TIME COURSES OF CELL ELECTROPORATION AS REVEALED BY SUBMICROSECOND IMAGING OF TRANSMEMBRANE POTENTIAL

Changes in the membrane conductance of sea urchin eggs, during the cou rse of electroporation, were investigated over the time range of 0.5 m us to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a re ctangular electric pulse of moderate intensity was applied across an e gg, a position-dependent potential developed synchronously with the pu lse, as theory predicts for a cell with an insulating membrane. From t he rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 muF/cm2 and the intracellular conductance 220 OMEGA . cm. Under an electric pulse of much higher intensity, the ris e of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequen t reversal of the potential development was ascribed to the introducti on of finite membrane conductance, or permeabilization of the membrane , by the action of the intense pulse (electroporation). Detailed analy sis indicated the following: already at 0.5 mus in the rectangular ele ctric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the o rder of 1 S/CM2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estima ted in a second electric pulse of a small amplitude. At least two reco very processes were distinguished, one with a time constant of 7 mus a nd the other 0.5 ms, at the end of which the membrane conductance was already <0.1 S/cm2.