The maintenance of postsynaptic acetylcholine receptors at high density clusters is critical for the effectiveness of synaptic transmission at th neuromuscular junction. Recent work from our lab has unexpectedly identified a new and important role for akap, a non-kinase anchoring protein, which is encoded within the calcium/calmodulin kinase II ? gene. We showed that ?kap protects acetylcholine receptors from degradation while in the secretory pathway. In both cultured muscle and heterologous cells the protective effect of ?kap is mediated by an ubiquitin dependent mechanism. In view of these results, we propose to study whether ?kap can protect AChR in living mice and determine the mechanistic link between ?kap and receptor stability. Using in vivo time-lapse imaging, quantitative fluorescence imaging, and electroporation approaches, the first specific aim will test whether the knockdown of ?kap in muscles of living wild type mice can alter the density, number or distribution of AChR. Conversely, we will test whether the overexpression of ?kap can rescue the drastically reduced levels of AChRs at the NMJs of ?-syntrophin and ?-dystrobrevin knockout mice and in surgically denervated muscles. The outcomes of these studies will provide new insight into mechanisms that can enhance the number of AChRs at synapses and will be relevant for many neuromuscular synapses where the number and density are compromised by diseases. Finally, this R21 project will establish the groundwork for future studies of how ?kap regulates the trafficking and stability of AChR or other associated proteins at the NMJ.
The efficiency of synaptic transmission relies on the maintenance of a high number of receptors at the postsynaptic membrane. Our preliminary results show that calmodulin kinase II-related anchoring protein ?kap) has a profound effect on the stability of acetylcholine receptors in cultured muscle cells and in heterologous cells in culture. This proposal will explore the role of ?kap in regulating receptors in muscles of living mice, and significantly extend our understanding of the underlying molecular mechanisms. These studies will add to our understanding of the basic mechanisms that underlie the stability of receptors and will be relevant to developing new approaches for treating neurological diseases where the number of receptors is compromised.