamine (100 mg IM) and pentobarbital sodium (100 mg IP), anticoagulated with heparin (700 U IV), and ventilated with 21% oxygen. The heart was removed and perfused by the Langendorff technique with the Joklik modification of Eagle's MEM (Sigma Chemical Co) containing 33 U/mL hyaluronidase (Sigma), 180 U/mL collagenase type III (Worthington Diagnostics), and 99 U/mL protease (Sigma) at 3 mL · g^-1 · min^-1 for 30 to 60 minutes. All solutions were kept at 37°C and gassed with 100% oxygen. After perfusion, the atria were removed and agitated in a solution containing Joklik medium, 10% bovine calf serum (HyClone Laboratories), penicillin G (75 U/mL), and streptomycin (0.05 mg/mL), filtered through a 250-µm nylon mesh (Tetko, Inc), and centrifuged at 400g for 5 minutes. Cells were resuspended in a solution containing Dulbecco's modified Eagle's medium (DMEM, HyClone), HAM's/F-12 (HyClone), 10% bovine calf serum, and antibiotics (as above). Half of the atrial tissue was initially agitated in serum-free medium and treated with DNAse (0.02 mg/mL; Sigma) for 15 minutes, centrifuged, and resuspended in the above solution. The cells were incubated at 37°C with humidified 5% CO₂. Only freshly isolated rod-shaped cells with clear striations were used.

Preparation of ßARK1 Peptides and Fusion Proteins
The peptides corresponding to amino acids ala⁵³¹-tyr⁵⁵³ (peptide A) and to trp⁶⁴³-ser⁶⁷⁰ (peptide G) of ßARK1 were prepared as their amino-terminal acylated and carboxyl-terminal amidated forms by methods of synthesis and purification previously described.²² The glutathione S-transferase (GST) and hexahistidine (His₆) fusion proteins (GST-ßARK1ct and His₆-ßARK1ct, respectively) containing the carboxyl-terminal 222 amino acids of ßARK1 (pro⁴⁶⁷-leu⁶⁸⁹) were prepared as described.^{17 27} Fig 1 shows the relation of these peptide and protein G_ß inhibitors to the carboxyl terminal segment of ßARK1.

View larger version (12K): [in this window] [in a new window]   Figure 1. Schematic illustrating the derivation of the protein and peptide inhibitors from ß-adrenergic kinase 1 (ßARK1). Scale bar represents the number of amino acid residues, starting from the amino terminal end at the left to the carboxyl terminal end at the right. The ßARK1 enzyme contains 689 amino acid residues. The experimentally determined Gß-binding domain lies between Q546 (glutamine at position 546) and S670 (serine at position 670). The GST-ßARK1ct (glutathione S-transferase ßARK1 carboxyl terminus) fusion protein contains the carboxyl terminal end of ßARK1, encompassing the residues between P467 (proline at position 467) and L689 (leucine at position 689), inclusive. His6-ßARK1ct contains the same segment from ßARK1 attached to a hexahistidine tag. Peptide G contains 28 amino acid residues at the carboxyl terminal end of the Gß binding site (trp643-ser670). Peptide A contains 23 residues lying outside the Gß-binding site (ala531-tyr553).

Solutions and Reagents
The bath solution consisted of (in mmol/L) KCl 140, MgCl₂ 2, EGTA-KOH 5, HEPES 5, Na₂ATP 0.2 to 0.4, and Na₃GTP 0.1. The solution was titrated with KOH to a pH of 7.3 at 35°C. Micropipettes were filled with a solution containing (in mmol/L) KCl 140, CaCl₂ 1, MgCl₂ 1, and HEPES 5, and ACh (Sigma) at 1 µmol/L. The pH was adjusted to 7.4 with KOH. The composition of the perfusion solution was identical to that of the bath solution. All solutions were filtered through a 0.22-µm filter. The peptide and protein inhibitors and G_ß were dissolved in the perfusion solution immediately before use. Peptide G and peptide A were used at a concentration of 100 µmol/L. His₆-ßARK1ct and GST-ßARK1ct fusion proteins had concentrations of 20 and 10 µmol/L, respectively. Bovine brain G_ß was prepared as previously described and used at a 20-nmol/L concentration.²⁹

