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 alpha 7 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 a7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound protei is at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques.