The M ₂ muscarinic receptor, in association to M ₁ , regulates the neuromuscular PKA molecular dynamics

Abstract

Muscarinic acetylcholine receptor 1 subtype (M1) and muscarinic acetylcholine receptor 2 subtype (M2) presynaptic muscarinic receptor subtypes increase and decrease, respectively, neurotransmitter release at neuromuscular junctions. M2 involves protein kinase A (PKA), although the muscarinic regulation to form and inactivate the PKA holoenzyme is unknown. Here, we show that M2 signaling inhibits PKA by downregulating Cβ subunit, upregulating RIIα/β and liberating RIβ and RIIα to the cytosol. This promotes PKA holoenzyme formation and reduces the phosphorylation of the transmitter release target synaptosome‐associated protein 25 and the gene regulator cAMP response element binding. Instead, M1 signaling, which is downregulated by M2, opposes to M2 by recruiting R subunits to the membrane. The M1 and M2 reciprocal actions are performed through the anchoring protein A kinase anchor protein 150 as a common node. Interestingly, M2 modulation on protein expression needs M1 signaling. Altogether, these results describe the dynamics of PKA subunits upon M2 muscarinic signaling in basal and under presynaptic nerve activity, uncover a specific involvement of the M1 receptor and reveal the M1/M2 balance to activate PKA to regulate neurotransmission. This provides a molecular mechanism to the PKA holoenzyme formation and inactivation which could be general to other synapses and cellular models.

M 1 and M 2 mAChRs activate PDK1 and regulate PKC βI and ε and the exocytotic apparatus at the NMJ

“…M 2 receptors are generally linked to Gi proteins, which inhibit adenylate cyclase and block PKA activity by downregulating cAMP production 58 . One of the consequences we recently demonstrated is that M 2 signaling decreases the PKA phosphorylation of SNAP‐25 Thr 138 at the NMJ 2 . Besides the PKA pathway, further studies revealed that PKC activity is also necessary for the M 2 muscarinic signaling in various neuromuscular models 1,74,75 …”

“…However, this essential signaling is highly complex because it involves multiple downstream transduction pathways and the crosstalk between receptor subtypes. An example of this complexity at the NMJ is that the M 2 muscarinic receptor needs the association to M 1 to regulate the neuromuscular PKA molecular dynamics 2 . Also, the selective inhibition of both mAChR subtypes induce PKC action on NMJ neurotransmission 1 in which PKC plays an essential role 3,4 .…”

“…An example of this complexity at the NMJ is that the M 2 muscarinic receptor needs the association to M 1 to regulate the neuromuscular PKA molecular dynamics. 2 Also, the selective inhibition of both mAChR subtypes induce PKC action on NMJ neurotransmission 1 in which PKC plays an essential role. 3,4 At the adult NMJ, PKC coupling to ACh release requires a stimulus like calcium, presynaptic stimulation or the modulation of mAChR.…”

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ

“…Primary antibodies were omitted from some samples during the procedure as controls, and they never revealed bands of the appropriate molecular weight. Furthermore, their specificity has been proved in previous publications [3,47,[50][51][52]. Membranes were revealed with Bio-Rad ECL kid on the ChemiDoc XRS+ machine (Bio-Rad, Hercules, CA, USA).…”

“…TrkB.FL, when it is not inhibited by TrkB.T1 heterodimerization [ 44 , 45 , 46 ], activates presynaptic protein kinases C (PKC) once they are phosphorylated by phosphoinositide-dependent kinase 1 (PDK1) [ 47 ], that modulate ACh release at the NMJ [ 3 , 48 , 49 ] by phosphorylating proteins of the exocytotic machinery such as Munc18-1 and synaptosomal nerve-associated protein 25 (SNAP-25) [ 50 , 51 ]. Furthermore, SNAP-25 is also phosphorylated by the cAMP-dependent protein kinase A (PKA) whose activity depends on muscarinic ACh receptors [ 52 ], which regulate the activation of TrkB [ 37 , 53 ]. Therefore, the optimization of this signaling pathway with exercise could strongly improve the functionality of NMJ.…”