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Glycerotoxin stimulates exocytosis and endocytosis by increasing intracellular Ca2+ in N-type calcium channels expressing cells

S. Cavaignac,1 M. Schenning,1 D. Proctor,1 M. Stafford,1 N. Lavidis,1 G.W. Zamponi,2 G. Schiavo3 and F.A. Meunier,1 1School of Biomedical Sciences, University of Queensland, St Lucia, Qld 4072, Australia, 2Department of Physiology and Biophisics, University of Calgary, Calgary, Alberta, Canada and 3Molecular Neuropathobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Field Laboratories, London, UK.

We recently purified a novel neurotoxin from Glycera convoluta named Glycerotoxin (GLTx), capable of stimulating neurotransmitter release from N-type Ca2+ channels expressing neurons for up to 24h (Schenning et al., 2006). Here, we have found that GLTx also stimulates compensatory endocytosis of synaptic vesicles using styryl dyes and electron microscopy. Furthermore, we have adapted a fluorescent-based assay to monitor intracellular Ca2+ flux from both rat brain synaptosomes and human embryonic kidney (HEK) cells over-expressing N, L, P/Q and R-type Ca2+ channels. GLTx triggers Ca2+ influx in HEK cells expressing rat or human N-type Ca2+ channels without affecting cells transfected with L, P/Q or R-type Ca2+ channels. In addition, GLTx promoted Ca2+ influx in rat brain synaptosomes and an increase in endogenous glutamate released with an EC50 of 50 pM. GLTx is therefore a unique tool available to unravel the mechanism controlling Ca2+-regulated exocytosis and compensatory endocytosis via the specific activation of N-type Ca2+ channels. Importantly, GLTx was found to act on both rat and human clones of N-type Ca2+ channels. GLTx or derivatives could therefore be useful in future human therapy strategies aiming at enhancing neurotransmitter release by selectively stimulating N-type Ca2+ channel-expressing neurons.

Schenning, M.P., Proctor, D.T., Ragnarsson, L., Barbier, J., Lavidis, N.A., Molgo, J.J., Zamponi, G.W., Schiavo, G. & Meunier, F.A. (2006) Journal of Neurochemistry 98: 894-904.