Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.
A biotinylated derivative of alpha-bungarotoxin and tetramethylrhodamine-labeled avidin were employed to fluorescence label the acetylcholine receptors (AcChoR) on the surface of rat myotubes in primary culture. Because of the multivalency of both the biotinylated bungarotoxin and the avidin, this treatment extensivey crosslinks the AcChoR. AcChoR crosslinking immobilizes more than 90% of the normally laterally mobile AcChoR as verified by the fluorescence photobleaching recovery technique; it also redistributes the AcChoR into visible micropatches. Biotinylated alpha-bungarotoxin/avidin-induced AcChoR crosslinking greatly accelerates the rate of internalization of surface AcChoR; this rapid internalization affects both the normally immobile AcChoR in areas of diffuse distribution and the normally immobile AcChoR in preexisting patches. The peculiar pattern of fluorescent avidin binding to AcChoR patches previously bound with biotinylated bungarotoxin suggests that almost all AcChoR patches are in very close contact (< 70 A) with the glass substrate. AcChoR immobilization leads to a partial immobilization of concanavalin A receptors in the myotube membrane.
D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002038:Bungarotoxins / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D008560:Membrane Fluidity; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000196:embryology / Q000378:metabolism*; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011952:Receptors, Concanavalin A / Q000187:drug effects
D002038:Bungarotoxins; D003432:Cross-Linking Reagents; D011950:Receptors, Cholinergic; D011952:Receptors, Concanavalin A; D001360:Avidin; D001710:Biotin