Measurement of exocytosis by amperometry in adrenal chromaffin cells: Effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics
Measurement of exocytosis by amperometry in adrenal chromaffin cells: Effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics
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DOI：
10.1016/s0300-9084(00)00196-6
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发表时间：
2000-05-01
期刊：
影响因子：
3.9
通讯作者：
Burgoyne, RD
中科院分区：
文献类型：
--
作者：
Graham, ME;Fisher, RJ;Burgoyne, RD
我们已经使用碳纤维安培法研究牛肾上腺嗜铬细胞中的单个分泌颗粒融合/释放事件的动力学。用编码肉毒杆菌神经毒素（BoNTs）轻链的质粒转染来研究突触融合蛋白或SNAP-25的切割对胞吐的影响。BoNT/C1或BoNT/E的表达抑制了由毛地黄皂苷/Ca 2+渗透和刺激单个嗜铬细胞引起的胞吐作用的程度。神经毒素表达后，残留的释放事件与对照细胞在其幅度和动力学上没有不同，从电流尖峰分析。与此相反，蛋白激酶C（PKC）的激活导致单颗粒释放事件的动力学的修改。佛波酯处理后，电流尖峰显示出显着减少，由于其平均半宽度的初始上升的速率增加，也下降到基线的尖峰，在其总电荷。这些变化被预防预处理与PKC抑制剂bisindolylmaleimide。这些结果表明，蛋白激酶C调节融合孔扩张的速度，也随后孔关闭或颗粒检索。因此，PKC介导的kiss-and-run融合调节可能控制单个分泌颗粒释放儿茶酚胺的程度。这里使用的实验方法可以提供进一步的信息的蛋白质成分和融合孔机械的调节。(C)2000年法国生物化学和生物分子学会/科学和医学版Elsevier SAS。
We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca2+ to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.