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 release at the frog neuromuscular junction, depleting small clear synaptic vesicles, 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 catecholamine 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-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced egocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 mu g/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense-core vesicles contained calcitonin gene-related peptide (CORP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CORP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release

Trachynilysin, a protein neurotoxin isolated from stonefish (Synanceia trachynis) venom, increases spontaneous quantal acetylcholine release from Torpedo marmorata neuromuscular junctions

Trachynilysin, a neurotoxin isolated from the venom of the stonefish (Synanceia trachynis, Scorpaenidae), produced a marked increase in the frequency of spontaneous miniature endplate potentials (MEPPs) at Torpedo neuromuscular junctions. The periods of high frequency MEPP discharges were of variable duration and were followed by periods of rest. In addition, trachynilysin increased as a function of time the proportion of larger than normal MEPPs, the so-called "giant" MEPPs. Trachynilysin did not affect the junctions when applied in Ca-free medium supplemented with EGTA, but the subsequent addition of Ca caused a rapid increase in MEPP frequency, even when the toxin was washed out of the Ca-free medium. Thus, trachynilysin binding to nerve terminals is not dependent on external Ca, but the cation is required for trachynilysin-elicited quantal transmitter release. The effect of trachynilysin on MEPP frequency was unaffected by the Ca channel blockers ω-conotoxin GVIA, ω-agatoxin IVA and Gd, which indicates that the toxin's action involves Ca entry via a pathway independent of voltage-sensitive Ca channels. Pre-treatment of the junctions with concanavalin-A prevented the trachynilysin-induced enhancement of quantal transmitter release, which suggests that the toxin interacts with or binds to a glycoprotein on the surface of motor nerve terminals