Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity

We report here the purification of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltage- dependent Ca2+ channels. By sequential and selective inhibition of various types of Ca2+ channels, we found that glycerotoxin was acting via Ca(v)2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, influx of Ca2+ sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca2+ transient in human embryonic kidney (HEK) cells over-expressing Ca(v)2.2 but not Ca(v)2.1 (P/Q-type). Whole-cell patch-clamp analysis of Ca(v)2.2 expressing HEK cells revealed an up-regulation of Ca2+ currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca(v)2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca2+-regulated exocytosis via the specific activation of Ca(v)2.2.