Membrane electroporation--fast molecular exchange by electroosmosis.
Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.
American Red Cross, Holland Laboratory/Cell Biology, Rockville, MD 20855.
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D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000818:Animals; D002463:Cell Membrane Permeability; D003911:Dextrans; D004560:Electricity; D004910:Erythrocyte Membrane / Q000502:physiology* / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005944:Glucosamine / Q000031:analogs & derivatives; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009994:Osmolar Concentration; D009995:Osmosis; D011817:Rabbits; D014743:Videotape Recording
D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; C015219:fluorescein isothiocyanate dextran; C045400:6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose; D009327:4-Chloro-7-nitrobenzofurazan; D016650:Fluorescein-5-isothiocyanate; D005944:Glucosamine