NEUROTOXINS can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45 Ca²⁺ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45 Ca²⁺ influx and they should be useful in studies involving Ca²⁺-dependent processes.

The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca²⁺-dependent glutamate release, but did not alter Ca²⁺-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with w-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.