AKT Signaling Regulates Agrin-Mediated Acetylcholine Receptor Surface Density

Background and Objectives: Acetylcholine receptors (AChRs) are ligand-gated ion channels concentrated at the postsynaptic membrane of skeletal muscle fibers, where their abundance is essential for efficient neuromuscular transmission. The serine/threonine kinase AKT is a central signaling node in muscle homeostasis, regulating metabolism, growth, and survival. However, its role in the Agrin-mediated regulation of postsynaptic AChRs remains incompletely defined. Here, we demonstrate a novel role of AKT in regulating Agrin-induced AChR accumulation in differentiated C2C12 myotubes. Materials and Methods: Differentiated C2C12 myotubes were stimulated with Agrin in the presence or absence of the AKT inhibitor MK2206 during either the formation or maintenance phase. AChR clustering was quantified using α-bungarotoxin labeling. Expression of AChR subunits and neuromuscular junction-associated genes was assessed. Proteasome involvement was examined using the inhibitor MG132. Results: Pharmacological inhibition of AKT using MK2206 during either the formation or maintenance phase of Agrin stimulation significantly reduced α-bungarotoxin-labeled AChR intensity. AKT inhibition also attenuated Agrin-induced expression of multiple AChR subunits and neuromuscular junction-associated genes. Importantly, inhibition of proteasome activity with MG132 restored AChR intensity in the presence of AKT inhibition, suggesting that AKT signaling limits proteasome-dependent AChR loss. Conclusions: these findings identify AKT as a regulator of Agrin-mediated AChR accumulation and maintenance in vitro. These findings identify AKT as a critical integrator of metabolic and synaptic signaling required for postsynaptic receptor stability, with implications for neuromuscular disorders and muscle atrophy.