Hausinger A, Volknandt W, Zimmermann H, Habermann E
Biozentrum der Johann-Wolfgang-Goethe-Universität, Frankfurt/Main, Germany.
Toxicon. 1995 Nov;33(11):1519-30. doi: 10.1016/0041-0101(95)00084-y.
Clostridial neurotoxins are known to inhibit regulated, i.e. calcium-dependent exocytosis. In the present study we have investigated their potential role in also inhibiting calcium-independent exocytosis. Synaptosomes from rat forebrain were preloaded with [3H]noradrenaline and permeabilized reversibly by freezing in Ca(2+)-free potassium glutamate containing dimethyl sulfoxide and the toxins to be assayed. Subsequently, outflow of radioactivity was measured in isotonic calcium-free potassium glutamate. The synaptic vesicle protein synaptobrevin-2/VAMP-2 and its toxin-dependent degradation were analysed by Western blotting. The light chain of tetanus toxin reduced the synaptosomal outflow of radioactivity, whereas the activity of the heavy chain was at the detection limit. The respective activities of the dichain toxins from Clostridium tetani and C. botulinum A, B and E were enhanced by pretreatment with dithiothreitol. Reduced single-chain tetanus toxin was less potent than reduced dichain tetanus toxin. Pretreatment with ethylene diamine tetraacetic acid as an inhibitor of Zn(2+)-proteases abolished the actions of the tetanus toxin light chain and of the reduced dichain toxins. Hydrolysis of synaptobrevin-2/VAMP-2 was obtained with tetanus toxin light chain, reduced dichain tetanus toxin and C. botulinum B toxin. Its hydrolysis by single-chain tetanus toxin was less pronounced, and it was absent with botulinum toxins A and E. It is concluded that clostridial neurotoxins can not only inhibit calcium-dependent release but also affect calcium-independent outflow from synaptosomes. Since this is accompanied by selective intrasynaptosomal proteolysis of synaptobrevin, calcium-independent outflow may at least in part involve the vesicular release apparatus.
已知梭菌神经毒素可抑制受调控的，即钙依赖性的胞吐作用。在本研究中，我们调查了它们在抑制非钙依赖性胞吐作用方面的潜在作用。将来自大鼠前脑的突触体预先装载[3H]去甲肾上腺素，并通过在含有二甲基亚砜和待检测毒素的无钙谷氨酸钾中冷冻进行可逆性通透化处理。随后，在等渗无钙谷氨酸钾中测量放射性流出。通过蛋白质印迹分析突触小泡蛋白突触结合蛋白-2/VAMP-2及其毒素依赖性降解。破伤风毒素轻链减少了突触体放射性流出，而重链的活性处于检测限。来自破伤风梭菌和肉毒梭菌A、B和E的双链毒素的各自活性通过二硫苏糖醇预处理而增强。还原的单链破伤风毒素比还原的双链破伤风毒素效力更低。用乙二胺四乙酸作为锌蛋白酶抑制剂进行预处理消除了破伤风毒素轻链和还原双链毒素的作用。破伤风毒素轻链、还原双链破伤风毒素和肉毒梭菌B毒素可导致突触结合蛋白-2/VAMP-2的水解。单链破伤风毒素对其水解作用较弱，肉毒毒素A和E则无此作用。结论是梭菌神经毒素不仅可以抑制钙依赖性释放，还可以影响突触体的非钙依赖性流出。由于这伴随着突触结合蛋白在突触体内的选择性蛋白水解，非钙依赖性流出可能至少部分涉及囊泡释放装置。
