title: Mutant alpha-latrotoxin (LTXN4C) does not form pores and causes secretion by receptor stimulation: this action does not require neurexins.
abstract: Alpha-latrotoxin (LTX) causes massive release of neurotransmitters via a complex mechanism involving (i) activation of receptor(s) and (ii) toxin insertion into the plasma membrane with (iii) subsequent pore formation. Using cryo-electron microscopy, electrophysiological and biochemical methods, we demonstrate here that the recently described toxin mutant (LTXN4C) is unable to insert into membranes and form pores due to its inability to assemble into tetramers. However, this mutant still binds to major LTX receptors (latrophilin and neurexin) and causes strong transmitter exocytosis in synaptosomes, hippocampal slice cultures, neuromuscular junctions, and chromaffin cells. In the absence of mutant incorporation into the membrane, receptor activation must be the only mechanism by which LTXN4C triggers exocytosis. An interesting feature of this receptor-mediated transmitter release is its dependence on extracellular Ca2+. Because Ca2+ is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTXN4C action. To test this hypothesis, we used conditions (substitution of Ca2+ in the medium with Sr2+) under which LTXN4C does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr2+ fully replaces Ca2+ in supporting the stimulatory effect of LTXN4C. These results indicate that LTXN4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.
authors.affiliation: Department of Biological Sciences, Imperial College London, London SW7 2AY, United Kingdom.
authors.forename: Kirill E
authors.initials: KE
authors.lastname: Volynski
authors.forename: Marco
authors.initials: M
authors.lastname: Capogna
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Ashton
authors.forename: Derek
authors.initials: D
authors.lastname: Thomson
authors.forename: Elena V
authors.initials: EV
authors.lastname: Orlova
authors.forename: Catherine F
authors.initials: CF
authors.lastname: Manser
authors.forename: Richard R
authors.initials: RR
authors.lastname: Ribchester
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Ushkaryov
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001740:Black Widow Spider; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002250:Carbon Radioisotopes; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism; D006224:Cricetinae; D005089:Exocytosis; D018698:Glutamic Acid / Q000493:pharmacokinetics; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009154:Mutation; D009469:Neuromuscular Junction / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics*; D013324:Strontium / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D002395:Catecholamines; D011956:Receptors, Cell Surface; D013111:Spider Venoms; D018698:Glutamic Acid; C020763:alpha-latrotoxin; D000109:Acetylcholine; D002118:Calcium; C038599:black widow spider venom; D013324:Strontium