M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gβγ-mediated inhibition of large conductance Ca2+-activated K+ channel activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M-2 and M-3 subtype. Stimulation of these receptors inhibits large conductance calcium-activated K+ (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M-2 or M-3 receptor (M2R or M3R). In M2R- but not (MR)-R-3-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M2R- induced inhibition was abolished by pertussis toxin treatment or overexpression of the G beta gamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nM purified G beta gamma decreased channel open probability by 55%. The physical interaction of G beta gamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (similar to 20%) extent. Mouse tracheal cells responded similarly to CCh, purified G beta gamma and phospholipase C/protein kinase C inhibition as M2R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, G beta gamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.