Sharp A A, Caldwell J H
Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Neurosci. 1996 Nov 1;16(21):6775-83. doi: 10.1523/JNEUROSCI.16-21-06775.1996.
Agrin is involved in signaling the formation of high concentrations of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). There are multiple isoforms of agrin attributable to alternative splicing, and these isoforms are differentially expressed during development and between tissues. The ability to cluster AChRs varies among the agrin isoforms. Sodium channels (NaChs) are also concentrated at the NMJ. We have tested various agrin isoforms for their ability to induce formation of clusters of NaChs. We grew cocultures of dissociated adult rat muscle fibers with chinese hamster ovary (CHO) cells that had been transfected with different isoforms of agrin. Using immunocytochemical techniques, we determined that after 1 d in culture, CHO cells synthesizing the neuronally expressed isoform with an eight amino acid insert (Agrin8) were able to form NaCh clusters at sites of contact between the CHO cell and muscle cell. Clusters of NaChs could be formed anywhere along a muscle fiber, but more clusters were detected close to the endplate where the endogenous level of NaChs was higher. None of the other neuronal-specific agrin isoforms was able to cluster NaChs. Because Agrin8 is the only agrin isoform that is upregulated at birth when NaChs begin to cluster at the NMJ, we conclude that Agrin8 expression by motor neurons is a signal for NaCh clustering at the NMJ during normal development.
集聚蛋白参与在神经肌肉接头（NMJ）处信号传导高浓度乙酰胆碱受体（AChR）的形成。由于选择性剪接，集聚蛋白存在多种同工型，并且这些同工型在发育过程中和不同组织之间差异表达。集聚蛋白同工型在聚集AChR的能力上有所不同。钠通道（NaCh）也集中在神经肌肉接头处。我们测试了各种集聚蛋白同工型诱导NaCh簇形成的能力。我们将成年大鼠解离的肌肉纤维与转染了不同集聚蛋白同工型的中国仓鼠卵巢（CHO）细胞进行共培养。使用免疫细胞化学技术，我们确定在培养1天后，合成具有八个氨基酸插入的神经元表达同工型（集聚蛋白8）的CHO细胞能够在CHO细胞与肌肉细胞的接触部位形成NaCh簇。NaCh簇可以在肌肉纤维的任何位置形成，但在终板附近检测到更多的簇，终板处NaCh的内源性水平较高。其他神经元特异性集聚蛋白同工型均不能使NaCh聚集。由于集聚蛋白8是出生时唯一上调的集聚蛋白同工型，此时NaCh开始在神经肌肉接头处聚集，我们得出结论，运动神经元表达的集聚蛋白8是正常发育过程中神经肌肉接头处NaCh聚集的信号。
