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 was found to bind on the average five 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 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). 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 two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) 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. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.