Cation selectivity of the plasma membrane of tobacco protoplasts in the electroporated state

AbstractCation selectivity of the cellular membrane of tobacco culture cells (cell line ‘bright yellow-2’) exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+40 or −40mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ±150mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ±600mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected (‘Intersection potential’). Using a modified version of the Goldmann–Hodgkin–Katz equation relative permeabilities to Ca2+ and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca2+>Li+>Rb+≈K+≈Na+>TEA+≈TBA+>Cl−. Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469–492) the effective average diameter of field induced pores was estimated to be about 1.8nm. Implications of these results for biotechnology and electroporation theory are discussed