Arnold S. Kreger

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 toxins 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 μg/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 (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin‐treated nerve endings exhibited CGRP‐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.

Effects of trachynilysin, a protein isolated from stonefish (Synanceia trachynis) venom, on frog atrial heart muscle

The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening.

Trachynilysin, a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom, forms nonselective pores in the membrane of NG108-15 cells.

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about −3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxins mode of action on motor nerve endings and neurosecretory cells.

Secretagogue Activity of Trachynilysin, a Neurotoxic Protein Isolated from Stonefish (Synanceia trachynis) Venom

Approximately 400 to 500 species of marine fish may be poisonous to humans after ingestion. Most poisonous fish are nonmigratory reef fish and can be either herbivores or carnivores. Some of them have tissues that are toxic at all times, others are poisonous during certain periods of the year or in certain geographical areas, and still others have only specific organs that are toxic, and their toxicity may vary with time, location, and habitat (reviewed in ref. 1).