Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF)
Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF)
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DOI：
10.1002/bem.20354
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发表时间：
2007-12-01
影响因子：
1.9
通讯作者：
Schoenbach, Karl H.
中科院分区：
文献类型：
--
作者：
Pakhomov, Andrei G.;Kolb, Juergen F.;Schoenbach, Karl H.
用全细胞膜片钳技术检测了nsPEF暴露的哺乳动物细胞质膜的屏障功能。开发了一种专门的装置，用于培养中单个细胞的nsPEF暴露，并表征了与膜片钳方法的无伪影兼容性。我们的研究第一次提供了实验证据，即使是12 kV/cm的单个60 ns脉冲也会导致细胞膜电阻（R-m）的深刻而持久（分钟）的降低，并伴随着膜电位的损失。在nsPEF暴露后80-120 s内测量的GH 3、PC-12和Jurkat细胞（但不在HeLa细胞中）的R-m降低约3倍，其逐渐恢复可能需要15 min。多脉冲增强透化作用，例如，GH 3细胞中的R-m在一系列5个脉冲后下降约10倍。在研究范围内，透化不依赖于移液管或浴溶液中存在的Ca 2+，Mg 2+，K+，Cs+，Cd 2+，EGTA，四乙基铵或4-氨基吡啶。我们的研究结果支持理论模型预测的质膜穿孔nsPEF。然而，R-m的延长降低（假设与孔的寿命有关）和单个细胞系的不同nsPEF敏感性尚未得到解释。长寿命的膜透化的现象提供了新的见解nsPEF开放的电导孔的性质和nsPEF生物效应的基础上的分子机制。
The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (R-m), accompanied by the loss of the membrane potential. R-m measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80-120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, R-m in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca2+, Mg2+, K+, Cs+, Cd2+, EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in R-m, assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects.