哺乳动物和植物细胞的跨膜电位测量

T. Berghofer, C. Eing, B. Flickinger, C. Gusbeth, R. Strassner, W. Frey, S. Schneider
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摘要

只提供摘要形式。脉冲激光荧光显微镜(PLFM)使用电压敏感染料ANNINE-6可以测量生物细胞的跨膜电位,时间分辨率为5纳秒。已经提出了几种理论来解释膜电导率在响应外电场时的变化,例如孔隙模型,由于电压缩引起的相变或脂筏引起的膜局部扰动。测量到的哺乳动物和植物细胞的跨膜电位的场强依赖性表现出饱和特征,这应该是由于纳米/微孔的形成而发生的。对于典型直径为15微米的哺乳动物细胞,在1千伏/厘米量级的外场强度下产生饱和效应。对于平均大小为哺乳动物细胞直径三倍的烟草原生质体(BY-2),在约为哺乳动物细胞饱和场强度的三分之一时,可以观察到相同的饱和效应。这证实了当前膜充电模型的一般预期。此外,原生质体在细胞的超极化半球和去极化半球表现出强烈的膜电荷不对称性,这是由于与哺乳动物细胞相比,原生质体具有更高的静息电位。据估计,孔隙形成起始的阈值跨膜电压的绝对值为+/-200 mV。此外,还研究了跨膜电位的方位分布随时间和场强的变化规律。跨膜电位响应电场脉冲的时间过程与理论预测一致。将介绍HEK293、HeLa和22Rv1细胞以及烟草原生质体的结果,并描述基本的测量设置。
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Transmembrane potential measurements of mammalian and plant cells
Summary form only given. Pulsed laser fluorescence microscopy (PLFM) using the voltage sensitive dye ANNINE-6 permits measurements of transmembrane potentials of biological cells with a time resolution of five nanoseconds. Several theories have been proposed to explain the change of the membrane conductivity in response to an external electric field, e.g. pore-models, phase transitions due to electro-compression or local disturbances in the membrane caused by lipid rafts. The measured field-strength-dependence of the transmembrane potential of both mammalian and plant cells exhibits a saturation character which is supposed to occur due to the formation of nano/micropores. For mammalian cells with a typical diameter of 15 mum saturation effects set in at external field strengths in the order of 1 kV/cm. For tobacco-protoplasts (BY-2) with an average size three times the diameter of mammalian cells, the same saturation effect can be observed at about one third of the saturation field strength, observed for mammalian cells. This confirms the general expectations of current membrane charging models. Furthermore, protoplasts exhibit a strong asymmetry of the membrane charging at the hyper- and depolarized hemisphere of the cell, which occurs due to the higher rest potential compared to mammalian cells. The threshold transmembrane voltage for the onset of pore formation has been estimated to an absolute value of +/-200 mV. In addition, time- and field-strength-dependencies of the transmembrane potential's azimuthal distribution have been examined. Time-courses of the transmembrane potential in response to an electric field pulse show a good agreement with theoretical predictions. Results for HEK293, HeLa and 22Rv1 cells as well as for tobacco-protoplasts will be presented and the underlying measurement setup will be described.
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