Isoprenaline can activate the acetylcholine-induced K⁺ current in canine atrial myocytes via G_s-derived βγ subunits

Abstract

1. G protein βγ subunits activate the acetylcholine-induced potassium current I_k,ach. There is no evidence of specificity at the level of the βγ subunits. Therefore all G protein-coupled receptors in atrial myocytes should be able to activate I_k,ach. Paradoxically, it is often stated that isoprenaline does not activate I_k,ach. Rationales to explain this negative result include insufficient concentrations of G_s in the atrium or restricted access of G_s-derived βγ subunits to the I_k,ach channel. We took advantage of a non-specific increase in G_s that results after infection with adenovirus.

2. Adenoviral infection unmasked a 1 μm isoprenaline-induced I_k,ach which was prevented by propranolol. Isoprenaline occasionally activated I_k,ach in uninfected and freshly dissociated atrial myocytes but the effect was larger and more consistent in infected myocytes.

3. Pertussis toxin pretreatment (100 ng ml⁻¹ overnight) did not block the effect of isoprenaline. The effect of isoprenaline became persistent if cells were pretreated with cholera toxin (200 ng nl⁻¹).

4. Signal transduction events distal to adenylyl cyclase were not involved in isoprenaline-induced I_k,ach. Forskolin (10 μm) did not activate I_k,ach. Inhibition of adenylyl cyclase with cytoplasmic application of 300 μm 2′-deoxyadenosine 3′-monophosphate did not prevent the activation of I_k,ach by isoprenaline.

5. Cytoplasmic application of a βγ binding peptide derived from the C terminus of β-adrenergic receptor kinase 1 (50 μm) prevented the effect of isoprenaline on I_k,ach. The peptide did not prevent the stimulation of the L-type calcium current by isoprenaline.

6. The results indicate that β-adrenoceptors can activate I_k,ach in atrial myocytes through the release of βγ subunits from G_s.

The atrial acetylcholine-induced potassium current (I_K,ACh) is an important regulator of cardiac rate, atrioventricular nodal conduction and force of contraction of the atrium (Loffelholz & Pappano, 1985). As the name implies, acetylcholine can activate I_K,ACh in native atrial myocytes. Adenosine is also effective (Kurachi et al. 1986). Both adenosine and acetylcholine are coupled to I_K,ACh by pertussis toxin-sensitive G proteins. There is some degree of receptor specificity for activation of I_K,ACh. It is often stated that β-adrenoceptor activation does not stimulate I_K,ACh (e.g. Hartzell, 1988, p. 215).

The channel for I_K,ACh is probably a heterotetramer composed of two Kir 3.1 (or GIRK1) and two Kir 3.4 (or CIR) subunits (Krapivinsky et al. 1995b; Tucker et al. 1996). The activation of I_K,ACh by adenosine or acetylcholine is dependent on pertussis toxin-sensitive GTP binding protein (G protein) (Sorota et al. 1985; Pfaffinger et al. 1985; Breitwieser & Szabo, 1985; Kurachi et al. 1986). G proteins are heterotrimeric proteins composed of two functional parts, an α subunit and a βγ dimer (Gilman, 1987). In the absence of agonists, the α subunits exist predominantly in the inactive GDP-liganded state which favours the association of αβγ (Gilman, 1987). Agonist-bound receptor promotes the dissociation of GDP from the α subunit. GTP then binds to the α subunit and the heterotrimer dissociates into α-GTP and free βγ dimer (Gilman, 1987). Both portions of the G protein are known to have important effects on downstream effector molecules (Gilman, 1987; Tang & Gilman, 1991; Pitcher et al. 1992; Taussig et al. 1993; Sternweis, 1994). Reconstitution studies have shown that I_K,ACh can be activated by GTPγS-liganded α subunits of the G_i family (but not G_s) (Yatani et al. 1987; Cerbai et al. 1988) or by βγ subunits (Logothetis et al. 1987; Cerbai et al. 1988). In addition to interaction with downstream effectors the α subunit and the βγ dimers have other essential roles. The α subunit is important for controlling the specificity of receptor-G protein interactions (Gilman, 1987). The βγ dimers are important for the distribution and proper functioning of α subunits (Florio & Sternweis, 1989; Sternweis, 1994).

Although evidence exists for a direct physical interaction between α subunits and an N-terminal region of Kir 3.1 (Huang et al. 1995), recent molecular studies on the mechanism for G protein activation of I_K,ACh have focused on the role of βγ subunits. Direct binding of βγ to Kir 3.1 and Kir 3.4 has been demonstrated (Krapivinsky et al. 1995a). Exogenous overexpression studies have shown that βγ subunits are effective activators of I_K,ACh subunits and that regions of the N and C terminus of Kir 3.1 are responsible for the activation by βγ subunits (Huang et al. 1995; Slesinger et al. 1995). The N-terminal βγ binding region from Kir 3.1 interacts efficiently with β₁ and β₂ subunits but not with γ subunits (Yan & Gautam, 1996). Consistent with an important role of βγ subunits in the physiological activation of I_K,ACh, a βγ binding peptide derived from the C terminus of β-adrenergic receptor kinase 1 (βARK-CT) (Koch et al. 1993) inhibited I_K,ACh channel activity in native atrial membranes (Nair et al. 1995).

Injection of 5-hydroxytryptamine receptor (5HT_1A) mRNA and total rat atrial mRNA in Xenopus oocytes resulted in the expression of a pertussis toxin-insensitive 5HT-induced inward rectifier (Dascal et al. 1993). If mRNA for G_iα2 was also injected, the 5HT-induced current became pertussis toxin sensitive. The induction of a similar current by an exogenous pertussis toxin-sensitive G protein and a pertussis toxin-insensitive G protein suggested that βγ subunits were responsible for current activation since the identity of the α subunit did not seem to be important in this exogenous overexpression system (Dascal et al. 1993). It has also been shown that coexpression of β₂-adrenoceptors, G_sα and Kir 3.1 can result in an isoprenaline-induced potassium current (Lim et al. 1995). It should be emphasized that stimulation of I_K,ACh via G_s-coupled receptors has never been shown to occur without exogenous overexpression of receptors, G proteins and potassium channel subunits, i.e. it has not been shown to occur in native cardiac myocytes.

Reconstitution studies using recombinant βγ subunits did not find important differences in the potency or efficacy of numerous βγ combinations to activate native I_K,ACh (Wickman et al. 1994). The studies of Wickman et al. (1994) included β₂, γ₅ and γ₇, which are probably the predominant β and γ subunits in native adult cardiac myocytes (Hansen et al. 1995). It therefore appears unlikely that receptor specificity for the activation of I_K,ACh arises at the level of the βγ subunits.

If βγ dimers are the physiological activators of I_K,ACh then any G protein-coupled receptor that is present in the atrium should be able to release βγ subunits and activate I_K,ACh. This prediction conflicts with the observation that β-adrenoceptor agonists are ineffective in activating native atrial I_K,ACh. The activation of I_K,ACh by β-adrenoceptors could be limited by several factors including: (1) spatial distribution of β-adrenoceptors and G_s relative to the channels, (2) an inadequate amount of βγ release from G_s compared with the amount of βγ released from pertussis toxin-sensitive G proteins, or (3) a significant role of the α subunit in the physiological activation of I_K,ACh. If factors (1) or (2) are involved, then increasing the amount of G_s present in the cell may permit activation of I_K,ACh by β-adrenoceptor agonist.

Infection of cardiac myocytes with replication-deficient adenovirus can non-specifically increase the expression of G_s (Novotny et al. 1994). The increased G_s is localized in the particulate fraction and is efficiently coupled to β-adrenoceptors (Novotny et al. 1994). Surprisingly, infection of cardiac myocytes with an adenoviral construct containing the Rous sarcoma virus (RSV) promoter upstream from the G_sα gene resulted in the additional expression of G_sα that was not efficiently coupled to β-adrenoceptors and was localized in the soluble fraction (Novotny et al. 1994). In the present study we took advantage of the non-specific effect of adenoviral infection to alter responsiveness to β-adrenoceptor agonist to determine whether this would permit the activation of I_K,ACh by isoprenaline.

METHODS

Cell isolation, infection with adenovirus and culture

Dog hearts were excised in accordance with the Guide for the Care and Use of Laboratory Animals (1996, National Academy of Sciences, Washington, DC, USA) after intravenous administration of 35 mg kg⁻¹ pentobarbital. Atrial myocytes were isolated using collagenase digestion of the right atrial free wall as previously described (Sorota & Hoffman, 1989). A replication-deficient adenovirus construct with the RSV promoter coupled to the β-galactosidase gene (Ad-β-Gal) was kindly provided by Dr C. S. H. Young (Department of Microbiology, College of Physicians and Surgeons, Columbia University, NY, USA). Cells were incubated in serum-free minimal essential medium (MEM; Life Technologies) plus 20 μg ml⁻¹ gentamicin for 2 h at 37°C without or with Ad-β-Gal (multiplicity of infection, 1000:1) in a humidified atmosphere of 95 % air, 5 % carbon dioxide. Virus-containing medium was removed, cells were suspended in MEM plus 10 % horse serum (Life Technologies), 1 mM cytosine arabinoside and 20 μg ml⁻¹ gentamicin and plated at 10⁴ cells ml⁻¹ onto laminin-coated 9 mm × 22 mm glass coverslips. Cells were incubated at 37°C in a humidified atmosphere of 95 % air, 5 % carbon dioxide for 2-5 days before being used for whole-cell patch clamp experiments. In some experiments cells were pretreated with pertussis toxin (List Biologicals, Campbell, CA, USA) by incubating cells overnight with 100 ng ml⁻¹ of the toxin. In another experiment cells were pretreated with 200 ng ml⁻¹ of cholera toxin (Calbiochem) for 3-6 h.

Histochemical staining of β-galactosidase activity

Cells were fixed for 5 min at 37°C with 0.5 % glutaraldehyde in a phosphate-buffered saline (PBS) containing (mM): 137 NaCl, 2.7 KCl, 10.58 Na₂HPO₄ and 1.54 KH₂PO₄ (pH to 7.2 with NaOH). Cells were stained for 4 h at 37°C in PBS with the following additions (mM): 5 K₄Fe(CN)₆, 5 K₃Fe(CN)₆, 2 MgCl₂, and 1 mg ml⁻¹ 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). Cells were washed in PBS and observed under a light microscope. There was no blue staining of control cells (not infected with Ad-β-Gal) under these conditions.

Electrical recording

The ruptured patch whole-cell configuration of the patch clamp technique was used for all electrical recordings (Hamill et al. 1981). Electrical activity was recorded as described previously (Du & Sorota, 1997a). Steady-state current-voltage relationships were measured with hyperpolarizing voltage ramps at -14 to -16.25 mV s⁻¹. A holding potential of -50, -10 or +10 mV was used. Steady-state currents and transmembrane potentials were digitized at a sampling interval of 40 ms. In one group of experiments the L-type calcium current was measured during voltage clamp steps from -50 to +5 mV for 310 ms. Caesium-containing solutions (see below) were used for L-type calcium current measurements to minimize contamination by potassium currents. For the calcium current measurements currents were digitized at a sampling interval of 0.02 ms for the first 10 ms and at a sampling interval of 0.6 ms for the next 300 ms.

Electrodes were pulled from 1.5 mm o.d., 0.86 mm i.d. borosilicate glass (Sutter Instruments). Initial electrode resistance ranged from 2 to 3.5 MΩ when filled with pipette solution (see below). Series resistance and cell membrane capacitance were calculated from the capacitance current during a 10 mV depolarizing voltage step as previously described (Sorota & Hoffman, 1989; Sorota, 1992). Series resistance during the experiments was 7 MΩ or less and was not compensated for electronically. The temperature for most experiments was 36 ± 0.5°C. Experiments using pressure-assisted dialysis of βARK-CT were performed at room temperature (21-23°C).

Solutions

The bath solutions for measurement of I_K,ACh (see below) contained 10 μm glibenclamide to block ATP-sensitive potassium currents. All bath solutions were hypertonic relative to the pipette solution to minimize swelling-induced chloride current. The measured junction potential between the bath solution and the pipette solution (10 mV) was subtracted from all measured voltages during analysis of the data.

The pipette solution used for experiments examining I_K,ACh contained (mM): 125 potassium aspartate, 15 KCl, 10 Hepes-KOH (pH 7.2), 5 disodium phosphocreatine, 4 EGTA, 3 MgATP, 1 MgCl₂ and 0.2 GTP. Two different potassium-containing bath solutions were used, one with 4 mM K⁺ and one with 25 mM K⁺. The 4 mM K⁺ bath solution contained (mM): 144 NaCl, 4 KCl, 10 Hepes-NaOH (pH 7.4), 1.8 CaCl₂, 1 MgCl₂, 5.5 dextrose, 50 mannitol and 0.01 glibenclamide. The 25 mM K⁺ bath solution contained 25 mM KCl and no mannitol but was otherwise identical in composition.

When the L-type calcium current was measured, the pipette solutions contained (mM): 20 CsCl, 20 TEA-Cl, 100 caesium aspartate, 10 Hepes-CsOH (pH 7.2), 5 disodium phosphocreatine, 4 EGTA, 3 MgATP, 1 MgCl₂ and 0.2 GTP. The bath solution contained (mM): 129 NaCl, 20 CsCl, 10 Hepes-NaOH (pH 7.4), 1.8 CaCl₂, 1 MgCl₂, 5.5 dextrose and 60 mannitol.

A stock of 10 mM isoprenaline in 10 mM HCl was prepared fresh on the day of an experiment. The isoprenaline stock solution was stored at 4°C. A final dilution to 1 μm isoprenaline was prepared within 5 min of cell superfusion. Cells were exposed once to isoprenaline. A new coverglass with naive cells was placed in the chamber after each isoprenaline superfusion. When present the concentration of carbachol was 10 μm.

All drugs and chemicals were from Sigma unless indicated otherwise.

Purification of βARK-CT

βARK-CT was purified from NM522 E. coli transformed with pGEX-2T (Pharmacia, Piscataway, NJ, USA) containing an insert coding for amino acids 546-670 of bovine βARK1 fused to glutathione-S-transferase (GST). Transformed NM522 E. coli were obtained from Robert J. Lefkowitz MD, Howard Hughes Medical Institute, Duke University Medical Center, USA.

A stab of the transformed NM522 E. coli was inoculated into 5 ml of LB-ampicillin medium containing (g l⁻¹): 10 bacto tryptone, 5 bacto yeast extract, 10 NaCl (pH 7.0), and 100 μg ml⁻¹ ampicillin. Cells were grown for 6-8 h at 37°C in a shaking incubator. The bacterial culture was diluted into 100 ml of fresh LB-ampicillin medium and grown overnight. The bacterial suspension was diluted to 900 ml with fresh LB-ampicillin medium and grown for 2-2.5 h until the OD at 600 nm was 0.6-0.8. To induce expression of the GST-βARK-CT fusion protein, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 μm. Cells were grown for 2.5-3 h after the addition of IPTG before harvesting by centrifugation at 5000 g for 10 min.

Cells were suspended in 20 ml of ice-cold PBS containing protease inhibitors (100 μm phenylmethylsulfonylfluoride, 154 μg ml⁻¹ benzamidine, 4 μg ml⁻¹ leupeptine, 5 μg ml⁻¹ aprotinin and 1 μg ml⁻¹ pepstatin A) and lysed by sonication (Branson sonicator model S-250, output level 4, 3 bursts of 1 min duration, 70 % duty cycle). Triton X-100 was added to the lysate at a final concentration of 1 % to minimize association of the GST fusion protein with bacterial proteins. The lysate was centrifuged at 10 000 g for 15 min at 4°C and the supernatant retained for subsequent purification.

The supernatant (25 ml) was applied to a 2 ml glutathione Sepharose 4B column (Pharmacia) that had been equilibrated with PBS. The column was washed with ice-cold PBS until the OD of the eluate at 280 nm was less than 0.01. Thrombin was used to release free βARK-CT by cleaving the fusion protein. One hundred units of thrombin were applied to the column in a total volume of 2 ml and the stopcock was closed. After overnight cleavage at room temperature, βARK-CT and thrombin were eluted from the column with 4 ml of PBS. GST remained bound to the column. Benzamidine Sepharose (2 mg; Sigma) was added to the eluate to bind thrombin. After a 2 h incubation at room temperature the slurry was centrifuged at 1500 g for 10 min. The supernatant was dialysed against the potassium-containing pipette solution (minus ATP and phosphocreatine) and was then concentrated using a Centricon-10 filter (Amicon, Beverly, MA, USA). The yield of βARK-CT was 5.5 mg of protein per 2 litres of bacterial culture. Protein concentration was determined by the method of Peterson (1977) with bovine albumin as the standard.