Recording Techniques
Recording micropipettes were fabricated from 1.5-mm-OD borosilicate glass tubing (6030, A-M Systems, Inc) by use of a Flaming-Brown P80/PC horizontal puller (Sutter Instrument Co), coated with Sylgard 184 elastomer (Dow Corning), and fire-polished immediately before use. Microelectrode resistances ranged from 8 to 10 M. Microelectrodes were coupled to the amplifier headstage via a Teflon-coated Ag/AgCl wire. The bath solution was grounded via a Ag/AgCl wire immersed in agar gel containing internal solution. Junction potentials were offset just before the formation of a gigaohm seal. The recording chamber, bath solution, and perfusion solution were all maintained at 33°C to 35°C with a bipolar temperature controller (TC-202, Medical Systems Corp). Gigaohm seals and inside-out patches were obtained by standard techniques.³⁰ Patches were held at -80 mV, and currents were measured with an EPC-7 patch-clamp amplifier (List Electronics). Perfusion micropipettes were fabricated from N-51A borosilicate glass (Drummond Scientific Co) with an aperture of 30 to 40 µm. Peptide and protein inhibitors were applied via a pressure injector (PV820 Pneumatic PicoPump, World Precision Instruments, Inc) through a perfusion micropipette positioned adjacent to the cell. Current signals were filtered at a corner frequency of 1.5 kHz with an eight-pole Bessel filter (902LPF, Frequency Devices) and sampled at 5 kHz.

Data Storage and Analysis
Data acquisition protocols were generated with a Compaq-Prolinea 3/25s personal computer (Compaq Computer Corp) with a TL-1 interface (Axon Instruments) and customized software. Data were acquired in 500-ms recording frames separated by 1 ms and digitized with an analog-to-digital interface with 12-bit resolution (DT 2821, Data Translation) and a Compaq 386-20 computer. Digitized data were analyzed off-line with a Sun 4/280 microcomputer (Sun Microsystems, Inc). An automatic threshold detection scheme was used with software developed in our laboratory. The open-state threshold was set at half the single-channel amplitude.³¹ The weighted least-squares fit to histograms of the open time was used to measure the closing rate. A bin width of four sampling intervals was used. A minimum ² procedure was used to assess the fit of the distribution to a single exponential. Open times were fit by one exponential, whereas closed times required two exponents. Data are expressed as mean±sample SD. Student's paired t test and ANOVA were used to make statistical comparisons. NP_o was calculated as the total number of channel openings multiplied by the channel opening probability.

	Results

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The current-voltage relation for I_K.ACh in cell-attached patches is shown in Fig 2. The mean chord conductance was 49±6.2 pS (n=12) and displayed strong inward rectification. I_K.ACh channel kinetics were examined in inside-out patches in control conditions (n=38). The internal and external solutions contained equivalent potassium concentrations. The open- and closed-time histograms from a representative patch are displayed in Fig 3A. The mean open time was 1.60±0.57 ms and was fit with a monoexponential function with a time constant (_o) of 1.59±0.53 ms. These values for conductance, mean open time, and _o are similar to those previously reported for I_K.ACh in rabbit atrial myocytes.^{32 33} The mean closed time was 3.02±1.35 ms and was best fit by a double exponential function. The fast time constant was 1.00 ms, and the slow time constant was 6.10 ms.

View larger version (20K): [in this window] [in a new window]   Figure 2. Recordings showing current-voltage (I-V) relation of the muscarinic activated K+ channel. A, Membrane currents recorded from a cell-attached membrane patch at various holding potentials (-80 to +20 mV). Current and time calibrations are shown at lower right. B, Mean single-channel current amplitude (n=12) with error bars (SD) is plotted against the holding potential. The least-squares fit gave a single-channel chord conductance of 49 pS. The extrapolated reversal potential was -3.8 mV.

View larger version (23K): [in this window] [in a new window]   Figure 3. Histograms of open and closed times under control conditions and with peptide G. A, Representative data from an excised patch in control conditions. Number of events per bin (800 µs) vs time. The continuous lines are derived from the density function f(t)= exp(-t) fitted to the observations (t indicates time; , rate constant). Since the area under the probability-density function should be 1, the plots are obtained by multiplying the function by the expected total number of events. The open time was fit by a single exponential (1.59 ms) and the closed time best fit by two exponentials (1 and 6.1 ms). Inset is a representative recording. B, Open- and closed-time histograms from an excised patch exposed to peptide G. The mean open time and the fast component of the closed-time distribution were similar to control conditions. However, the slow component of the closed time was prolonged (14.21 ms). Inset shows a representative recording.

The GTP dependence of I_K.ACh in this experimental preparation was confirmed (Fig 4A). Patches were excised in GTP-free solution, resulting in gradual rundown of channel activity. Compared with NP_o when cell-attached, the relative NP_o in excised patches at 1, 3, and 6 minutes was 0.26, 0.08, and 0.06, respectively. Perfusion with 100 µmol/L GTP consistently reactivated patches. Washout of GTP returned the channels to baseline activity (n=6). There was no significant difference in either NP_o, open time, or closed time between cell-attached conditions and inside-out patches with GTP.

