Zusammenfassung
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 protein-activated inwardly rectifying K(+) channels (GIRKs) via
pertussis toxin-sensitive G 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
Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition
was significantly reduced, suggesting competition between the receptor-mediated
inhibition and the large pool of available Gbetagamma 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) mAChR
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) mAChR and alpha(1A) adrenergic receptors
also caused the inhibition, whereas other G protein-coupled receptors
including A(1) and A(3) 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 Gbetagamma-activated GIRK currents in
a membrane-delimited fashion.
Nutzer