Activity-dependent muscarinic signalling regulates presynaptic PKC pathway and neurotransmission machinery at the neuromuscular junction.

BACKGROUND: Acetylcholine (ACh) signalling mediated by muscarinic acetylcholine receptors (mAChRs) significantly influences various physiological functions, including muscle contraction. Among these receptors, the M(1) and M(2) subtypes are key modulators of neurotransmission at the neuromuscular junction (NMJ), acting through the protein kinase C (PKC) signalling pathway, critical for synaptic function. PKC phosphorylates essential synaptic vesicle-associated proteins such as Mammalian uncoordinated-18 (Munc18-1) and Synaptosome-associated protein of 25 kDa (SNAP-25), facilitating synaptic vesicle fusion and neurotransmitter release. Although neurotransmitter release at the NMJ is known to be coordinately regulated by both presynaptic nerve stimulation and retrograde signalling from muscle contraction, the role of these activities in controlling M(1) and M(2) receptor-mediated PKC pathways has not been previously explored. METHODS: To differentiate the effects of presynaptic nerve stimulation from muscle contraction on M(1) and M(2) receptor pathways, rat phrenic nerves were electrically stimulated at 1 Hz for 30 min, both with and without muscle contraction, which was abolished with μ-conotoxin GIIIB. Selective inhibitors Pirenzepine (M(1) receptor antagonist) and Methoctramine (M(2) receptor antagonist) were used to differentiate the muscarinic receptor-specific PKC pathways. Protein levels and phosphorylation states were analysed through Western blotting. Immunohistochemical analysis was used to locate specific molecules at the NMJ. RESULTS: We observed that presynaptic nerve stimulation resulted in a downregulation of M(2) receptor expression, while nerve-induced muscle contraction upregulated both M(1) and M(2) receptors. Interestingly, M(1) receptor activity was consistently inhibited by the M(2) receptor signalling. Regarding PKC signalling, our data revealed that M(1) receptor activity selectively downregulated PKCβ1, whereas M(2) receptor activity reduced PKCε expression under both presynaptic stimulation and nerve-induced muscle contraction. These changes influenced the phosphorylation of key PKC substrates, Munc18-1 and SNAP-25, essential for synaptic vesicle dynamics. Specifically, the M(1)-PKCβ1 axis was critical for Munc18-1 phosphorylation during presynaptic activity, thus regulating synaptic vesicle fusion. Meanwhile, the M(2)-PKCε pathway, modulated by M(1) receptor activity, controlled SNAP-25 phosphorylation during both presynaptic activity and nerve-induced muscle contraction, ultimately impacting neurotransmitter release. CONCLUSION: Our findings reveal a detailed molecular mechanism underlying the activity-dependent interplay between M(1) and M(2) mAChRs and their respective PKC isoforms, highlighting their combined roles in optimizing acetylcholine release at the NMJ.