Effect of Calmodulin Kinase II-Related Anchoring Protein on the Stability of AChR

描述（由申请人提供）：突触后乙酰胆碱受体维持在高密度簇对于神经肌肉接头处突触传递的有效性至关重要。我们实验室最近的工作意外地发现了akap的一个新的重要作用，akap是一种非激酶锚定蛋白，它在钙/钙调蛋白激酶II基因中编码。我们发现，kap保护乙酰胆碱受体在分泌途径中免于降解。在培养的肌肉和异源细胞中，kap的保护作用是由泛素依赖性机制介导的。鉴于这些结果，我们建议研究<$kap是否可以保护活体小鼠的AChR，并确定<$kap和受体稳定性之间的机制联系。使用体内时间推移成像，定量荧光成像和电穿孔方法，第一个具体目标将测试 在活的野生型小鼠肌肉中敲低kap是否可以改变AChR的密度、数量或分布。相反，我们将测试<$kap的过表达是否可以挽救<$-syntrophin和<$-dystrobrevin敲除小鼠和手术失神经肌肉的NMJ处AChR水平的急剧下降。这些研究的结果将提供新的见解机制，可以增加突触AChR的数量，并将与许多神经肌肉突触的数量和密度受到疾病的影响。最后，这个R21项目将为未来研究kap如何在NMJ调节AChR或其他相关蛋白质的运输和稳定性奠定基础。

DESCRIPTION (provided by applicant): 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.