PDF(Pubmed)
Abstract：
With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to -400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (\"Ca2+ plumes\") faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to -50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.
摘要：
经过50多年的电穿孔研究，细胞膜透化的性质仍然难以捉摸。分子模型中的电孔寿命仅限于纳秒或微秒，而电穿孔细胞的透化可以持续几分钟。这项研究旨在解决关于长期透化是否是由于细胞中长寿命孔的形成而引起的长期争论。我们开发了一种动态监测和电导测量单个电孔的方法。这通过在加载有CAL-520染料并放置在氧化铟锡(ITO)表面上的HEK细胞中的延时全内反射荧光(TIRF)成像来实现。应用1-ms，贴片移液管和ITO之间的0至-400mV脉冲诱发了确定单个电孔的局灶性Ca2+瞬变。一些瞬变在毫秒内消失，但另一些则持续超过一分钟。持续瞬变（“Ca2羽流”）随着时间的推移逐渐消失到稳定或随机波动的水平，其中可能包括完全静止的时期。单孔电导,用0到-50mV测量，在电穿孔后30和60s时50ms的步长，范围从80到200pS。这些实验证明了细胞中的电孔寿命，与分子模拟和脂质双层中的许多发现形成鲜明对比。