Pressure-assisted dialysis with βARK-CT

In one group of experiments βARK-CT was introduced into the cytoplasm of cells by pressure-assisted dialysis. The pipette tips for these experiments were capillary filled for 5 s with peptide-free solution before backfilling with pipette solution containing 50 μmβARK-CT. Three minutes after patch rupture, peptide solution was injected into the cells by the transient application of positive pressure to the patch electrode. No drugs were applied to the cell for at least 10 min after injection to allow for diffusion of peptide and for cells to shrink back to their initial volume. To ensure that cells did not remain swollen after injection of the peptide, 20 mM mannitol was added to the 25 mM K⁺ bath solution for experiments examining the effect of isoprenaline on I_K,ACh.

Statistics

Statistical analysis was performed using Sigmastat software (Jandel Scientific, San Rafael, CA, USA). Averaged data are presented as means ±s.e.m. Single statistical comparisons were made using Student's unpaired t test. When two current-voltage relationships were compared, a two way analysis of variance was followed by multiple comparisons using the Student-Neuman-Keuls test.

RESULTS

Adenoviral infection unmasks isoprenaline-induced I_K,ACh

Exposure of primary cultures of dog atrial myocytes to Ad-β-Gal at a multiplicity of 1000:1 resulted in infection of 80-90 % of the myocytes as determined with X-Gal staining (not shown). Electrophysiological experiments with 4 mM extracellular K⁺ (K⁺_o) revealed an isoprenaline-induced inward rectifier in infected cells. An example is shown in Fig. 1A and B. In this cell isoprenaline activated a current with a reversal potential and current-voltage relationship that were similar to carbachol-induced I_K,ACh. With the superfusion system that we used (approximately 30 s tubing lag), the time course of activation by carbachol and isoprenaline appeared similar. The effect of isoprenaline subsided on washout. An isoprenaline-induced inward rectifier was observed in 7/9 infected cells studied in 4 mM K⁺_o. Surprisingly, we could also detect an isoprenaline-induced inward rectifier in some of the uninfected cells (2/9). Isoprenaline induced a larger increase in inward slope conductance in infected cells compared with cells not exposed to adenovirus (infected, 81 ± 34 pS pF⁻¹; uninfected, 37 ± 25 pS pF⁻¹; P < 0.05). In contrast to the effect on isoprenaline-induced current, the carbachol-induced increase in inward slope conductance was not significantly different between the two groups (infected, 557 ± 191 pS pF⁻¹; uninfected, 455 ± 141 pS pF⁻¹).

Figure 1. Isoprenaline activates a current similar to I_K,ACh in Ad-β-Gal-infected cells via a pertussis toxin-insensitive pathway.

K⁺_o was 4 mM in these experiments. A, steady-state whole-cell currents recorded in the absence (•) and presence of 1 μm isoprenaline (▪). B, comparison of the 1 μm isoprenaline- (▪) and 10 μm carbachol-induced (•) difference currents from the same myocyte. The currents reverse at approximately -85 mV which is slightly positive to the calculated potassium equilibrium potential (E_K, -94 mV) under our recording conditions. C, pertussis toxin pretreatment does not prevent the effect of isoprenaline on steady-state whole-cell current. Whole-cell currents recorded from an Ad-β-Gal-infected cell that was pretreated overnight with 100 ng ml⁻¹ pertussis toxin. The toxin treatment completely prevented the effect of carbachol but not the effect of isoprenaline. •, control; ▪, isoprenaline; ▴, washout of isoprenaline; ♦, carbachol.

The activation of I_K,ACh by isoprenaline could be due to non-specific activation of pertussis toxin-sensitive G proteins. To test this possibility we looked at the isoprenaline and carbachol responses of Ad-β-Gal-infected cells that had been pretreated with pertussis toxin. An example is shown in Fig. 1C. In this cell isoprenaline was able to activate I_K,ACh although pertussis toxin treatment abolished the effect of carbachol on whole-cell current. Similar results were seen in seven cells. These results indicate that the activation of I_K,ACh by isoprenaline is not mediated by pertussis toxin-sensitive G proteins.

Effect of elevated K⁺_o on isoprenaline-induced I_K,ACh

The currents induced by isoprenaline with 4 mM K⁺_o were small. We increased K⁺_o to 25 mM to enhance the amplitude of I_K,ACh. This improved the signal-to-noise ratio and permitted more reliable measurement of isoprenaline-induced I_K,ACh. Examples of the results in an infected cell and an uninfected cell are shown in Fig. 2A and B, respectively. In both cells there is a readily detectable isoprenaline-induced I_K,ACh. Mean isoprenaline-induced I_K,ACh current densities were larger in infected cells than in uninfected cells (Fig. 2C). Carbachol-induced I_K,ACh current densities were similar in infected and uninfected cells (Fig. 2D). Although adenoviral infection was not required in order to observe isoprenaline-induced I_K,ACh, viral infection resulted in a larger and more consistent effect of isoprenaline. An isoprenaline-induced I_K,ACh was detectable in 6/6 infected cells versus 4/6 uninfected cells. Therefore the remaining experiments were performed on Ad-β-Gal-infected cells.

Figure 2. Effect of Ad-β-Gal infection on isoprenaline- and carbachol-induced I_K,ACh in 25 mM K⁺_o.

