Mouslim_2012_J.Neurosci_32_5177

alphakap, a muscle specific anchoring protein encoded within the Camk2a gene, is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of alphakap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of alphakap expression with shRNA significantly enhanced the degradation of AChR alpha-subunits (AChRalpha), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that alphakap promoted AChRalpha stability by a ubiquitin-dependent mechanism. In the absence of alphakap, AChRalpha was heavily ubiquitinated, and the number of AChRalpha was increased by proteasome inhibitors. However, in the presence of alphakap, AChRalpha was less ubiquitinated and proteasome inhibitors had almost no effect on AChRalpha accumulation. The major sites of AChRalpha ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRalpha stability in the absence of alphakap. These results provide an unexpected mechanism by which alphakap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function.