Heterologous facilitation of G protein-activated K+ channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-acti vated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K current, I-KaCh, was recorded from adult rat atrial cardiomyocytes using t he 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 simultan eously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, an d 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 we re abolished by preincubation in 50 mu M H89 {N-[2-((p-bromocinnamyl) amino ) ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This hetero logous facilitation of GIRK current by beta-adrenergic stimulation was furt her studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic recep tors, m(2)-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited G IRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, bu t not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK cu rrents was a result of the phosphorylation of the P-adrenergic receptor (be ta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the resid ues for PKA-mediated modulation were mutated. Overexpression of the ct subu nit of G proteins (Ga,) led to an increase in basal as well as agonist-indu ced GIRK1/GIRK4 currents (inhibited by H89), At higher levels of expressed G alpha(s), GIRK currents were inhibited, presumably due to sequestration o f the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIR K1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulat ed 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, P-adrenergic stimulation potentiates the ACh-evoked GIR K channels Via a pathway that involves PKA-catalyzed phosphorylation downst ream from beta(2)AR.