title: 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 (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, 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 (25 mM) did not affect currents elicited by 10 ms-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.
authors.affiliation: Karlsruhe Institute of Technology, Institute for Pulsed Power and Microwave Technology (IHM), Campus North, 76344 Eggenstein-Leopoldshafen, Germany; Karlsruhe Institute of Technology, Botanical Institute I-Molecular Cell Biology, Campus South, 76131 Karlsruhe, Germany. Lars.Wegner@ihm.fzk.de
authors.forename: Lars H
authors.initials: LH
authors.lastname: Wegner
authors.forename: Bianca
authors.initials: B
authors.lastname: Flickinger
authors.forename: Christian
authors.initials: C
authors.lastname: Eing
authors.forename: Thomas
authors.initials: T
authors.lastname: Berghöfer
authors.forename: Petra
authors.initials: P
authors.lastname: Hohenberger
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Frey
authors.forename: Peter
authors.initials: P
authors.lastname: Nick
mesh_terms: D000465:Algorithms; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D005942:Gluconates / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology*; D011523:Protoplasts / Q000166:cytology / Q000502:physiology*; D019789:Tetraethylammonium / Q000494:pharmacology; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology
chemical_list: D005942:Gluconates; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D019789:Tetraethylammonium; C030691:gluconic acid; D011188:Potassium