ALPHA-LATROTOXIN ACTION PROBED WITH RECOMBINANT TOXIN - RECEPTORS RECRUIT ALPHA-LATROTOXIN BUT DO NOT TRANSDUCE AN EXOCYTOTIC SIGNAL

alpha-Latrotoxin stimulates neurotransmitter release probably by bindi ng to two receptors, CIRL/latrophilin 1 (CL1) and neurexin I alpha, We have now produced recombinant alpha-latrotoxin (Ltx(WT)) that is as a ctive as native alpha-latrotoxin in triggering synaptic release of glu tamate, GABA and norepinephrine, We have also generated three alpha-la trotoxin mutants with substitutions in conserved cysteine residues, an d a fourth mutant with a four-residue insertion. All four alpha-latrot oxin 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 I alpha as w ell as Ltx(WT), and similarly stimulated synaptic hydrolysis of phosph atidylinositolphosphates. Therefore, receptor binding by alpha-latroto xin and stimulation of phospholipase C are insufficient to trigger exo cytosis, This conclusion was confirmed in experiments with La3+ and Cd 2+. La3+ blocked release triggered by Ltx(WT), whereas Cd2+ enhanced i t, Both cations, however, had no effect on the stimulation by Ltx(WT) of phosphatidylinositolphosphate hydrolysis, Our data show that recept or binding by alpha-latrotoxin and activation of phospholipase C do no t by themselves trigger exocytosis, Thus receptors recruit alpha-latro toxin to its point of action without activating exocytosis, Exocytosis probably requires an additional receptor-independent activity of alph a-latrotoxin that is selectively inhibited by the Ltx(N4C) mutation an d by La3+.