Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios

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 a7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophorelabeled 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 a7 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.