Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(+)-dependent and Ca2(+)-independent effects mediated by the toxin.

The potent neurotoxin alpha-latrotoxin (alpha LTx), from black widow spider venom, induces neurotransmitter release in both Ca2(+)-containing and Ca2(+)-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of alpha LTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 x 10(-10) M) for alpha LTx, with approximately 50% of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells alpha LTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 x 10(-10) M). The use of La3+ as an inhibitor of alpha LTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 microM) was shown to inhibit Ca2+ influx, both Ca2(+)-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by alpha LTx. At the same time, La3+ did not block alpha LTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 microM) or the phorbol ester tetradecanoylphorbol acetate (100 nM). La3+ also blocked the influx of Mn2+ ions through the alpha LTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by alpha LTx. Our conclusions with regard to the mode of action of alpha LTx are as follows. (i) All the effects of alpha LTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2(+)-insensitive alpha LTx receptor. (ii) alpha LTx-induced PPI hydrolysis is most likely due to the activation of a Ca2(+)-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2(+)-containing medium, and not via direct coupling of the alpha LTx receptor to the enzyme. (iii) Toxin-evoked Ca2(+)-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.