Black J D, Dolly J O
J Cell Biol. 1986 Aug;103(2):521-34. doi: 10.1083/jcb.103.2.521.
The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.
运用电子显微镜放射自显影技术，研究了放射性碘化A型和B型肉毒杆菌神经毒素（125I - BoNT）在脊椎动物神经肌肉接头处产生的标记模式。所获数据可得出以下结论。将125I - BoNT A型在体内或体外应用于小鼠膈肌或青蛙胸皮肌时，仅与运动神经末梢发生饱和性相互作用；银颗粒出现在质膜上、突触小体内以及神经干的轴浆中，提示毒素或其片段发生内化及轴突逆行运输。将125I - BoNT B型体外应用于小鼠神经肌肉接头时，同样与运动神经末梢相互作用，但内化放射性的比例较低。这一结果与这些毒素类型相似但不完全相同的药理作用相符。每种情况下标记的饱和性提示有受体参与；用代谢抑制剂阻止内化步骤后，其确切位置变得明显。在神经末梢膜的所有无髓鞘区域均发现了它们，包括终末前轴突和突触小体。尽管125I - BoNT A型与新生大鼠的发育中终末特异性相互作用，但神经干的无髓鞘质膜未被标记，表明这些受体是局限于神经末梢区域的独特成分。A型和B型肉毒杆菌神经毒素在终末膜上有不同的受体。在优化了这些结合位点饱和的条件并校准放射自显影程序后，我们发现A型和B型受体的密度分别约为150和630个/微米2膜面积。有人提出，这些膜受体将肉毒杆菌神经毒素靶向神经末梢，并介导其递送至细胞内位点，从而有助于毒素对神经递质释放的选择性抑制作用。
