Forsayeth J R, Franco A, Rossi A B, Lansman J B, Hall Z W
Department of Physiology, School of Medicine, University of California, San Francisco 94143-0444.
J Neurosci. 1990 Aug;10(8):2771-9. doi: 10.1523/JNEUROSCI.10-08-02771.1990.
CHO cells were transfected with cDNAs for all 4 subunits of the mouse muscle acetylcholine receptor (AChR) to obtain a stable cell line (CHO-AR) expressing the AChR on its surface. Immunoprecipitation experiments established that the AChR contained alpha- and beta-subunits assembled with gamma- and/or delta-subunits. In addition, one of the toxin-binding sites of the AChR was blocked by a myasthenic serum that specifically recognizes AChR containing the gamma-subunit. AChR from the CHO-AR cells had the same sedimentation rate, association rate constant for the binding of alpha-bungarotoxin (alpha BTX), and the same metabolic half-life as the AChR in myotubes of the mouse muscle cell line C2C12. Electrophysiological assay of CHO-AR cells by single-channel recording showed the presence of ACh-responsive ion channels with the characteristics of the embryonic AChR (gamma = 40 pS, tau = 5.6 msec). In some patches a smaller conductance channel was also seen that may represent partially assembled receptor. Fluorescence microscopy of fixed, permeabilized cells stained with rhodamine-alpha-BTX demonstrated both perinuclear and diffuse surface staining. The expression of fully assembled, functional mammalian muscle AChR in nonmuscle cells will allow detailed investigation of its properties and interactions with other cellular components.
将小鼠肌肉乙酰胆碱受体（AChR）的所有4个亚基的cDNA转染至CHO细胞，以获得在其表面表达AChR的稳定细胞系（CHO-AR）。免疫沉淀实验证实，AChR包含与γ和/或δ亚基组装在一起的α和β亚基。此外，AChR的一个毒素结合位点被一种重症肌无力血清所阻断，该血清特异性识别含有γ亚基的AChR。来自CHO-AR细胞的AChR与小鼠肌肉细胞系C2C12的肌管中的AChR具有相同的沉降速率、α-银环蛇毒素（αBTX）结合的缔合速率常数以及相同的代谢半衰期。通过单通道记录对CHO-AR细胞进行电生理分析，结果显示存在具有胚胎型AChR特征（γ = 40 pS，τ = 5.6毫秒）的ACh反应性离子通道。在一些膜片中还观察到较小电导的通道，其可能代表部分组装的受体。用罗丹明-α-银环蛇毒素对固定、透化的细胞进行荧光显微镜检查，结果显示核周和弥散性表面染色。在非肌肉细胞中表达完全组装的、有功能的哺乳动物肌肉AChR，将有助于对其特性以及与其他细胞成分的相互作用进行详细研究。
