title: Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios.
abstract: 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.
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Simonson
authors.forename: Hannah A
authors.initials: HA
authors.lastname: Deberg
authors.forename: Pinghua
authors.initials: P
authors.lastname: Ge
authors.forename: John K
authors.initials: JK
authors.lastname: Alexander
authors.forename: Okunola
authors.initials: O
authors.lastname: Jeyifous
authors.forename: William N
authors.initials: WN
authors.lastname: Green
authors.forename: Paul R
authors.initials: PR
authors.lastname: Selvin
mesh_terms: D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009469:Neuromuscular Junction / Q000378:metabolism; D038761:Photobleaching; D011978:Receptors, Nicotinic / Q000378:metabolism*; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; C579167:Chrna7 protein, human; D005456:Fluorescent Dyes; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor