Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of K(v)7 channels via M-1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M-1 receptor agonist oxotremorine M did not correlate with the extent of K(v)7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M-1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the K(v)7 channel inhibition by the muscarinic agonist unchanged. M-1 receptor-induced depolarisations were also altered when extra- or intracellular Cl- concentrations were changed, as were depolarising responses to gamma-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl- channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca2+-activated Cl- and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca2+-activated Cl- channels in addition to the well-known inhibition of K(v)7 channels.