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

Abstract

The receptor for a-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-a-latrotoxin antibodies . Experiments on rat brain synaptosomes (where the existence of a-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 ' 25 1-a-latrotoxin (Kd = 5 x 10-10 M; borax = 1 .36 ± 0.16 [SEJ x 109 sites/mg tissue, dry weight) . Nonpermeabilized muscles were first treated with a-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti -a-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 a-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 alatrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by a-latrotoxin in this preparation . When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to a-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the a-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/um 2.The presynaptic membrane is the portion of the neuronal plasmalemma that surrounds synaptic terminals, where the release of neurotransmitters occurs by fusion of synaptic vesicles (exocytosis) . A number of other important functions (e .g., the recycling of the vesicle membrane, the high-affinity uptake processes of neurotransmitters or transmitter precursors, and the initiation of retrograde transport) are also localized at this level. It is generally agreed that these functional specializations of the presynaptic membrane occur as a consequence ofits molecular dissimilarity with respect to the rest 124 F.

α-Latrotoxin Receptor, Latrophilin, Is a Novel Member of the Secretin Family of G Protein-coupled Receptors

“…3A). We have also previously shown that latrophilin is greatly enriched in synaptosomal plasma membranes (9). This and the strictly presynaptic mode of toxin action suggests that latrophilin is a presynaptic protein.…”

“…In addition, to be able to act, LTX requires specific membrane receptors, which are found in the plasma membrane of neurons and some endocrine cells (2). In neuromuscular junctions the toxin receptors are localized presynaptically (9). The existence of two types of receptors, Ca 2ϩ -dependent and -independent, has long been recognized (6), the latter ones being solely responsible for the toxin action in the absence of Ca 2ϩ .…”

High Affinity Binding of α-Latrotoxin to Recombinant Neurexin Iα

“…␣-Latrotoxin binds to specific membrane receptors that are found only in the nervous system (8,9). Immunofluorescence localization of bound ␣-latrotoxin at the neuromuscular junction suggested that the binding sites are localized to the presynaptic plasma membrane (10). Together, these studies suggest that ␣-latrotoxin acts by binding to presynaptic receptors, which it either activates directly or which serves to target its insertion into the presynaptic plasma membrane.…”

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

“…Previous studies of the effect of temperature on the action of black widow spider venom or a-LTx showed that binding occurs at temperatures near 0 'C (Valtorta, Madeddu, Meldolesi & Ceccarelli, 1984), but quantal release is small when moderate doses of these agents were used (Rubin, 1976;Pumplin & Reese, 1977;Fritz, 1980). However, we have found that high concentrations of oLTx (2 ,ug/ml) raise MEPP rates to several hundred per second at temperatures of 1-3 'C and cause a profound depletion of vesicles after 2 h.…”