α‐Latrotoxin action probed with recombinant toxin: receptors recruit α‐latrotoxin but do not transduce an exocytotic signal

α‐Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Iα. We have now produced recombinant α‐latrotoxin (Ltx WT ) that is as active as native α‐latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three α‐latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four‐residue insertion. All four α‐latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant Ltx N4C exhibited receptor‐binding affinities identical to wild‐type Ltx WT , bound to CL1 and neurexin Iα as well as Ltx WT , and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by α‐latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La 3+ and Cd 2+ . La 3+ blocked release triggered by Ltx WT , whereas Cd 2+ enhanced it. Both cations, however, had no effect on the stimulation by Ltx WT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by α‐latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit α‐latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor‐independent activity of α‐latrotoxin that is selectively inhibited by the Ltx N4C mutation and by La 3+ .