Transmembrane potential measurements of mammalian and plant cells

Abstract

Summary form only given. Pulsed laser fluorescence microscopy (PLFM) using the voltage sensitive dye ANNINE-6 permits measurements of transmembrane potentials of biological cells with a time resolution of five nanoseconds. Several theories have been proposed to explain the change of the membrane conductivity in response to an external electric field, e.g. pore-models, phase transitions due to electro-compression or local disturbances in the membrane caused by lipid rafts. The measured field-strength-dependence of the transmembrane potential of both mammalian and plant cells exhibits a saturation character which is supposed to occur due to the formation of nano/micropores. For mammalian cells with a typical diameter of 15 mum saturation effects set in at external field strengths in the order of 1 kV/cm. For tobacco-protoplasts (BY-2) with an average size three times the diameter of mammalian cells, the same saturation effect can be observed at about one third of the saturation field strength, observed for mammalian cells. This confirms the general expectations of current membrane charging models. Furthermore, protoplasts exhibit a strong asymmetry of the membrane charging at the hyper- and depolarized hemisphere of the cell, which occurs due to the higher rest potential compared to mammalian cells. The threshold transmembrane voltage for the onset of pore formation has been estimated to an absolute value of +/-200 mV. In addition, time- and field-strength-dependencies of the transmembrane potential's azimuthal distribution have been examined. Time-courses of the transmembrane potential in response to an electric field pulse show a good agreement with theoretical predictions. Results for HEK293, HeLa and 22Rv1 cells as well as for tobacco-protoplasts will be presented and the underlying measurement setup will be described.