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 Ca(2+) channels. By sequential and selective inhibition of various types of Ca(2+) 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 Ca(2+) sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca(2+) 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 Ca(2+) 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 Ca(2+)-regulated exocytosis via the specific activation of Ca(v)2.2.
Meunier
Frédéric A
FA
Molecular Neuropathobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. fmeunier@mrc-lmb.cam.ac.uk
Feng
Zhong-Ping
ZP
Molgó
Jordi
J
Zamponi
Gerald W
GW
Schiavo
Giampietro
G
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D019439:Chromaffin Cells; D004594:Electrophysiology; D005089:Exocytosis; D015801:Helminth Proteins; D006801:Humans; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009469:Neuromuscular Junction / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011077:Polychaeta / Q000378:metabolism*; D011233:Precipitin Tests; D011893:Rana esculenta; D012910:Snake Venoms; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D014688:Venoms; D020960:omega-Conotoxins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D020864:Calcium Channels, N-Type; D015801:Helminth Proteins; D009498:Neurotoxins; D012910:Snake Venoms; D014688:Venoms; D020960:omega-Conotoxins; C042564:glycerotoxin; D002118:Calcium