ACTIVITY-DEPENDENT MUSCARINIC SIGNALLING CONTROLS PKC PHOSPHORYLATION OF MUNC18-1 AND SNAP-25 TO REGULATE NEUROTRANSMISSION AT THE NEUROMUSCULAR JUNCTION

Muscarinic acetylcholine receptors (mAChRs) regulate neurotransmission at the neuromuscular junction (NMJ). M₁/M₂ mAChRs modulate protein kinase C (PKC) signalling, essential for synaptic function. PKC phosphorylates vesicle-release proteins including Mammalian uncoordinated-18 (Munc18-1) and Synaptosome-associated protein of 25 kDa (SNAP-25), promoting vesicle fusion and acetylcholine release. Neurotransmitter release is coordinated by both nerve activity and muscle-induced signals, but it remains unclear whether these activities differentially regulate M₁/M₂-PKC-dependent pathways. To separate the effect of presynaptic activity from that of the resulting muscle contraction, rat diaphragm with phrenic nerve was stimulated (1Hz,30min) with or without muscle contraction (abolished by µ-conotoxin GIIIB). Pirenzepine (M₁-antagonist) and methoctramine (M₂-antagonist) were used to differentiate receptor-specific PKC signalling during each condition. Protein abundance and phosphorylation levels were measured by Western blotting, and NMJ localization was assessed by immunohistochemistry. We demonstrate that presynaptic activity downregulated M₂, whereas nerve-induced muscle contraction increased both M₁ and M₂ mAChRs levels. Across conditions, M₂ signalling consistently inhibited M₁ activity. At the PKC level, M₁ activity selectively downregulated PKCβ1, while M₂ activity reduced PKCε under both stimulation conditions. The M₁-PKCβ1 axis promoted Munc18-1 phosphorylation during presynaptic activity, supporting regulation of synaptic vesicle fusion. In parallel, the M₂-PKCε pathway, modulated by M₁ activity, controlled SNAP-25 phosphorylation under both stimulation conditions, implicating this axis in the regulation of neurotransmitter release. Activity-dependent coupling between M₁/M₂ mAChRs and distinct presynaptic PKC isoforms coordinates phosphorylation of Munc18-1 and SNAP-25, highlighting their role in balancing the optimal process of ACh release, with M₁ enhancing ACh release and M₂ decreasing it.
Funding:2021SGR01214,PID2019-106332GB-I00,PID2022-141252NB-I00,PRE2020-092084,2023PMF-PIPF-15,2021-FI-B00755.