Mutant -Latrotoxin (LTX N4C ) Does Not Form Pores and Causes Secretion by Receptor Stimulation

-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 (LTX) 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 LTX triggers exocytosis. An interesting feature of this receptormediated transmitter release is its dependence on extracellular Ca . Because Ca is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTX action. To test this hypothesis, we used conditions (substitution of Ca in the medium with Sr ) under which LTX does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr fully replaces Ca in supporting the stimulatory effect of LTX. These results indicate that LTX can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.