View larger version (27K): [in this window] [in a new window]   Figure 4. Recordings showing GTP dependence of IK.ACh channel activity. A, Channel activity with and without GTP. In this and all subsequent figures, the top row gives the conditions for each recording (CA indicates cell-attached; IO, inside-out excised patch; and W, wash), and the lower row indicates the time lapse since the onset of the condition shown at the arrow. After excision, channel activity progressively diminished. By 6 minutes, opening probability (NPo) had dropped to an average of 6% of control activity. GTP (100 µmol/L) activated the patch to a level greater than or equal to that under cell-attached conditions. Washout returned channel activity to the previous baseline level. B, Stability of IK.ACh activity in excised patches with GTP in the bath. Channel activity is stable under recording conditions for up to 25 minutes after patch excision. Calibration scale applies to both panels.

The stability of inside-out patches in this system was established by excising patches in GTP-containing solution (Fig 4B). Stable channel activity was demonstrated up to 25 minutes after excision (mean, 10.5 minutes; n=4).

To examine the effect of G_ß inhibitors on I_K.ACh kinetics, various peptide and protein constructs with G_ß binding activity were perfused onto the inside-out patches. Peptide G contains 28-amino-acid residues derived from the carboxyl terminus of ßARK1. Application of peptide G (100 µmol/L) consistently resulted in a decrement in channel activity (Fig 5A). The mean NP_o decreased to 29% of the activity during control conditions (P<.001), as seen in Fig 6. The mean closed time increased from 3.52 to 10.04 ms (P<.001, n=13). The time constant for the closed time had a fast component of 0.96 ms and a slow component of 14.21 ms (Fig 3B). There was no significant change in the mean open time (Fig 3B) or in the current amplitude. Washout of peptide G did not result in a return to baseline activity in any of the trials. However, application of G_ß (20 nmol/L) consistently reactivated channel activity (n=3), demonstrating continued patch viability (Fig 5B).

View larger version (26K): [in this window] [in a new window]   Figure 5. Recordings showing that IK.ACh activity is diminished by application of peptide G but not peptide A. A, Perfusion of peptide G (100 µmol/L) onto an excised inside-out (IO) patch markedly reduces channel activity, and it remains suppressed despite a washout (W) phase. Channel activity was reduced in all patches tested (n=13). The opening probability (NPo) diminished by an average of 71% and remained at this level after washout. B, After inhibition with peptide G, the patch can be reactivated with Gß (20 nmol/L). C, Application of peptide A (100 µmol/L) does not affect IK.ACh channel activity, even with a prolonged exposure. Calibration scale applies to B and C.

View larger version (42K): [in this window] [in a new window]   Figure 6. Bar graph showing relative opening probability (NPo) for each peptide and the fusion proteins. The NPo for each patch during perfusion with the respective compound was compared with its own control by Student's paired t test and normalized to the control NPo. Standard deviation bars are shown. *Significant differences compared with controls. GST-ßARK1ct and His6-ßARK1ct represent the fusion proteins glutathione S-transferase ß-adrenergic receptor kinase 1 carboxyl terminus and the hexahistidine ß-adrenergic receptor kinase 1 carboxyl terminus, respectively.

To confirm that the effects of peptide G were due to its G_ß-binding capacity and not a nonspecific inhibitory property, a second peptide was also examined. Peptide A is derived from an upstream region of ßARK1 and contains a similar number of amino acid residues. It has previously been shown that peptide A does not inhibit G_ß activation of ßARK1.²² Perfusion of peptide A (100 µmol/L) onto excised patches did not significantly affect channel activity (Fig 5C). Mean closed time was 2.56 ms in control conditions and 2.82 ms with peptide A (P=NS, n=5). There was also no significant change in NP_o (Fig 6), mean open time, or current amplitude. These results indicate that peptide G does specifically bind G_ß, resulting in a significant reduction in the activation of I_K.ACh.

To further explore the role of G_ß in signal transduction, we also used fusion protein inhibitors of G_ß: GST-ßARK1ct and His₆-ßARK1ct. In four patches, GST-ßARK1ct (10 µmol/L) produced a decrease in channel activation (Fig 7A). The mean closed time increased from 2.24 to 7.4 ms (P=.037). The relative NP_o decreased to 33% of control conditions (P=.03, Fig 6). Application of His₆-ßARK1ct (20 µmol/L) also resulted in a decrement in channel activity (n=6, Fig 7B). The mean closed time increased from 2.60 to 4.32 ms (P=.006), and the relative NP_o decreased to 52% of control activity (P=.008, Fig 6). There was no significant change in current amplitude or mean open time for either of these protein constructs.

