Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane.

Abstract
The receptor for .alpha.-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-.alpha.-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of .alpha.-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex binds on the average 5 antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-.alpha.-latrotoxin (Kd = 5 .times. 10-10 M; bmax = 1.36 .+-. 0.16 [SE] .times. 109 sites/mg tissue, dry wt). Nonpermeabilized muscles were first treated with .alpha.-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-.alpha.-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated .alpha.-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by 2 sets of findings: fluorescent endplate images were not seen in muscles that had been denervated, and the distribution of fluorescence in many fibers treated with .alpha.-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by .alpha.-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to .alpha.-latrotoxin, the specific fluorescent endplate images failed to appear. The .alpha.-latrotoxin receptor is evidently an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction is .apprx. 2400/.mu.m2.

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