Electric field-mediated glycophorin insertion in cell membrane is a localized event

Purified soluble glycophorin, an intrinsic protein, can be back 'electroinserted' in the membrane of Chinese hamster ovary cells by submitting the cell/protein mixture to short electric field pulses. Previous studies showed that this complex between pulsed cells and proteins, which is detected only when the cell membrane is electropermeabilized, was very stable. This strongly suggested that the protein was indeed inserted in the membrane. The basic processes involved in this phenomena are studied in the present work. The association is observed at the single cell level by means of videoimmunofluorescence. Electric field-mediated insertion occurs firstly in a limited patch of the cell surface, which size is in agreement with the prediction of Electropermeabilization theory. A free diffusion of the inserted proteins then follows on the cell surface. The diffusion coefficient is computed to be less than 10(-10) cm2/s as observed for transmembranous proteins. This slow process gives an homogeneous distribution of the inserted protein.