Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle

Abstract

1. alpha-Bungarotoxin was labelled with fluorescent dyes and used as a stain for visualizing the distribution of acetylcholine receptors in vertebrate skeletal muscle fibres.2. Dye-toxin conjugates had the same pharmacological properties as native toxin, but their potencies were lower.3. Fluorescent staining was examined in teased muscle fibres. The stain was found to be confined to the neuromuscular junction and associated with the subsynaptic membrane.4. Staining intensity was reduced by curare and even more so by carbachol, but not by atropine or neostigmine. Pre-treatment of muscles with unlabelled alpha-bungarotoxin entirely prevented staining.5. The staining at amphibian neuromuscular junctions was characterized by a pattern of intense transverse bands occurring at intervals of approximately 0.5-1 mum, with fluorescence of lower intensity between them. Fluorescent staining was not detected on adjacent, extrasynaptic, muscle membrane. In side views the staining appeared as a fine line with small protuberances occurring at the same intervals as the intense bands seen face-on. These results indicate that acetylcholine receptors are associated with the entire subsynaptic membrane, including the membrane of the junctional folds and that their density changes abruptly at the border between synaptic and extrasynaptic muscle membrane.