Heterologous Facilitation of G Protein-Activated K⁺ Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase

To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K⁺ channels (GIRK), acetylcholine (ACh)-evoked K⁺ current, I_KACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I_KACh was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited I_KACh was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β₂-adrenergic receptors, m₂ -receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β₂-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β₂AR) by PKA was excluded by using a mutant β₂AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gα_s) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gα_s, GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β₂AR.