title: Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells.
abstract: Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D002463:Cell Membrane Permeability; D003911:Dextrans; D004058:Diffusion; D004560:Electricity; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D014176:Protein Biosynthesis
chemical_list: D003911:Dextrans; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate