Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Biophys J. 2010 Nov 17;99(10):L81-3. doi: 10.1016/j.bpj.2010.08.076.
Nicotinic acetylcholine receptors are some of the most studied synaptic proteins; however, many questions remain that can only be answered using single molecule approaches. Here we report our results from single α7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophore-labeled bungarotoxin, we can image individual receptors and count the number of bungarotoxin-binding sites in receptors expressed in HEK 293 cells. Our results indicate that there are two bungarotoxin-binding sites in neuromuscular junction receptors, as expected, and five in α7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound proteins at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques.
烟碱型乙酰胆碱受体是研究最为深入的突触蛋白之一；然而，仍有许多问题需要通过单分子方法才能得到解答。在这里，我们报告了在哺乳动物细胞膜中单 α7 和神经肌肉接头型烟碱型乙酰胆碱受体的研究结果。通过用荧光标记的金环蛇毒素对受体进行标记，我们可以对单个受体进行成像，并对在 HEK 293 细胞中表达的受体的金环蛇毒素结合位点的数量进行计数。我们的结果表明，神经肌肉接头受体中有两个金环蛇毒素结合位点，这与预期相符，而 α7 受体中有五个，这澄清了先前的不确定性。这表明，该技术在单分子水平上对膜结合蛋白的亚基进行计数具有重要价值，其光学要求不高，并且与基于荧光蛋白的先前技术相比，具有更高的信号/噪声比。
