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.

Abstract A novel procedure of α-latrotoxin (αLTX) purification has been developed. Pure αLTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca 2+ , Mg 2+ or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified αLTX loses the ability to tetramerise spontaneously; the addition of Mg 2+ or Ca 2+ restores this ability. This suggests that αLTX 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 αLTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both αLTX 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 αLTX pore formation, suggesting that the tetramers are the pore-forming species of αLTX. The toxin actions mediated by the pore include: 1) Ca 2+ entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca 2+ . The Ca 2+ -dependent αLTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca 2+ stores and functional phospholipase C (PLC). The Ca 2+ -dependent effect of the toxin is stronger when dimeric αLTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca 2+ -independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.