View larger version (34K): [in this window] [in a new window]   Figure 7. Recordings showing fusion protein inhibition of IK.ACh activity in excised patches. A, GST-ßARK1ct (10 µmol/L) produced progressive attenuation in channel openings without a significant change after washout (W) (n=4). IO indicates inside-out patch. B, Application of His6-ßARK1ct (20 µmol/L) resulted in a similar though less pronounced effect on IK.ACh channels (n=6). The opening probability (NPo) was significantly reduced for both preparations. GST-ßARK1ct and His6-ßARK1ct represent the fusion proteins glutathione S-transferase ß-adrenergic receptor kinase 1 carboxyl terminus and the hexahistidine ß-adrenergic receptor kinase 1 carboxyl terminus, respectively. Calibration scale applies to both panels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The physiological role of G and G_ß in regulating I_K.ACh has been under continuing investigation. Heterogeneous G_-GTPS preparations,^{5 6 8} as well as G_ß,^{9 10 11 12} have been demonstrated to activate I_K.ACh. G_-GTPS can activate I_K.ACh in picomolar concentrations, whereas G_ß requires concentrations in the nanomolar range. However, it is expected that a permanently active form of the subunit would be more potent than a more physiologically appropriate transient form that is continuously cycling between its GDP- and GTP-bound states. A more relevant though less feasible comparison would be between G_-GTP and G_ß.¹⁶

It is unclear whether both G and G_ß participate in activating I_K.ACh in vivo or whether only one unique subunit is responsible. Evidence for the role of G as the physiological activator derives from two primary sources: (1) diminished agonist activation of I_K.ACh with a monoclonal antibody to the transducin -subunit⁷ and (2) inhibition of I_K.ACh activation with G_ß.²¹ However, further investigation revealed that the above antibody actually disrupts the link between receptor and G protein and not the one between the ion channel and the G protein subunit.³⁴ In addition, an inhibitory effect of G_ß has not been corroborated by other investigators. Evidence for the physiological role of G_ß in activating I_K.ACh has come from several different approaches: (1) Bovine G_ß^{9 10 11 12} as well as recombinant G_ß³⁵ activates I_K.ACh. (2) Both GTP-S and G_ß activate I_K.ACh in a positive cooperative manner with a similar Hill coefficient.³⁶ (3) Multiple heterogeneous G_-GDP preparations and recombinant G_-GDP diminish I_K.ACh activation, presumably by forming an inactive trimer of the G- and G_ß-subunits.^{11 28 35} (4) Recent investigation using heterogeneous G_-GTPS preparations at 10 pmol/L to 10 nmol/L concentrations has shown only weak, inconsistent activation of I_K.ACh, with maximal channel activation only 20% of that induced by GTP-s or G_ß.¹¹

We examined the physiological significance of G_ß in I_K.ACh activation by using specific G_ß inhibitors in an isolated cell membrane patch. The inhibitors are derived from the carboxyl terminal segment of ßARK1. Peptide G is a 28-amino-acid segment that appears to contain the specific residues critical for G_ß binding to ßARK1. It inhibits G_ß activation of ßARK1 in rod outer segment membrane phosphorylation assays with a mean IC₅₀ of 76 µmol/L.²² We showed that peptide G significantly inhibits I_K.ACh in inside-out membrane patches at a concentration of 100 µmol/L. NP_o is decreased by 71% (P<.001), and closed time is increased approximately threefold (P<.001). Peptide G appears to bind G_ß irreversibly, since there is no return to baseline activity with washout. However, its inhibitory properties are not due to a nonspecific membrane effect, since application of G_ß results in a vigorous return of channel activity. In addition, we examined the properties of a control peptide derived from a neighboring region of ßARK1 (peptide A), which has no in vitro effect on G_ß activation of ßARK1.²² This peptide did not change I_K.ACh channel activity.

