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J. Biol. Chem., Vol. 278, Issue 33, 31058-31066, August 15, 2003

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

THIS ACTION DOES NOT REQUIRE NEUREXINS^*

Kirill E. Volynski , Marco Capogna , Anthony C. Ashton , Derek Thomson ¶, Elena V. Orlova  ||, Catherine F. Manser , Richard R. Ribchester ¶ and Yuri A. Ushkaryov  **

From the Department of Biological Sciences, Imperial College London, London SW7 2AY, United Kingdom, the Medical Research Council, Anatomical Neuropharmacology Unit, Oxford OX1 3TH, United Kingdom, and the ^¶Department of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, United Kingdom

-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^N4C) 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^N4C 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 LTX^N4C action. To test this hypothesis, we used conditions (substitution of Ca²⁺ in the medium with Sr²⁺) under which LTX^N4C 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 LTX^N4C. These results indicate that LTX^N4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.

Received for publication, October 10, 2002 , and in revised form, May 19, 2003.

^* This work was supported by the Wellcome Trust (Senior European Research Fellowship (to Y. A. U.), project grant (to R. R. R.)), Medical Research Council (to M. C.), and Biotechnology and Biological Sciences Research Council (Research Grant 28/B14085 (to Y. A. U.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Current address: Dept. of Crystallography, Birkbeck College, London WC1E 7HX, UK.

^** To whom correspondence should be addressed. Tel.: 44-(0)20-7594-5237; Fax: 44-(0)20-7594-5207; E-mail: y.ushkaryov{at}imperial.ac.uk.

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