The effect of transfection with Botulinum neurotoxin C1 light chain on exocytosis measured in cell populations and by single-cell amperometry in PC12 cells
The effect of transfection with Botulinum neurotoxin C1 light chain on exocytosis measured in cell populations and by single-cell amperometry in PC12 cells
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DOI：
10.1007/s004240050842
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发表时间：
1999-04-01
影响因子：
4.5
通讯作者：
Burgoyne, RD
中科院分区：
文献类型：
--
作者：
Fisher, RJ;Burgoyne, RD
我们检测了瞬时转染特异性内源性蛋白酶肉毒杆菌神经毒素C1轻链（BoNT/C1）对PC12神经内分泌细胞胞吐的影响，该酶可裂解syntaxin和SNAP-25。毒素表达对基础胞吐和诱发胞吐的影响是通过细胞群测量和单细胞转染-电流法测定的。在人生长激素（hGH）作为分泌颗粒标记物的转染细胞中，BoNT/C1的go表达导致嘌呤能激动剂ATP或55 mM K+去极化引起的hGH释放抑制95%。此外，基础生长激素释放也受到相同程度的抑制。这种抑制程度所揭示的高水平共转染效率在高分辨率单细胞实验中得到了利用，该实验基于增强绿色荧光蛋白（EGFP）表达的细胞检测和使用碳纤维微电极的安培法分析诱发多巴胺释放。单独表达EGFP的细胞表现出群体反应和单细胞安培反应，与未转染的对照细胞没有区别。相反，BoNT/C1与EGFP的共表达导致ATP引起的胞外分泌几乎完全抑制电流瞬变。这些结果建立并验证了用于分析特定蛋白质对胞吐作用的单细胞转染-安培法分析。
We examined the effect on exocytosis in PC12 neuroendocrine cells of transient transfection with the specific endoprotease Botulinum neurotoxin C1 light chain (BoNT/C1), which cleaves syntaxin and SNAP-25. The effects of toxin expression on basal and evoked exocytosis were determined in cell population measurements and also in a single-cell transfection-amperometry assay. Go-expression of BoNT/C1 with human growth hormone (hGH) as a marker of secretory granules in transfected cells resulted in a 95% inhibition of hGH release evoked either by the purinergic agonist ATP or by depolarization with 55 mM K+. In addition, basal hGH release was also inhibited to the same extent. The high level of co-transfection efficiency revealed by this extent of inhibition was exploited in a high-resolution single-cell assay based on cell detection by expression of enhanced green fluorescent protein (EGFP) and analysis of evoked dopamine release by amperometry using a carbon fibre microelectrode. Cells expressing EGFP alone showed population responses and single-cell amperometric responses indistinguishable from those of control non-transfected cells. In contrast, co-expression of BoNT/C1 with EGFP resulted in an almost complete inhibition of current transients due to exocytosis evoked by ATP. These results establish and validate a single-cell assay of transfection-amperometry for analysing the effects of specific proteins on exocytosis.