Planta

Planta (1981)152:314-318

9 Springer-Verlag 1981

High electric field effects on the cell membranes of

Halicystis parvula

A charge pulse study R. Benz 1 and U. Zimmermann 2 1 Fakultfit ffir Biologie, Universit~it Konstanz, Postfach 5560, D-7750 Konstanz, and 2 Arbeitsgruppe Membranforschung am Institut ftir Medizin der Kernforschungsanlage Jiilich, Postfach 1913, D-5170 Jiilich, Federal Republic of Germany

Abstract. The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage Vc. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 gs and 100 ~ts. Below 10 ~ts the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 gs the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvuIa. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains). Key words: Cell fusion - Charge pulse technique Electrical breakdown - Halicystis - Membrane potential - Pulse length dependence.

Introduction A high conductance state of biological and artifical membranes is observed when the membranes are po-

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larized very rapidly to potential differences in the order of 1 V (Zimmermann et al. 1976; Benz et al. 1979). In analogy to similar phenomena in solid state physics, this high-conductance state has been termed electrical (dielectric) breakdown (Zimmermann et al. 1973). The changes in permeability and resistance of the membranes during breakdown are reversible. After a certain time the original resistance and permeability of the membrane are restored. The electrical breakdown can be repeated several times on a given artificial or biological membrane without irreversible changes of the membrane properties (Zimmermann et al. 1976; Benz et al. 1979). Electrical breakdown of membranes of suspended cells has been demonstrated to be an important tool for the encapsulation of normally impermeable lowmolecular weight substances and even macromolecules (such as proteins and DNA) within the cell (Zimmermann 1981). It was suggested that erythrocytes and lymphocytes, loaded with drugs by means of this electrical breakdown technique, could be used for targeting drugs to specific sites in the organisms for controlled drug release in time and space (Zimmermann 1981). Furthermore, we demonstrated recently that fusion could be induced by electrical breakdown of the cell membrane of two cells which were brought into close membrane contact by an alternating inhomogeneous field (Zimmermann and Scheurich 1981; Scheurich and Zimmermann 1981). The electrically-induced fusion process is synchronous, a preselected number of multipletes can be fused, and the yield is very high, up to 100%. The electric field intensity and duration (pulse length) required for breakdown of cell membranes are critical factors and vary considerably from species to species. In order to arrive at a general prediction for the optimum conditions for entrapment of impermeable substances and for cell-to-cell fusion, the pulse length dependence of the breakdown voltage, in particular,

R. Benz and U. Z i m m e r m a n n : Field effects in Halicystis

has to be known. So far, we only know that the breakdown voltage of artificial lipid bilayer membranes (made from oxidized cholesterol) and of the membranes of Valonia utricularis increases below a pulse length of about 10 ~ts, and reaches a plateau value below a pulse length of about 0.8 las (Zimmermann and Benz 1980; Benz and Zimmermann 1980). In this communication we describe the pulse length dependence of the membrane breakdown voltage derived from experiments on the marine alga Halicystis parvula. These algal cells are large enough for direct measurement of the breakdown voltage of the two membranes (tonoplast and plasmalemma, arranged in series) by using the charge pulse technique. The charge pulse technique can be used to take the membrane potential very rapidly to a desired value and the actual membrane potential can be measured within a short time after the end of the charge pulse (Benz and Lfiuger 1976; Benz et al. 1976). Cells of H. parvula exhibit a very low elastic modulus of the cell wall (Zimmermann and Htisken 1980; Graves and Gutknecht 1976). Thus, cells of H. parvula show a volume relaxation in response to external osmotic stress rather than a turgor relaxation (Zimmermann and Hiisken 1980). In this respect, H. parvula exhibits a behavior which may be more similar to that found in algal cells lacking a cell wall and in animal cells (Kauss 1978 ; Gimmler et al. 1977). Hence, the results from breakdown experiments should be more relevant for designing electrical entrapment and cell-to-cell fusion experiments in wall-less cells.

