Tetramerisation of α-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca2+-dependent vesicular exocytosis from synaptosomes

A novel procedure of a-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca2+, Mg2+ or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified aLTX loses the ability to tetramerise spontaneously; the addition of Mg2+ or Ca2+ restores this ability. This suggests that alpha LTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 Angstrom-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To Study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca2+ entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca2+. The Ca2+-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca2+ stores and functional phospholipase C (PLC). The Ca2+-dependent effect of the toxin is stronger when dimeric aLTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca2+-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.