title: alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal.
abstract: alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.
authors.affiliation: Center for Basic Neuroscience and Department of Molecular Genetics, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ichtchenko
authors.forename: M
authors.initials: M
authors.lastname: Khvotchev
authors.forename: N
authors.initials: N
authors.lastname: Kiyatkin
authors.forename: L
authors.initials: L
authors.lastname: Simpson
authors.forename: S
authors.initials: S
authors.lastname: Sugita
authors.forename: T C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D006023:Glycoproteins; D007811:Lanthanum / Q000494:pharmacology; D051379:Mice; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D018000:Receptors, Peptide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D010716:Phosphatidylinositols; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C112162:neurexophilin; D002104:Cadmium; C020763:alpha-latrotoxin; D007811:Lanthanum; D010738:Type C Phospholipases; D003545:Cysteine