Nanoelectropulse-driven membrane perturbation and small molecule permeabilization
Nanoelectropulse-driven membrane perturbation and small molecule permeabilization
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DOI：
10.1186/1471-2121-7-37
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发表时间：
2006-10-19
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影响因子：
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通讯作者：
Gundersen, Martin A.
中科院分区：
文献类型：
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作者：
Vernier, P. Thomas;Sun, Yinghua;Gundersen, Martin A.
背景：纳秒、兆伏每米脉冲电场扰乱膜磷脂，释放细胞内钙，并诱导细胞凋亡。流式细胞术和荧光显微镜证据表明磷脂重排与纳米电脉冲暴露直接相关，并支持电脉冲期间跨脂质双层产生的电势驱动磷脂酰丝氨酸 (PS) 外化的假设。结果：在这项工作中，我们将对暴露于持续时间为 30 ns 和 7 ns 的电脉冲的细胞的观察扩展到更窄的脉冲 我们发现即使是 3 ns 的脉冲也足以产生与之前报道的类似的响应。我们在此表明，与单极脉冲相比，单极脉冲仅在电池的阳极侧扰乱膜磷脂顺序，用 FMI-43 荧光跟踪，双极脉冲在阳极和阴极上重新分布磷脂，这与阴离子 PS 头基在跨膜场中的迁移一致。此外，我们证明，正如膜充电假说所预测的那样，一串较短的脉冲需要更高的场来产生与时间等效的较长脉冲串产生的磷脂扰乱相当的效果（对于给定的应用场，30、4 ns 脉冲产生的响应比 4、30 ns 脉冲更弱）。最后，我们发现，在 Jurkat T 淋巴母细胞暴露于足够大量的脉冲后，观察到 YO-PRO-1（一种用于检测早期细胞凋亡和嘌呤能 P2X(7) 受体通道激活的荧光染料）的流入，这表明当电场足够高时，即使使用纳秒脉冲也会发生膜穿孔。碘化丙啶进入是电穿孔的传统指标，在脉冲计数更高时发生。结论：短至 3 ns 的每米兆伏电脉冲会改变质膜的结构，并使细胞透化为小分子。细胞对持续时间为 3 ns 至 30 ns 的单极和双极脉冲的剂量响应支持这样的假设：膜电介质的场驱动充电导致纳秒时间尺度上的孔的形成，并且阴离子磷脂 PS 沿着这些孔的壁以电泳方式迁移到膜的外表面。
Background: Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization.Results: In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FMI-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X(7) receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts.Conclusion: Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane.