A patch clamp study on the electro-permeabilization of higher plant cells: Supra-physiological voltages induce a high-conductance, K+ selective state of the plasma membrane

Abstract

Permeabilization of biological membranes by pulsed electric fields (“electroporation”) is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current–voltage relations of protoplasts derived from the tobacco culture cell line “Bright yellow-2”. Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1nS/pF) to a high-conductance state (~5nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around −250 to −300mV and +200 to +250mV, respectively. Current–voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5–10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25mM) did not affect currents elicited by 10ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced “regular” K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages.