Tetramerisation of alpha-latrotoxin by divalent cations is responsible fortoxin-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 develop ed. Pure alpha LTX has been demonstrated to exist as a very stable homodime r. Such dimers further assemble into tetramers, and Ca2+, Mg2+ or higher to xin concentrations facilitate this process. However, when the venom is trea ted with EDTA, purified aLTX loses the ability to tetramerise spontaneously ; the addition of Mg2+ or Ca2+ restores this ability. This suggests that al pha LTX has some intrinsically bound divalent cation(s) that normally suppo rt its tetramerisation. Single-particle cryoelectron microscopy and statist ical image analysis have shown that: 1) the toxin has a non-compact, branch ing 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 brai n sections. To Study the effects of the dimers and tetramers on norepinephr ine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a pre paration correlates with alpha LTX pore formation, suggesting that the tetr amers 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) p assive efflux of neurotransmitters via the pore that occurs independently o f Ca2+. The Ca2+-dependent alpha LTX-stimulated secretion conforms to all c riteria 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 m easured biochemically represents leakage of neurotransmitters through the t oxin pore. These results are discussed in relation to the previously publis hed observations. (C) 2000 Societe francaise de biochimie et biologie molec ulaire / Editions scientifiques et medicales Elsevier SAS.