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

The atrial acetylcholine-induced potassium current (IK,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 IK,ACh in native atrial myocytes. Adenosine is also effective (Kurachi et al. 1986). Both adenosine and acetylcholine are coupled to IK,ACh by pertussis toxin-sensitive G proteins. There is some degree of receptor specificity for activation of IK,ACh. It is often stated that β-adrenoceptor activation does not stimulate IK,ACh (e.g. Hartzell, 1988, p. 215). The channel for IK,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 IK,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 IK,ACh can be activated by GTPγS-liganded α subunits of the Gi family (but not Gs) (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 IK,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 IK,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 IK,ACh, a βγ binding peptide derived from the C terminus of β-adrenergic receptor kinase 1 (βARK-CT) (Koch et al. 1993) inhibited IK,ACh channel activity in native atrial membranes (Nair et al. 1995). Injection of 5-hydroxytryptamine receptor (5HT1A) 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 Giα2 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, Gsα and Kir 3.1 can result in an isoprenaline-induced potassium current (Lim et al. 1995). It should be emphasized that stimulation of IK,ACh via Gs-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 IK,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 IK,ACh arises at the level of the βγ subunits. If βγ dimers are the physiological activators of IK,ACh then any G protein-coupled receptor that is present in the atrium should be able to release βγ subunits and activate IK,ACh. This prediction conflicts with the observation that β-adrenoceptor agonists are ineffective in activating native atrial IK,ACh. The activation of IK,ACh by β-adrenoceptors could be limited by several factors including: (1) spatial distribution of β-adrenoceptors and Gs relative to the channels, (2) an inadequate amount of βγ release from Gs 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 IK,ACh. If factors (1) or (2) are involved, then increasing the amount of Gs present in the cell may permit activation of IK,ACh by β-adrenoceptor agonist. Infection of cardiac myocytes with replication-deficient adenovirus can non-specifically increase the expression of Gs (Novotny et al. 1994). The increased Gs 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 Gsα gene resulted in the additional expression of Gsα 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 IK,ACh by isoprenaline.