Novel inhibition of Gβγ-activated potassium currents induced by M2 muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M-2 muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate (G) under bar protein-activated inwardly rectifying (K) under bar(+) channels (GIRKs) via pertussis toxin-sensitive Cf proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M-2 receptors not only activate but also reversibly inhibit these K+ currents when stimulated with agonist for up to 2 min. The M-2 mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M-2 mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous G beta gamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available G beta gamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M-2 mAChn was not required because a phosphorylation- and internalization-deficient mutant of the M-2 mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M-4 mAChn and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A3 adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M-2 and M-4 mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of G beta gamma-activated GIRK currents in a membrane-delimited fashion.