alpha-latrotoxin triggers transmitter release via direct insertion into the presynaptic plasma membrane

alpha-latrotoxin, a component of black widow spider venom, binds to presyna ptic nerve terminals and stimulates massive neurotransmitter release. Previ ous studies have demonstrated that alpha-latrotoxin first binds to two high -affinity receptors on nerve terminals, neurexins and CLs (CIRLs and latrop hilins), and then executes a critical, second step of unknown nature that s timulates neurotransmitter release. We now demonstrate that incubation of a lpha-latrotoxin with synaptosomes at 0 Y C results in its peripheral membra ne association. Incubation at 37 Y C, however, converts the toxin into an o perationally integral membrane protein, and induces generation of a proteas e-resistant N-terminal domain or alpha-latrotoxin and becomes protease sens itive after lysis of synaptosomes, Our data suggest that alpha-latrotoxin i nserts into the presynaptic plasma membrane after receptor binding, resulti ng in an intracellular location of the N-terminal sequences. Membrane inser tion of the N-terminal domain of alpha-latrotoxin occurs spontaneously, ind ependently of membrane recycling or transmembrane ion gradients. We postula te that alpha-latrotoxin acts intracellularly in triggering release, and pr opose that non-selective cation channels induced by alpha-latrotoxin may be a by-product of membrane insertion.