Material and methods H. parvula (Derbesia tenuissima) was obtained by courtesy of Dr. J. Gutknecht, Duke University, Marine Laboratory, Beaufort, N o r t h Carolina. The cells were maintained and cultivated at 17 ~ C in water from the N o r t h Sea supplied by the Biomaris Company, Bremen, F R G . A detailed description of the life cycle of H. parvula (D. tenuissima) may be found elsewhere (Graves and Gutknecht 1976; Gutknecht et al. 1978). Cells of the gametophyte stage were used for the investigations reported here. The cells were shaped like rotational ellipsoids with a cell volume between 30 and 50 m m 3 (and thus a surface area between 45 and 70 mm2). The normal turgor pressure of the cells used in this study measured with the pressure probe ( Z i m m e r m a n n and Steudle 1978) was about 0.3 to 0.5 bar and thus very low compared to other algal and plant cells. The pressure probe and the charge pulse technique are described in detail elsewhere ( Z i m m e r m a n n and Steudle 1978; Zimm e r m a n n and Benz 1980). For clarity, only a brief outline of the charge pulse technique is given here (see Fig. 1). A fast commercial pulse generator (Hewlett Packard 214 B) with a rise time of 10 ns and a m a x i m u m output voltage of 100 V at 50f2 was connected to the internal current electrode (platinized platinum wire) through a diode with a reverse voltage resistance larger than 10 l~ O. The actual voltage across the m e m b r a n e was measured using a second internal electrode (silver/silver chloride wire) connected to a Tektronic 7633 (7AI 3 amplifier, 1 M f2 input resistance,

315 voltage source Vo >> Vm

1 ,

fast electronic switch membrane capacitance Cm RE

resistance of electrolyte and electrodes

Rm membrane resistance

Vm Fig. 1. Principle of the charge pulse relaxation technique. The membrane capacity C,, is charged to an initial voltage V,, by a short current pulse through a fast electronic switch (output voltage Vo). At the end of the pulse, the external circuit is switched to a high resistance. The decay of the m e m b r a n e voltage with time is then only caused by conductance processes in the m e m b r a n e (relaxation time z = R.,. C.,)

band-width 80 MHz) storage oscilloscope. The external electrode (silver/silver chloride) of large area was used as both current and voltage electrode. Breakdown experiments were performed at constant output voltage between 3 and 100 V at 50 ~2 and at increasing charging times (pulse lengths) in a preselected pulse length range. The apparatus was tested carefully with d u m m y circuits as described earlier ( Z i m m e r m a n n and Benz 1980). The time resolution of the instrumentation was approx. 500 ns throughout all the experiments. The temperature was kept at 17 ~ C.

Results

When the two membranes, tonoplast and plasmalemma (which are arranged in series), are charged to very low voltages, say 2 to 200 mV, by a short charge pulse of 50 ns duration, the potential difference, V,,, built up across the membranes is a linear function of the injected charge, Q. This is expected by the well-known capacitor equation Q = C . 1/,,, whereby C is the membrane capacitance. The validity of this equation can also be approximately demonstrated by traces 1 and 2 in Fig. 2A, where a H. parvula cell is charged by charge pulses of 5 ~ts and 10 ~ts duration (injected charge 8.5-10 -8 A s and 1.6-10 -7 A s) to voltages of 320 and 560 mV, respectively. The initial voltage of trace 2 as compared to that calculated from the capacitor equation (600 mV) is a little lower, because the conductance of the two membranes cannot

