Studies on the Molecular Mechanism of Neuromuscular Junction Formation

我们之前已经证明，在肌肉发育过程中，γ亚基被epsilon亚基取代是导致乙酰胆碱受体功能改变的原因。为了建立乙酰胆碱受体亚单位发生转换的体外系统，对成肌细胞系(L6和C2C12)和原代培养成肌细胞在分化过程中乙酰胆碱受体亚单位的表达进行了检测。在从成肌细胞到肌管的发育过程中，α、β、γ和增量亚单位的mRNAs显著增加，而epsilon亚基只有在原代培养细胞发育的后期才能检测到，但在细胞系中没有检测到。注射牛α、β和增量亚单位特异性mRNAs的非洲爪蛙卵母细胞中表达的乙酰胆碱受体(简称α-β-AChR)是不寻常的，因为它的通道在没有激动剂的情况下会以很高的频率自发开放。因此，γ和epsilon亚基对于稳定未连接受体通道的关闭状态是重要的。此外，乙酰胆碱激活的α-AChR的平均通道开放时间明显长于含有γ-或epsilon亚基的完整受体。它的容易组装增加了这样一种可能性，即α-AChR与发育过程中发生的受体特性的一些变化有关。

We have previously shown that the replacement of the gamma-subunit by the epsilon-subunit is responsible for the functional alteration of the acetylcholine receptor during muscle development. In an attempt to establish an in vitro system in which the switch of the acetylcholine receptor subunits takes place, myogenic cells lines (L6 and C2C12) and primary culture of myoblasts were tested for expression of the acetylcholine receptor subunits during differentiation. The alpha-, beta-, gamma-and delta-subunit mRNAs increased prominently during development from myoblasts to myotubes, whereas the epsilon-subunit was detected only in late stage of the development of the primary cultured cells, but not in the cell lines.The acetylcholine receptor expressed in Xenopus oocytes injected with the bovine alpha-, beta- and delta-subunit-specific mRNAs (referred to as alphabetadelta-AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. Thus, the gamma-and epsilon-subunits are important for stabilizing the closed state of the unliganded receptor channel. Furthermore, the mean channel open time of acetylcholine-activated alphabetadelta-AChR is considerably longer than that of the complete receptors containing either the gamma- or epsilon-subunit. The ease with which it is assembled raises the possibility that the alphabetadelta-AChR contributes to some of the variation in receptor properties that occur during development.