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
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
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DOI：
10.1111/j.1460-9568.1996.tb00736.x
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发表时间：
1996-10-01
影响因子：
3.4
通讯作者：
Molgo, J
中科院分区：
文献类型：
--
作者：
Colasante, C;Meunier, FA;Molgo, J
我们以前的观察，低浓度的石鱼（Synanceia trachynis）毒液引起自发的量子乙酰胆碱释放脊椎动物运动神经末梢促使我们目前的研究纯化的量子发射器释放毒素存在于毒液和表征毒素的能力，改变超微结构和免疫反应性的青蛙运动神经末梢。S.通过连续的阴离子交换快速蛋白液相色谱（FPLC）和尺寸排阻FPLC对trachynis毒液进行纯化，得到了高度纯化的膜扰动（溶血）蛋白毒素制剂，命名为trachynilysin。Trachynilysin（2-20 μ g/ml）显着增加自发的量子乙酰胆碱释放从运动末梢，通过记录从离体蛙皮心神经肌肉制剂的微型终板电位检测。超微结构分析的神经末梢，其中量子乙酰胆碱释放刺激到耗尽3小时暴露于trachynilysin显示肿胀的神经末梢和显着的小清晰的突触囊泡耗尽。然而，trachynilysin并没有诱导大的致密核心囊泡的平行消耗。大的致密核心囊泡含有降钙素基因相关肽（CORP），所揭示的胶体金免疫染色，和trachynilysin处理的神经末梢表现出类似于未经处理的终端的CORP样免疫荧光。我们的研究结果表明，石首鱼毒液引起脊椎动物运动神经末梢自发量子乙酰胆碱释放的能力是trachynilysin的功能，它选择性地刺激释放的小清晰的突触囊泡和损害的小清晰的突触囊泡的回收，但不影响大致密核心囊泡的释放。Trachynilysin可能是一个有价值的工具，用于在其他分泌终端区分神经递质和神经肽的释放。
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.