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The Journal of Neuroscience, November 2, 2005, 25(44):10188-10197; doi:10.1523/JNEUROSCI.3560-05.2005

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Cellular/Molecular
N-Terminal Insertion and C-Terminal Ankyrin-Like Repeats of -Latrotoxin Are Critical for Ca²+-Dependent Exocytosis

Gang Li,^1,2 David Lee,² Li Wang,^1,2 Mikhail Khvotchev,³ Soon Kwang Chiew,^1,2 Lakshmanan Arunachalam,^1,2 Tony Collins,¹ Zhong-Ping Feng,² and Shuzo Sugita^1,2

¹Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada M5T 2S8, ²Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8, and ³Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390

-Latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms of its actions are poorly understood. Here, we developed a new method to generate active recombinant -latrotoxin and conducted a structure/function analysis of the toxin in stimulating Ca²⁺-dependent exocytosis. -Latrotoxin consists of a conserved N-terminal domain and C-terminal ankyrin-like repeats. After cleavage of an N-terminally fused purification tag of glutathione S-transferase (GST), the recombinant toxin strongly stimulated exocytosis, whereas the GST-fused toxin was much less potent. The GST-fused toxin bound to the receptors [neurexin 1; CL1 (CIRL/latrophilin 1)] as efficiently as did the GST-cleaved toxin but was much less effective in inserting into the plasma membrane and inducing cation conductance. The toxin with deletion of the last two ankyrin-like repeats still bound the receptors but could neither stimulate exocytosis nor induce cation conductance efficiently. The abilities of the mutated toxins to stimulate exocytosis correlated well with their abilities to induce cation conductance, but not their binding to the receptors. Our results indicate that (1) C-terminal ankyrin-like repeats and a free (unfused) N terminus are both required for the toxin to form pores, which is essential for Ca²⁺-dependent exocytosis, and (2) receptor binding alone is not sufficient to stimulate Ca²⁺-dependent exocytosis.

Key words: -latrotoxin; exocytosis; neurexin 1; CL1; cation conductance; PC12 cell

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Received May 2, 2005; accepted September 19, 2005.

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