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

Abstract

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 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 Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl− concentrations were changed, as were depolarising responses to γ-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 Kv7 channels.

Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate

“…Primary cultures of dissociated superior cervical ganglion (SCG) neurons from newborn rats were prepared as described previously (Salzer et al 2014). Newborn Sprague-Dawley rats were kept and killed 5-8 days after birth by decapitation.…”

“…Recordings of currents through Kv7 channels were performed at room temperature (20-24°C) using the perforated patch-clamp technique, which prevents their rundown (Salzer et al 2014). Patch pipettes were fabricated from borosilicate glass capillaries (GB150-8P, Science Products, Hofheim, Germany) using a Sutter P97 puller (Sutter Instruments, Novato, CA, USA) and front filled with a solution containing (in mM): potassium gluconate 133; NaCl 5.9; CaCl 2 1.0; MgCl 2 0.7; Hepes 10; EGTA 10; KOH 29.4 (pH 7.4).…”

Electrophysiological Investigation of the Subcellular Fine Tuning of Sympathetic Neurons by Hydrogen Sulfide

“…In particular, K υ 7 and K Ca 1 channels have been documented to represent major determinants of membrane excitability in SCG neurons (Jones et al, 1995 ; Vivas et al, 2014 ). Recently, Ca 2+ activated Cl − channels have been evidenced to contribute to the regulation of excitability in SCG neurons as well (Salzer et al, 2014 ). At membrane voltages corresponding to the resting membrane potential of SCG neurons, NaHS triggered outward currents that were (i) similar to currents elicited by diazoxide and (ii) blocked by tolbutamide, activators and inhibitors of K ATP channels, respectively (Foster and Coetzee, 2015 ).…”

Regulation of neural ion channels by muscarinic receptors

“…The effect of the latter was replicated by PKC activation and prevented by PKC inhibitors. In the experiments by Salzer et al (2014), the current appeared as a more sustained inward current on application of a muscarinic agonist, which did not require a priming Ca 2+ charge, but responded directly to PKC activation (so might have been sensitized to resting Ca 2+ levels). It was identified as a Cl(Ca) current, probably carried by anoctamine channels, because it was inhibited by TMEM15A blockers.…”

“…M1R stimulation can also enhance or activate a Ca 2+ -dependent Clcurrent in sympathetic neurons (Marsh et al, 1995;Salzer et al, 2014). In the former case, the current appeared as a delayed but transient inward (depolarizing) current after a priming Ca 2+ charge delivered through voltage-gated Ca 2+ channels or nicotinic acetylcholine receptors, which was then strongly enhanced by M1R stimulation.…”