Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.

Abstract

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 the biotinylated bungarotoxin and avidin, this treatment extensively crosslinks the AcChoR. AcChoR crosslinking immobilizes > 90% of the normally laterally mobile AcChoR as verified by the fluorescence photobleaching recovery technique; it 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 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 large AcChoR patches are in close contact (< 70 .ANG.) with the glass substrate. AcChoR immobilization leads to a partial immobilization of concanavalin A receptors in the myotube membrane.