A, whole-cell currents recorded from an Ad-β-Gal-infected cell. •, control; ▪, isoprenaline; ▴, washout of isoprenaline; ♦, carbachol. B, whole-cell currents recorded from an uninfected cell from the same cell isolation and culture. C, the isoprenaline-induced inwardly rectifying current is larger in Ad-β-Gal-infected (▪) compared with uninfected cells (•). Averaged isoprenaline-induced currents are presented. Currents were normalized to cell membrane capacitance prior to averaging. The average current density at a given test potential differs significantly (P < 0.05) for the two groups at all potentials negative to -62 mV. D, carbachol-induced I_K,ACh is not affected by Ad-β-Gal infection. Averaged carbachol-induced current densities are presented. Number of cells (n) = 6 for each group. Vertical lines in C and D represent s.e.m. and are plotted at 1 mV intervals.

At the most positive voltages, there is a subtle difference in the shape of the current-voltage relationship for the isoprenaline-induced compared with the carbachol-induced current (Fig. 2C and D). We believe that this reflects a small isoprenaline-induced stimulation of the outwardly rectifying swelling-induced chloride current (Du & Sorota, 1997b). The stimulation of swelling-induced chloride current is dependent on elevation of cAMP levels and can be prevented by intracellular dialysis with an inhibitor of adenylyl cyclase (see below) (Du & Sorota, 1997b). We could also minimize this contaminating current by working at room temperature (see below), which slows the activation of swelling-induced chloride current (Sorota & Du, 1998).

Effect of isoprenaline is mediated by β-adrenoceptors

Cells were exposed to isoprenaline in the absence and presence of propranolol (1 μm) to determine whether activation of β-adrenoceptors was a critical step in the activation of I_K,ACh by isoprenaline. For these experiments Ad-β-Gal-infected cells were equilibrated with propranolol for 5 min before exposure to isoprenaline. After coapplication of propranolol and isoprenaline both drugs were washed out and the cell was challenged a second time with isoprenaline alone. The results of one experiment are shown in Fig. 3A. In this cell isoprenaline did not activate I_K,ACh in the presence of propranolol. After propranolol was washed out isoprenaline was able to activate I_K,ACh. Similar results were seen in two out of two cells.

Figure 3. Isoprenaline activates I_K,ACh via β-adrenoceptors and G_s but independently of adenylyl cyclase.

A, whole-cell currents recorded from an Ad-β-Gal-infected cell. K⁺_o was 25 mM. The cell was preincubated with 1 μm propranolol for 5 min prior to superfusion with the combination of propranolol and 1 μm isoprenaline. Both drugs were washed out for 5 min prior to a second challenge with isoprenaline alone. •, propranolol; ▴, propranolol + isoprenaline; ♦, washout; ▪, isoprenaline alone. B, the effect of isoprenaline is persistent after cholera toxin pretreatment. Averaged results are presented from infected cells that were pretreated with 200 ng ml⁻¹ cholera toxin for 3-6 h. Cholera toxin alone did not fully activate I_K,ACh under basal conditions (○) in Ad-β-Gal-infected dog atrial cells. Isoprenaline (▪) increased I_K,ACh and the effect persisted after isoprenaline was washed out (□) for 4-5 min (n= 4). In control cells from the same preparation, the effect of isoprenaline always subsided after 2-3 min of washout (n= 4, not shown). C, forskolin does not activate I_K,ACh. Averaged drug-induced currents from 4 cells. The effect of forskolin (•, 10 μm) is compared with the effect of carbachol (▪). K⁺_o was 25 mM. D, the adenylyl cyclase inhibitor 2′-dAMP does not prevent the activation of I_K,ACh by isoprenaline. The pipette solution contained 300 μm 2′-dAMP. Averaged drug-induced currents from 4 cells. The effects of carbachol (▪) and isoprenaline (•) are compared. Both drugs activated I_K,ACh under these conditions. K⁺_o was 25 mM. Vertical lines in B-D represent s.e.m.

The effect of isoprenaline is mediated by G_s

Cholera toxin pretreatment was used to determine whether the effect of isoprenaline was mediated by G_s. This toxin specifically ADP-ribosylates the α subunit of G_s and inhibits the deactivation of G_s. Cholera toxin pretreatment tended to increase the basal inward whole-cell current at negative test potentials when compared with control cells from the same preparation but this effect did not achieve statistical significance (n= 4 each group, not shown). There was, however, one marked effect of cholera toxin. In toxin-pretreated cells, the isoprenaline-induced I_K,ACh did not subside on washout of isoprenaline (Fig. 3B). These results are consistent with a slow rate of basal GDP-GTP exchange in our cells, resulting in little activation of G_s by cholera toxin alone. Exposure to isoprenaline would be expected to increase the rate of dissociation of GDP from G_sα and result in persistent activation of ADP-ribosylated G_sα.

Adenylyl cyclase is not involved in isoprenaline activation of I_K,ACh

The best-known effects of isoprenaline on cardiac myocytes are attributable to activation of adenylyl cyclase and subsequent protein kinase A (PKA)-dependent phosphorylation. To determine whether activation of adenylyl cyclase could account for the activation of I_K,ACh by isoprenaline, we looked at whether forskolin, a compound that can directly activate adenylyl cyclase without activating G proteins, could mimic the effect of isoprenaline on I_K,ACh. Forskolin (10 μm) was unable to activate I_K,ACh in Ad-β-Gal-infected cells (Fig. 3C). As shown in Fig. 2C isoprenaline consistently activated I_K,ACh under these experimental conditions.

