Membranes electric breakdown

In recent years work was started in which this sort of phenomena are smdied at the cell level, particularly the electrical breakdown of cell membranes and the fusion of neighboring cells under the influence of electric fields. These studies are of great value, in particular, for the further development of cell engineering and gene technology. 

In the following, a physical fusion method is described based on the electrical breakdown of membranes. No attempt is made to explain this procedure in full detail (for an extensive review see 61)), but rather a qualitative picture is given in order to provide a general understanding of the process. 

During the fusion process the relative surface area decreases with increasing volume indicating a loss of membrane material (about 22% in Fig. 51). In analogy to the fusion process of protoplasts it can be assumed that the excess lipid is removed in form of small, submicroscopic vesicles (Fig. 52). The electric breakdown in the membrane contact zone leads to the formation of several pores in which lipid molecules are randomly oriented (Fig. 52 b). The molecules reorient forming submicroscopic vesicles and the new membrane of the fused vesicle (Fig. 52c). Thus, fused giant liposomes should contain small, submicroscopic vesicles. This could possibly be proven by using fluorescence-labelled lipids for liposome fusion. 

Fig. 52a-c. Scheme of the fusion process of giant liposomes and the formation of small unilamellar vesicles (SUV) at the interface, a) lipid bilayers in contact b) pores generated by electric breakdown and lipid reorientation forming SUVs c) reconstitution of lipid membranes formation of a fused giant liposome and SUVs . 

Electrical breakdown of the membrane sets an upper limit on the fields that can be applied. Controlled electrical breakdown of the membrane is a fairly well understood process which is used in the techniques of electrofusion and electroporation but it is clearly undesirable in the devices under discussion. Surprisingly, fibroblasts can be cultivated for days with no apparent adverse effects in fields inducing trans-membrane potentials of 80% of the breakdown voltage (unpublished data). 

Bioelectric Organization of Nervous Tissue. The membrane potential of 70 mV is developed across the lipid bilayer of the cell membrane. This layer is approximately 40 8 thick, so that the transmembrane electric gradient is of the order of 105 V/cm. This extraordinary dielectric strength is not easily replicated in artificial materials. It is noteworthy that the resting membrane potential maintains this dielectric bilayer within a factor of two of electrical breakdown (19). Release of neural transmitter substances from synaptic terminals on the nerve cell surface transiently shifts the membrane potential at the site of release by a few millivolts. In terms of an altered transmembrane gradient, this shift is of the order of 1.0 kV/cm. 

Coster, H.G. and Zimmermann, U., Mechanisms of electrical breakdown in membrane of valonia utricularis, J. Memb. Biol., 22, 73, 1975. 

Crowley, J.M., Electrical breakdown of bimolecular hpid membranes as an electromechanical instabihty, Biophys. J., 13, 711, 1973. 

Y.A., Pastushenko, V.F., and Tarasevich, M.R. (1979) Electrical breakdown of bilayer lipid-membranes. 1. Main experimental facts and their qualitative disaission. Bioelectrochemistry and Bioenergetics, 6 (1), 37-52. 

Coster, H.G.L. and Zimmerman, U. 1975. The mechanisms of electrical breakdown in the membrane of Valonia utricularis. Journal of Membrane Biology 22 73-90. 

In contrast, pore models consider the pore-expanding forces that are parallel to the membrane and that arise from normal forces acting on pores as U is increased. Pore models can quantitatively account for both rupture and reversible electrical breakdown (REB) in planar membranes and reversible behavior in cells. For this reason, our discussion will emphasize localized electroconformational changes, particularly hydrophilic pores. The modeling of electroporation using such pores was first presented in an impressive series of seven back-to-back papers only the first paper is cited here (10). 

Reversible electrical breakdown (REB) is particularly striking. (REB is actually a rapid discharge due to the high ionic conductivity caused by the gentle structural membrane rearrangement of multiple pore formation.) In our models, subcritical pores (i.e., nonrupture-causing pores) are responsible for this high conductance state (II). Our first quantitative description of REB (33, 34) correctly predicted many key features of U(t) and G(t) but had... 

Figure 1A. Short time scale (0-1 pus) behavior of the transmembrane voltage [U(t)] predicted by a recent version of the theoretical model for a planar bilayer membrane exposed to a single very short (0.4-fis) pulse that is, charge injection conditions (16). The key features of reversible electrical breakdown (REB) are predicted by the model, as is the occurrence of incomplete reversible electrical breakdown. In the case of incomplete reversible electrical breakdown, the membrane discharge is incomplete because U(t) does not reach zero after the pulse. Each curve is labeled by the corresponding value of the injected charge Q. The curves for Q = 25 and 20 nC show REB, whereas the other...

