Muscarinic receptor modulation of protein kinase A, protein kinase C and exocytotic proteins at the neuromuscular junction

Identifier:  TDX:4002

Handle: https://hdl.handle.net/20.500.11797/TDX4002

Authors:  Cilleros Mañé, Víctor

Abstract:
The neuromuscular junction (NMJ) uses plastic mechanisms to adjust the release of acetylcholine (ACh) to an incredibly dynamic environment. Muscarinic acetylcholine receptors (mAChRs) participate as autoreceptors, tuning neurotransmission. The M1 subtype activates protein kinase C (PKC) to enhance the release, whereas M2 inhibits protein kinase A (PKA) to decrease it. The captivating research in the past decade has provided extensive electrophysiological knowledge about muscarinic signaling. However, the molecular data accompanying this knowledge was limited; and the role of some second messengers remained elusive. Therefore, the present thesis aimed to characterize how M1 and M2 mAChRs regulate the multiple PKA and PKC subunits, their scaffolds and exocytotic targets. We analyzed the muscarinic cascade at the rat diaphragm muscle by testing selective and general inhibitors of M1 and M2 mAChR, nPKCε, cPKCβI, PKA and PDK1 and analyzed each alteration mainly by Western blotting as well as subcellular fractionation and co-immunoprecipitation. We also made use of immunohistochemical and confocal techniques to corroborate the presynaptic location of our molecules of interest. Our results show that M1 receptors are downregulated by the M2 pathway. Regarding PKA signaling, M2 inhibits PKA activity by downregulating Cβ subunit, upregulating RIIα/β and liberating RIβ and RIIα to the cytosol, which reduces the phosphorylation of SNAP-25 on Thr138 and CREB. M1 signaling crosstalks with M2/PKA by recruiting R subunits to the membrane. Regarding PKC signaling, both M1 and M2 mAChR activate the master kinase PDK1, which promotes the priming of the presynaptic PKCβI and PKCε isoforms. M1 recruits both primed PKCs to the membrane and promotes Munc18-1, SNAP-25 and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and its PKC-phosphorylation. The results demonstrate that M1 and M2 mAChRs perform a coordinated and interdependent signaling to modulate neurotransmission at the NMJ.