We also tested whether activation of adenylyl cyclase was important for the effect of isoprenaline on I_K,ACh using conditions that inhibit adenylyl cyclase. 2′-Deoxyadenosine 3′-monophosphate (2′-dAMP) inhibits adenylyl cyclase through interaction with the so-called P-site of adenylyl cyclase (Fain & Malbon, 1979). Intracellular dialysis of dog atrial cells with 300 μm 2′-dAMP completely blocks the stimulation of dog atrial cell L-type calcium current by isoprenaline (not shown). When adenylyl cyclase was inhibited with 2′-dAMP, I_K,ACh could still be activated by isoprenaline (Fig. 3D). Under these conditions the carbachol- and isoprenaline-induced current-voltage relationships had similar shapes.

Effect of βARK-CT on isoprenaline-induced I_K,ACh

A 125 amino acid βγ binding protein, βARK-CT (Koch et al. 1993), was used to determine whether the activation of I_K,ACh by isoprenaline was due to release of βγ subunits from G_s. For this experiment cells that were dialysed with βARK-CT for 10 min were compared with parallel time control cells with no βARK-CT in the pipette solution. The addition of 50 μmβARK-CT to the pipette solution fully inhibited isoprenaline-induced I_K,ACh (Fig. 4A). There was no detectable isoprenaline-induced I_K,ACh in any of the individual cells in the βARK-CT group. This concentration of βARK-CT partially blocked the effect of carbachol on I_K,ACh (Fig. 4B).

Figure 4. βARK-CT peptide fully inhibits isoprenaline-induced I_K,ACh without preventing isoprenaline-dependent stimulation of the L-type calcium current.

A, βARK-CT peptide prevents the activation of I_K,ACh by isoprenaline. Average isoprenaline-induced currents are plotted. Pipette solution contained either 50 μmβARK-CT peptide (▪) or no peptide (•). All cells were transiently inflated 3 min after patch rupture to inject pipette solution. Cell volume returned to control over a 10 min recovery period because the bath solution was hypertonic (see Methods). After the 10 min recovery period isoprenaline-induced currents were measured. B, βARK-CT peptide partially inhibits the activation of I_K,ACh by carbachol. Carbachol-induced I_K,ACh was measured in the same cells studied in A. After isoprenaline was washed out carbachol-induced currents were recorded. K⁺_o was 25 mM. Vertical bars represent s.e.m.n= 6 for each group. C, transient currents evoked by voltage clamp steps from -50 to +5 mV with a 10 s interpulse interval. Caesium-containing pipette and bath solutions were used. Pipette solution contained either no peptide (Control) or 50 μmβARK-CT peptide. Under either condition isoprenaline enhanced the amplitude of the L-type calcium current. All cells were transiently inflated 3 min after patch rupture to inject pipette solution. Cell volume returned to control over a 10 min recovery period because the bath solution was hypertonic (see Methods). After the 10 min recovery period the effect of isoprenaline on L-type calcium currents was determined. Calibration bars represent 50 ms and 3 pA pF⁻¹. The horizontal calibration bar represents the zero current level. Currents were not leak subtracted. D, average peak L-type calcium current density for cells dialysed with βARK-CT peptide or peptide-free pipette solution (Control). Neither basal nor isoprenaline-stimulated current density differed significantly between the two groups. Error bars represent s.e.m.n= 3 for each group.

Although βARK-CT binds to the βγ subunits of heterotrimeric G proteins, responses that are inhibited by βARK-CT are not necessarily attributable to a direct effect of βγ subunits. This is because the function of α subunits of heterotrimeric G proteins is critically dependent on the presence of βγ subunits (Florio & Sternweis, 1989; Sternweis, 1994). Therefore functions that are mediated by the α subunit could be blocked by sequestration of βγ subunits. To determine whether cell dialysis with 50 μmβARK-CT was interfering with the function of G_sα subunits we looked at whether the ability of isoprenaline to stimulate the L-type calcium current was affected. For these studies potassium currents were blocked using intracellular caesium and tetraethylammonium, and extracellular caesium. Isoprenaline could still stimulate L-type calcium currents in cells dialysed with 50 μmβARK-CT (Fig. 4C). Although not statistically significant with a sample size of three in each group, there may be a modest decrease in peak L-type calcium current compared with time control cells without βARK-CT in the pipette solution (Fig. 4D). The important point, however, is that a concentration of βARK-CT that fully prevents isoprenaline-stimulated I_K,ACh does not completely block the effect of isoprenaline on the calcium current. We cannot rigorously rule out the possibility that G_sα-dependent effects on I_K,ACh are more sensitive to βARK-CT than G_sα-dependent stimulation of I_Ca,L. However, we believe that a simpler interpretation is that under our conditions dialysis with 50 μmβARK-CT results in selective inhibition of βγ function and that responses that are dependent on G_sα are either unaffected or only slightly inhibited.

Isoprenaline activates I_K,ACh in freshly dissociated cells

Although we used cultured myocytes for most of the experiments in the present report, it was not necessary to place cells in culture or expose cells to adenovirus in order to observe an effect of isoprenaline on I_K,ACh. An example is shown in Fig. 5. Elevated K⁺_o (25 mM) was used. In this cell an isoprenaline-induced I_K,ACh was readily observed. A larger I_K,ACh was activated by carbachol. This effect was not seen in all cells, however. In 25 mM K⁺_o, an isoprenaline-induced inward rectifier that subsided on washout of isoprenaline was detected in three out of eight cells examined.

Figure 5. Isoprenaline activates I_K,ACh in a freshly dissociated dog atrial cell.

Whole-cell currents are plotted. K⁺_o was 25 mM. Similar results were observed in 3/8 freshly dissociated dog atrial cells. •, control; ▪, isoprenaline; ▴, washout of isoprenaline; ♦, carbachol.