It should be noted that an electric field promotes pore formation in the membrane. This has been clearly demonstrated in studiesof the electric breakdown of the BLM which involves formation and development of such pores. 

About 10 years ago, a new, easy and versatile technique for the introduction of larger macromolecules into eukaryotic and prokaryotic cells was established (Neumann et al., 1982 Knight, 1981) it is now commonly known as electroporation (Weaver, 1993). It is mainly a physical process, based on the transient permeabiliza-tion of cell membranes by pulses of sufficiently high electric fields. The underlying membrane phenomenon, called reversible electrical breakdown (REB) followed by transient pore formation, occurs if the transmembrane potential reaches values of 0.5 -1.5 V. Membrane pores are generated and molecules are transported through these pores by diffusion, electrical drift, and electroosmosis. Electroporation seems to be a rather universal process in most natural membranes. 

The study of ionic conduction, pore formers, defects, electric breakdown in membranes, their interaction and fusion, the influence of an... 

Experimental studies on a two planar bilayer model made it possible to describe in detail all the phases of fusion and confirm the main premises of the stalk mechanism (for so-called dry" membranes without a solvent). After the membranes are brought into close contact a trilaminary structure is formed spontaneously, and its lifetime depends on the field inside the membrane. By sending a voltage impulse, an electric breakdown can be caused, which brings about complete fusion. 

Benz, F. Beckers, and U. Zimmermann, Reversible electrical breakdown of lipid bilayer membranes a charge-pulse relaxation study,... 

F. Pastushenko, Yu. A. Chizmadzhev, and V. B. Arakelyan, Electric breakdown of bilayer lipid membranes. Calculation of the membrane lifetime in the steady-state diffusion approximation, Bioelectrochem. Bioenerget. , 6 53 (1979). 

Crowley JM (1973) Electrical breakdown of bimolec-ular lipid membranes as an eleedromechanical instability. Biophys J 13 711-724... 

Abidor IG, Arakelyan VB, Chemomoidik LV, Chizmadzhev Y, Pastushenko VF, Tarasevich MR (1979) Electric breakdown of bilayer lipid membranes. I the main experimental facts and their qualitative discussion. Bioelectrochem Bioenerg 6 37-52... 

Chemomordik LV, Sukharev SI, Popov SV, Pastushenko VF, Sokirko AV, Abidor IG, Chizmadzhev Y (1987) The electric breakdown of cell and lipid membranes the similarity of phenome-nologies. Biochim Biophys Acta 902 360-373... 

If two cells adhere to each other during the field pulse, electrical breakdown occurs in the contact zone between the two cells. If the membrane contact is close enough (1-2 nm), lipid molecules are able to diffuse from one membrane into the other forming bridges between both membranes. After turning off the field pulse, the fusion of the two cells into one sphere is energetically favoured. Thus, close contact of membranes has to be achieved before fusion can be induced by an electrical field pulse. 

The principle of electrofiision is the simultaneous electric breakdown of plasma membranes at the contact points with adjacent cells, when a short pulse of supercritical voltage for electric breakdown is applied to the cell pair. A lumen is formed between the coaxial pores at the breakdown site, which eventually enlarges to connect the cytoplasm of the fusion partner cells. The force involved in making cell-cell contact before, during and after pulse application, and the force causing the simultaneous membrane breakdown, can be calculated from the conductivities and dielectric constants of the cytoplasm, the membrane, and the external medium. When all parameters are known precisely, the outcome of electrofiision can be predicted with certainty. 

Okahata, Y Hachiya, S. Ariga, K. Seki, T. Functional capsule membranes. Part 22. The electrical breakdown and permeability control of a bilayer-corked capsule membrane in an external electric field. J. Am. Chem. Soc. 1986, 108, 2863-2869. 

Electroporation. When bacteria are exposed to an electric field a number of physical and biochemical changes occur. The bacterial membrane becomes polarized at low electric field. When the membrane potential reaches a critical value of 200—300 mV, areas of reversible local disorganization and transient breakdown occur resulting in a permeable membrane. This results in both molecular influx and efflux. The nature of the membrane disturbance is not clearly understood but bacteria, yeast, and fungi are capable of DNA uptake (see Yeasts). This method, called electroporation, has been used to transform a variety of bacterial and yeast strains that are recalcitrant to other methods (2). Apparatus for electroporation is commercially available, and constant improvements in the design are being made. 

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