ID: 2195

  • Title:
    Proving the persistence of discrete electropores in live cells by tracking Ca2+ puffs and pore currents

    Silkunas, Mantas - Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA, 23508 and Institute for Digestive System Research, Lithuanian University of Health Sciences, 44307 Kaunas, Lithuania
    Pakhomov, Andrei G.- Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA, 23508

    In artificial lipid bilayers and molecular models, the lifetime of membrane pores after an electric shock is limited to nano- or microseconds. In contrast, permeabilized state of electroporated cells can last minutes. This discrepancy raises a question whether electropores are indeed responsible for the persistent membrane permeabilization, or if it is caused by a diffuse impairment of the membrane barrier function due to lipid oxidation or other mechanisms.

    An unequivocal answer to this would involve time-resolved observation of individual pores by optical microscopy or some alternative method. However, with electropore diameters in the nanometer range, they are below the resolution limit of optical microscopy. Instead, we employed a novel technique that combines the total internal reflection fluorescence (TIRF) microscopy to monitor “puffs” of Ca2+ entering the cell with the whole-cell patch clamp to control the transmembrane potential and measure pore currents. Human epithelial kidney (HEK 293) cells were cultured and imaged on glass coverslips with an electrically conductive but optically transparent indium tin oxide (ITO) layer. Electroporation was accomplished by applying brief hyperpolarizing pulses (1 to 25 ms, to at least -220 mV) between the patch clamp pipette and the ITO layer. Following electroporation, cells were kept at zero membrane potential, with periodic pulses to -50 mV to measure the membrane resistance. The cells were loaded with a Ca2+-sensitive fluorescence indicator Cal 520 through a patch clamp pipette. The extremely low field depth of TIRF imaging (<100 nm) enabled the efficient separation of discrete electroporation sites, not achievable by other microscopy methods. Time-lapse TIRF imaging at approximately 200 frames/s was synchronized with electroporation pulses and electric current measurements. While HEK cells typically do not express any voltage-gated ion channels, the pipette and bath solutions were formulated to block such channels if any were present.

    For the first time, we were able to observe and monitor individual membrane electroporation lesions in live cells. TIRF images of cells prior to electroporation showed no fluorescent features. Applying a brief pulse of a supra-physiological amplitude caused a rapid appearance of one or multiple bright spots, representing discrete points of Ca2+ entry into cell. Their brightness declined with time, but some Ca2+ puffs persisted over a minute and kept changing their brightness in response to test voltage steps. A single lesion electrical conductance could be in the nanosiemens range immediately after the electroporation, and dropped with time to 80-200 pS. These values fall within a conductance range for most endogenous ion channels, consistent with expectations that each fluorescence puff reflects Ca2+ flow through a single, discrete electropore.

    Our results unequivocally demonstrate that discrete electroporation lesions (membrane pores) can stay open in live cells for tens of seconds after an electroporation shock. We also measured the kinetics of individual pore conductance changes with time and their dependence on the electroporation voltage.

    membrane, patch clamp, electroporation, TIRF microscopy


    Topic 1:
    2. Biophysics and biochemistry of interaction mechanisms

    Topic 2:
    1. Biological responses (molecular, subcellular, cellular and intercellular)

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