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 (Synance ia trachynis) venom elicit spontaneous quantal acetylcholine release f rom vertebrate motor nerve terminals prompted our present study to pur ify the quantal transmitter-releasing toxin present in the venom and t o characterize the toxin's ability to alter the ultrastructure and imm unoreactivity of frog motor nerve terminals. Fractionation of S. trach ynis venom by sequential anion exchange fast protein-liquid chromatogr aphy (FPLC) and size-exclusion FPLC yielded a highly purified preparat ion of a membrane-perturbing (haemolytic) protein toxin, named trachyn ilysin. Trachynilysin (2-20 mu g/ml) significantly increased spontaneo us quantal acetylcholine release from motor endings, as detected by re cording miniature endplate potentials from isolated frog cutaneous pec toris neuromuscular preparations. Ultrastructural analysis of nerve te rminals in which quantal acetylcholine release was stimulated to exhau stion by 3 h exposure to trachynilysin revealed swelling of nerve term inals 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 trac hynilysin-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 acetylcholin e release from vertebrate motor nerve terminals is a function of trach ynilysin, which selectively stimulates the release of small clear syna ptic vesicles and impairs the recycling of small clear synaptic vesicl es but does not affect the release of large dense-core vesicles. Trach ynilysin may be a valuable tool for use in other secretory terminals t o discriminate between neurotransmitter and neuropeptide release.