Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter r elease at the frog neuromuscular junction, depleting small clear synaptic v esicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamin e release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-depe ndent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely o n Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, tra chynilysin elicited a localized increase in intracellular fluorescence moni tored with fluo-3/AM, that precisely co-localized with the increase of fluo rescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-i nduced egocytosis. Thus, the observed requirement of external Ca2+ for stim ulation of large dense core vesicles exocytosis from chromaffin cells impli cates plasma membrane channels that signal efflux of Ca2+ from intracellula r stores. This study also suggests that the bases of exocytosis of large de nse core vesicles from motor nerve terminals and neuroendocrine cells are d istinct.