DISCUSSION

Isoprenaline activates I_K,ACh in native atrial cells

Prior exogenous expression studies had indicated that it was possible for βγ subunits derived from G_s to activate a current that was similar to I_K,ACh (Dascal et al. 1993; Lim et al. 1995). The present manuscript is the first report demonstrating that atrial myocytes have the capacity to activate native I_K,ACh by release of βγ subunits from endogenous G_s. This is an important distinction for three reasons. First, there is the possibility of promiscuous interactions in reconstitution and exogenous overexpression experiments. Second, the exogenously expressed potassium channels may not have identical subunit composition to native channels (Lim et al. 1995; Krapivinsky et al. 1995b). Finally, from a physiological perspective, the present data suggest that β-adrenoceptor agonists might be able to activate I_K,AChin situ and that altered expression of components of the β-adrenoceptor-G_s-I_K,ACh system can affect the amplitude of this response.

We have shown in this manuscript that isoprenaline can activate a whole-cell current with a current-voltage relationship and a reversal potential that are similar to carbachol-induced I_K,ACh. The reversal potentials for the isoprenaline- and carbachol-induced currents were close to the potassium equilibrium potential with either 4 or 25 mM K⁺_o. The absence of a region of negative slope conductance for the isoprenaline-induced current rules out an effect on the background inward rectifier potassium current, I_K1. Other potassium currents that could possibly be modulated by isoprenaline include I_K,ATP and the slow component of the delayed rectifier, I_KS. A contribution of these currents to our isoprenaline-induced steady-state currents can be excluded by either the recording conditions, the shape of the isoprenaline-induced current-voltage relationship or the voltage protocols used to record steady-state currents. Glibenclamide was present to inhibit I_K,ATP. The slow hyperpolarizing voltage clamps used in the present study would cause deactivation of I_KS. We can also rule out I_KS as a major contributor to the isoprenaline-induced currents because we could record an isoprenaline-induced inward rectifier when the holding potential was -50 mV and because I_KS does not exhibit inward rectification.

In 4 mM K⁺_o an isoprenaline-induced I_K,ACh was small and inconsistent in uninfected cells. This observation is in accordance with previous statements that isoprenaline does not stimulate I_K,ACh (Hartzell, 1988). However, in elevated K⁺_o we did find a clear isoprenaline-induced I_K,ACh in a small percentage of freshly dissociated dog atrial cells, indicating that neither viral infection nor time in culture are required. The effect of isoprenaline is enhanced by infection with adenovirus presumably by increasing membrane-associated G_s (Novotny et al. 1994). It should be noted, however, that we have only shown an enhanced effect of isoprenaline after infection in this report, we have not directly shown that G_sα levels are increased in our cells. The effect of isoprenaline was more readily seen with elevated extracellular potassium due to an effect of extracellular potassium on I_K,ACh amplitude. Most of our studies were conducted on cultured adenovirus-infected myocytes with 25 mM K⁺_o because an isoprenaline-induced I_K,ACh was larger and more consistently observed under these conditions.

Isoprenaline stimulates I_K,ACh via release of βγ from G_s

The isoprenaline-induced I_K,ACh was prevented by propranolol but not pertussis toxin pretreatment. The effect of isoprenaline subsided on washout, but was persistent if cells were pretreated with cholera toxin. These observations suggest that the effect is dependent on activation of β-adrenoceptors and G_s. A prior report indicated that isoprenaline could enhance muscarinic agonist-dependent I_K,ACh by PKA-dependent phosphorylation of the channel or a closely associated regulatory protein (Kim, 1990). However PKA-dependent phosphorylation cannot explain the present results. Kim (1990) reported that PKA-dependent phosphorylation alone could not activate I_K,ACh. In the present report, the lack of effect of forskolin and the presence of an isoprenaline-induced I_K,ACh when adenylyl cyclase was effectively inhibited by cytoplasmic 2′-dAMP indicate that cAMP accumulation and PKA stimulation are not involved in the stimulation of I_K,ACh by isoprenaline. Prior reconstitution studies have shown that GTPγS-liganded G_sα does not support the activation of I_K,ACh in inside-out atrial membrane patches (Yatani et al. 1987). Therefore the most likely mediator of the isoprenaline response is G_s-derived βγ. In support of an essential role for βγ, βARK-CT peptide fully prevented the effect of isoprenaline on I_K,ACh. Importantly, the selectivity of the βARK-CT for βγ subunits versus G_sα was demonstrated by showing that the stimulation of the L-type calcium current by isoprenaline was largely unaffected under our recording conditions.

βγ dimers are a physiological activator of I_K,ACh

Exogenous expression and reconstitution studies have documented that βγ dimers can activate I_K,ACh (Logothetis et al. 1987; Cerbai et al. 1988; Dascal et al. 1993; Wickman et al. 1994; Huang et al. 1995; Slesinger et al. 1995). The ability of βARK-CT peptides to inhibit ACh-induced I_K,ACh in inside-out rabbit atrial membrane patches supports a role for βγ subunits in the physiological activation of I_K,ACh in native myocytes (Nair et al. 1995). There is little specificity in the ability of different recombinant βγ combinations to activate I_K,ACh (Wickman et al. 1994), suggesting that any G protein-coupled receptor should activate I_K,ACh by releasing βγ. One problem with the proposal that βγ dimers are the unique activator of I_K,ACh was that G_s-derived βγ was not believed to be effective in activating I_K,ACh in native atrial myocytes (Hartzell, 1988). The data presented in this manuscript show that βγ released from G_s can activate I_K,ACh in native atrial myocytes. Our data add further support to the widely accepted notion that βγ subunits are a physiological activator of I_K,ACh. The data also suggest that the reason why isoprenaline-stimulated I_K,ACh is often small or undetectable in native atrial myocytes is that there is either a limited amount of G_s relative to G_o and G_i (Robishaw & Foster, 1989) or there is restricted access of βγ released from G_s to the I_K,ACh channel.

