Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse

Time courses of electropermeabilization were analyzed during the electric f ield application using a rapid fluorescent imaging system. Exchanges of cal cium ions through electropermeabilized membrane of Chinese hamster ovary ce lls were found to be asymmetrical. Entry of calcium ions during a milliseco nd pulse occurred on the anode-facing cell hemisphere. Entry through the re gion facing the cathode was observed only after the pulse. Leakage of intra cellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitiv e to the structural alteration of membrane, with cell membrane was asymmetr ical during electropermeabilization. Localized enhancement of the dye fluor escence was observed during and after the pulsation on the cell surface. Sp ecific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increase d during and immediately after:the pulse whereas the geometry of the staini ng was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membra ne on which structural alterations occurred was defined by the field streng th. The density of defects was governed by the pulse duration. Electroperme abilization is a localized but asymmetrical process. The membrane defects a re created unequally on the two cell sides during the pulse, implying a vec torial effect of the electric field on the membrane.