Patch Clamp in Use of Electroporation Mechanisms Studies

Abstract

Exposure of cells to strong pulsed electric fields shifts the membrane potential to values far outside the physiological voltage range and elicits membrane permeabilization, purportedly due to aqueous pore formation (“electroporation”). These pores allow passage of usually membrane-impermeable solutes, offering various biomedical applications. In this tutorial essay, the reader is introduced to patch clamp techniques (both “classical” manual patch clamp and more advanced automated versions). Benefits as well as potential drawbacks of these techniques are explained with respect to studying membrane properties at poorly studied “supraphysiological voltages” that are hardly accessible by any other method. Using a glass microelectrode, the cellular membrane is homogenously charged to defined voltages (known as “voltage clamp”). The current response elicited by a voltage pulse provides information on dielectric properties of the membrane and on membrane permeabilization by putative membrane pores. Recent scientific progress in this field is briefly summarized. Analysis of current voltage relations covering a broad voltage range allows to exactly determine the threshold potentials of pore formation (separately at both positive and negative voltages). In a more refined biophysical approach, data are interpreted in terms of a transition between a low-conductance, non-porated and a high -conductance, porated state of the membrane described by Boltzmann distributions. Ion selectivity of electropores can be probed by polarizing the membrane to induce pore formation and subsequently shifting the membrane voltage to opposite polarity at a fast, approximately constant rate (voltage ramp protocols). These experiments indicated cation selectivity of electropores. By using cations of various molecular diameter, a pore size of about 1.8 nm was extrapolated. Moreover, post-pulse recordings with the patch clamp technique revealed that the membrane remains permeabilized for a long time (in the range of minutes) after field exposure. Among other things, this effect is amplified by repetitive pulsing, suggesting that a memory effect is involved.