We also used fusion proteins containing a much larger portion of the ßARK1 carboxyl terminus. His₆-ßARK1ct and GST-ßARK1ct each contain the terminal 222-amino-acid residues of this segment, linked to a hexahistidine or GST tag, respectively. The hexahistidine tag is a small polypeptide, whereas GST is a large protein. These tags enhance purification methods and increase protein stability. The use of two different tags mitigates, to a degree, their potential to confound the interpretation of inhibitory effect of the protein. Use of the fusion proteins allows the entire carboxyl terminus binding domain to be evaluated. In addition, we can compare our results with those of other investigations using these protein segments. Previous work has shown that the cellular expression of the carboxyl terminus of ßARK1 inhibits G_ß activation of phospholipase C-ß and type II adenylyl cyclase in COS-7 cells.²⁶ His₆-ßARK1ct has also been demonstrated to inhibit G_ß activation of type II adenylyl cyclase in a permeabilized human embryonic kidney cell system²⁷ as well as a cloned rat muscarinic potassium channel (GIRK) in Xenopus oocytes.²⁸

We found that GST-ßARK1ct (10 µmol/L) and His₆-ßARK1ct (20 µmol/L) both affected I_K.ACh activity by significantly decreasing NP_o and increasing the closed time. These protein constructs were used at concentrations close to their mean IC₅₀, as assessed by their inhibition of G_ß activation of ßARK1 phosphorylation activity.²² At these concentrations, their ability to inhibit I_K.ACh was comparable to the activity of peptide G, which was used at a concentration near its IC₅₀. However, since dose-response curves were not performed, we cannot draw definite conclusions regarding the relative potency of these compounds in attenuating I_K.ACh channel activity.

One potential drawback to the use of these fusion proteins relates to their large molecular weight. The diffusion and penetration of these fusion proteins to the active membrane site may not be as efficient as with the smaller peptides. Further, the His₆ and GST tags could potentially alter the potency of the fusion protein in inhibiting G_ß, either by steric hindrance or via an intrinsic property of the tag itself.

This is the first study to assess the effect of peptide G and GST-ßARK1ct on I_K.ACh. Although channel activity was reduced, it was not abolished. Overall channel activity would depend on the concentration of agonist and GTP, the rate of hydrolysis of G_-GTP, and the binding affinity of the inhibitor to G_ß. Given the persistence of some residual I_K.ACh activity, it is not possible to definitively conclude that G_ß is the only activator. However, since multiple investigators have recently demonstrated the inhibitory capacity of G_-GDP,^{11 28 35} residual I_K.ACh activity is most likely due to incomplete suppression of Gß.

One previous study assessed the effect of His₆-ßARK1ct on the activity of a cloned rat muscarinic potassium channel (GIRK1), expressed in Xenopus oocytes along with ß₁ and ₂.²⁸ NP_o was markedly diminished upon application of this fusion protein to an excised patch. This effect was more complete than was seen with His₆-ßARK1ct in our preparation (87% versus 48% reduction compared with control). The concentrations used in the two studies were similar. There may be a difference in the affinity of His₆-ßARK1ct to ß₁₂ compared with rabbit atrial G_ß. Kleuss et al³⁷ demonstrated that different ß subtypes vary in their ability to interact with their assigned effector. They concluded that G protein structural differences are reflective of a functional diversity. Similarly, different G_ß subunits may vary in their ability to bind the carboxyl terminal segment of ßARK1. However, the subtype composition of G_ß in rabbit atrial tissue is unknown at present.

It is theoretically possible that these inhibitors diminished I_K.ACh activity by acting directly on the channel. However, we previously showed that peptide G and the fusion proteins specifically bind G_ß.²² In addition, as previously discussed, the ßARK1 carboxyl terminus attenuates the G_ß activation of a variety of other intracellular effectors, including type II adenylyl cyclase and phospholipase C-ß. It is unlikely that the inhibitors prevent effector activation by binding G_ß in some subcellular signaling systems and act directly on the effector in others. The most consistent interpretation involves direct inhibition of G_ß.

In summary, application of peptide and fusion protein G_ß inhibitors results in a significant decrease in I_K.ACh channel activity. Peptide G and the ßARK1 carboxyl terminal fusion proteins all significantly increased the closed time and decreased NP_o compared with control conditions. Although a similar result was demonstrated with a cloned muscarinic potassium channel expressed with heterologous G_ß, this is the first study to show inhibition of G_ß-I_K.ACh coupling in an intact membrane system. This represents an important step in enhancing our understanding of G protein signaling and provides further evidence for the role of G_ß as a physiological activator of I_K.ACh.

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid from the American Heart Association, North Carolina affiliate (Dr Kwatra), and grants HL-32708 and HL-17670 from the National Institutes of Health. We wish to express our appreciation to Ronda Baldwin for her expertise in cell isolation techniques.

Received November 3, 1994; accepted January 23, 1995.

	References

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