The receptor for α-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-α-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of α-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-α-latrotoxin (K(d) = 5 x 10-10 M; b(max) = 1.36 ± 0.16 [SE] x 109 sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with α-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-α-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 α-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by 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 α-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by α-latrotoxin in this preparation. When muscles were treated with proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to α-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together with these findings indicate that the α-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 ~ 2,400/μm2.
|Number of pages||9|
|Journal||Journal of Cell Biology|
|Issue number||1 I|
|Publication status||Published - 1984|
ASJC Scopus subject areas
- Cell Biology