Are βγ dimers the unique activators of I_K,ACh?

Although there is substantial evidence in favour of a role for βγ subunits in the activation of I_K,ACh it is difficult to dismiss completely reconstitution studies demonstrating that picomolar concentrations of GTPγS-liganded G_iα could activate I_K,ACh in inside-out atrial membrane patches (Yatani et al. 1987; Cerbai et al. 1988). There may be free GTPγS contamination of GTPγS-G_iα preparations but the amount of free GTPγS cannot explain the activation of I_K,ACh by picomolar concentrations of GTPγS-G_iα (Wickman et al. 1994). Since G_iα subunits do bind to I_K,ACh channel subunits (Huang et al. 1995) it is possible that both α and βγ subunits are involved in the physiological activation of I_K,ACh. The main evidence against a role for G_iα comes from studies in which it was shown that GDP-G_iα could inhibit GTPγS-stimulated I_K,ACh channel activity in inside-out atrial membrane patches (Wickman et al. 1994). It is difficult to exclude the possibility that GDP-G_iα competed with GTPγS-G_iα for binding to the I_K,ACh channel.

In the present study the effect of isoprenaline was shown to be exclusively mediated by βγ subunits since βARK-CT peptide fully inhibited the current under conditions that had little effect on G_sα. The effect of isoprenaline was always smaller than the effect of carbachol (except in the pertussis toxin-pretreated cells). This observation would be consistent with either of two possibilities: (1) the amount of βγ released from G_s was smaller than the amount released from G proteins coupled to muscarinic receptors and was the limiting factor for the amount of current, or (2) a requirement for G_iα plus βγ in order to observe full activation of I_K,ACh in native cells.

The inhibitory effect of βARK-CT peptides on native muscarinic agonist-induced I_K,ACh in the study of Nair et al. (1995) was only partial. The reason for the incomplete inhibition was not clear but could have been due either to insufficient concentration of peptide or to a significant role of α subunits in the activation of I_K,ACh by muscarinic agonist. Nair et al. (1995) did not demonstrate the specificity of βARK-CT peptides under their experimental conditions. Since the effects of α subunits are critically dependent on interactions of α subunits with βγ dimers (Florio & Sternweis, 1989; Sternweis, 1994) it is even possible that some of the inhibition of I_K,ACh in the study of Nair et al. was due to non-specific effects of βARK-CT peptides on the function of α subunits.

The interpretation of the effect of βARK-CT on carbachol-induced I_K,ACh in the present study is also problematic. Like Nair et al. (1995), we saw a partial inhibition of muscarinic receptor-dependent I_K,ACh. While we believe that the inhibitory effect of βARK-CT on carbachol-induced I_K,ACh in the present study is likely to reflect a significant role for βγ subunits derived from pertussis toxin-sensitive G proteins, we cannot exclude the possibility that βARK-CT inhibited the function of G_iα under our experimental conditions. Although we have shown that 50 μmβARK-CT had little effect on a G_sα-dependent function we cannot be certain that G_iα is unaffected. It is difficult to devise a functional test to exclude an effect of βARK-CT on G_iα because there are no known actions of atrial muscarinic receptors which can unequivocally be attributed solely to G_iα with no role for βγ subunits.

Physiological relevance of isoprenaline-stimulated I_K,ACh

The results presented in this manuscript are consistent with the observations of a propranolol-sensitive noradrenaline-induced hyperpolarization of dog coronary sinus (Boyden et al. 1983). The hyperpolarization was not blocked by acetylstrophanthidin or by potassium-free extracellular solution, indicating that sodium-potassium pump activity was not required (Boyden et al. 1983). Boyden et al. (1983) found that carbachol and isoprenaline increased potassium conductance. Although in the present report the effect of isoprenaline on freshly dissociated cells was small and variable, dog atrial cells have a high input resistance. The high input resistance will enhance the effect of small changes in resting current on the resting potential, and cell-to-cell variability will be damped in the electrically coupled cells of intact atrial tissue. It is possible that isoprenaline activates sufficient I_K,ACh to hyperpolarize atrial trabeculae. Besides moving resting potential towards the potassium equilibrium potential, the activation of I_K,ACh by β-adrenoceptor agonist would be expected to shorten action potential duration (APD). Thus in the atrium APD shortening by catecholamines may be due to stimulation of I_K,ACh in addition to the well-known enhancement of I_KS.

Our results show that it is possible for atrial myocytes to undergo changes in the responsiveness of I_K,ACh to β-adrenoceptor stimulation, presumably due to altered expression of endogenous genes. We used infection with adenovirus to cause a non-specific increase in the level of G_sα (Novotny et al. 1994) and unmask an isoprenaline-stimulated I_K,ACh. It is possible that increases in β-adrenoceptor responsiveness can also occur in situ during certain clinical conditions. For example, increased responsiveness to β-adrenoceptor agonists can occur with elevated thyroid hormone levels. Increases in β-adrenoceptors (Bilezikian & Loeb, 1983) as well as membrane-associated G_s (Bahouth, 1995) can be produced by elevated thyroid hormone. We would predict that these changes would enhance the ability of isoprenaline to activate I_K,ACh. Elevated thyroid hormone levels are often associated with a shortened APD (Freedberg et al. 1970) and atrial fibrillation (Woeber, 1992). It remains to be determined whether catecholamine-dependent activation of I_K,ACh is one of the factors that contributes to APD shortening and atrial fibrillation in patients with elevated thyroid hormone levels.