The M₂ muscarinic receptor, in association to M₁ , regulates the neuromuscular PKA molecular dynamics

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.