M2 muscarinic receptor-dependent contractions of airway smooth muscle are mediated by inhibition of Kv7 channels

Cholinergic tone is elevated in the airways of patients with COPD and asthma. Acetylcholine contracts the airways by binding to M2 and M3 muscarinic receptors on airway smooth muscle cells. The purpose of the present study was to investigate if M2R-dependent contractions of ASM involved inhibition of Kv7 channels. Real-time qPCR and immunocytochemistry revealed that Kv7.5 was the highest expressed Kv7 subtype in mouse ASM, followed by Kv7.4. Cholinergic nerve-evoked contractions of mouse bronchial rings were induced by electrical field stimulation (EFS). Reducing the stimulus interval from 100 to 10s augments EFS-evoked contractions via an M2R-dependent pathway. Inhibition of Kv7 channels with XE991 increased the amplitude of EFS responses at 100 s intervals, but not at 10 s. The Kv7 channel openers ICA069673 and ML213 inhibited M2R-dependent responses and similar effects were obtained using the adenylate cyclase activator forskolin and the PKA activator 6MB-cAMP. The effects of M2R activation on Kv7 currents was determined by examining the effect of carbachol on HEK293 cells co-expressing M2Rs with Kv7.4 or Kv7.5. Activation of M2Rs reduced the amplitude of both Kv7.4 and Kv7.5 currents. ICA069673 reversed the inhibitory effects of M2R activation on Kv7.4, while ML213 reversed the effects on both Kv7.4 and Kv7.5. Inclusion of PIP2diC8 in the pipette prevented the inhibitory effects of carbachol on Kv7.4, but not Kv7.5, whereas 6MB-cAMP potentiated Kv7.5 currents but not Kv7.4. These data suggest that M2Rs exert their contractile effects on ASM by inhibiting Kv7 channels via pathways that deplete PIP2 and reduce PKA activity.