Control by Low Levels of Calcium of Mammalian Cell Membrane Electropermeabilization.

Abstract

Electric pulses, when applied to a cell suspension, induce a reversible permeabilization of the plasma membrane. This permeabilized state is a long-lived process (minutes). The biophysical molecular mechanisms supporting the membrane reorganization associated to its permeabilization remain poorly understood. Modeling the transmembrane structures as toroidal lipidic pores cannot explain why they are long-lived and why their resealing is under the control of the ATP level. Our results describe the effect of the level of free Calcium ions. Permeabilization induces a Ca2+ burst as previously shown by imaging of cells loaded with Fluo-3. But this sharp increase is reversible even when Calcium is present at a millimolar concentration. Viability is preserved to a larger extent when submillimolar concentrations are used. The effect of calcium ions is occurring during the resealing step not during the creation of permeabilization as the same effect is observed if Ca2+ is added in the few seconds following the pulses. The resealing time is faster when Ca2+ is present in a dose-dependent manner. Mg2+ is observed to play a competitive role. These observations suggest that Ca2+ is acting not on the external leaflet of the plasma membrane but due to its increased concentration in the cytoplasm. Exocytosis will be enhanced by this Ca2+ burst (but hindered by Mg2+) and occurs in the electropermeabilized part of the cell surface. This description is supported by previous theoretical and experimental results. The associated fusion of vesicles will be the support of resealing.