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 Ca(2+) transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients ("Ca(2+) 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.