Abstract
Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se, and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.
Highlights
Synapse functionality is the result of several signaling pathways converging on intracellular kinases, which phosphorylate protein targets to perform adequate adaptive changes (Tomàs et al, 2014)
These results indicate that conventional protein kinase C beta I (cPKCβI) is necessary for the increase of mammalian uncoordinated18 (Munc18)-1 phosphorylation caused by nerve stimulation but not for the increase of Munc18-1 levels (Figure 4C, last column)
The results demonstrate that phosphorylation of Munc18-1 at the neuromuscular junction (NMJ) is increased in response to a signaling mechanism initiated with synaptic activity and directly mediated by novel protein kinase C epsilon (nPKCε) while cPKCβI and tropomyosin receptor kinase B (TrkB) activities work to prevent this synaptic activity–induced Munc18-1 phosphorylation
Summary
Synapse functionality is the result of several signaling pathways converging on intracellular kinases, which phosphorylate protein targets to perform adequate adaptive changes (Tomàs et al, 2014). Munc tightly binds to syntaxin and holds it in a closed conformation to prevent SNARE assembly together with synaptobrevin and SNAP-25 (Hata et al, 1993; Dulubova et al, 1999; Misura et al, 2000; Yang et al, 2000; Liu et al, 2004; Khvotchev et al, 2007; Medine et al, 2007; Shen et al, 2007; Südhof and Rothman, 2009; Smyth et al, 2010a,b). It works as a chaperone by delivering syntaxin to the plasma membrane (Arunachalam et al, 2007; Han et al, 2009) and, when associated with the SNARE complex, favors lipid mixing between membranes (Huang et al, 2011; Südhof, 2013)
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