Electro-permeabilization of cell membranes: Effect of the resting membrane potential

Abstract

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.