316

R. Benz and U. Zimmermann: Field effects in Halicystis

Fig. 2A, B. Oscillographic records of breakdown experiments on a H. parvula cell (surface area 46 mmZ). A Five charge pulses of increasing length (5, 10, 20, 30 and 40 ps) were applied to the cell (injected charge between 8.5.10-SA s and 4.2.10-?A s). The upper trace corresponds to the voltage at the internal electrode during the charging process and represents a superimposition of five single pulses. Traces 1 to 5 reflect the corresponding voltage relaxations in response to the charge pulses (5 to 40 gs duration). The maximum voltage to which the membrane could be polarized (trace 4) indicates the breakdown. Vc=l V (30 gs); T=17 ~ C. B Four charge pulses of increasing length (1.5, 2.5, 4.5 and 6 gs) were applied to the same cell (injected charge between 1.6.10 .7 A s and 6.4.10-7 A s). The extrapolation of the discharge process to the end of the third charge pulse (2,5 gs duration) gave a maximum voltage (Vc) of about 1.9 V; T=17 ~ C

be neglected in the considered time range. The decay o f the m e m b r a n e voltage after the end o f the charge pulse as given by traces 1 and 2 (Fig. 2 A ) reflects the R C - t i m e constant o f the two m e m b r a n e s (whereby R is the total m e m b r a n e resistance). Whereas the relation between injected charge and resulting voltage is nearly linear for small charge injections (as predicted by the capacitor equation), there are very large deviations f r o m linearity at higher charge injections (see traces 3, 4 and 5 with injected charges o f 2 . 7 - 1 0 - 7 A s, 3.6-10 . 7 A s and 4.2.10 . 7 A s, respectively). U n d e r these experimental conditions electrical breakd o w n o f the m e m b r a n e s is induced, resulting in a d r a m a t i c decrease in m e m b r a n e resistance (from a b o u t 500 Q cm 2 to 1-2 f2 cm 2). Thus it is n o t possible to keep the m e m b r a n e charged and the initial voltage built up across the m e m b r a n e in response to the charge pulse decreases (see for example trace 5). The initial m a x i m u m voltage which c a n n o t be exceeded by increasing the a m o u n t of charge injected is defined as the b r e a k d o w n voltage, V~, of the cell membrane. V~ has, in the experiments presented in Fig. 2A, the value o f a b o u t 1 V (30 ps pulse length). Vo is not independent of the pulse length and increases with decreasing charging times. This can be seen f r o m Fig. 2B, where m e m b r a n e b r e a k d o w n o f the same algal cell was induced within 1.5 gs. The m a x i m u m voltage (trace 3) is a b o u t 1.5 V. However, because o f the delay time o f the measuring system, the m a x i m u m potential is underestimated. The initial

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Fig. 3. Breakdown voltage, Vc, as a function of the charging time of the membranes of H. parvula to breakdown. Vc is defined as the maximum voltage the membranes can be charged to. The data were taken from eight different cells; T= 17~ C value o f the m e m b r a n e potential o f the experiment shown in Fig. 2B is calculated to be a b o u t 1.9 V by extrapolation to zero time ( = end o f the charge pulse), assuming an exponential decay of the m e m b r a n e voltage during the time interval o f 1 to 2 l~s. The maxim u m initial voltage at a given pulse length is defined as the b r e a k d o w n voltage, Vc. Figure 3 shows the b r e a k d o w n voltage, Vc, of 8 cells measured at 17 ~ C as a function of the duration of the charge pulses. With a charging time o f 200 gs, a b r e a k d o w n voltage of a b o u t 600 mV is recorded. With smaller pulse

R. Benz and U. Zimmermann: Field effects in Halicystis lengths between 100 ~ts and 8 gs a strong increase of V~ (by about a factor of 3) is observed (from 0.6 to 1.9 V). Below pulse lengths of 0.8 gs, Vc seems to reach a plateau value of about 1.9 V. Discussion

The extreme values of the m e m b r a n e breakdown voltage of H. parvula at very short pulse lengths (ns-range) and at very long pulse lengths (10 to 100 gs-range) are similar to those measured with cells of V. utricularis ( Z i m m e r m a n n and Benz 1980). If we make the assumption that the two membranes exhibit the same breakdown voltage, the corresponding values of the breakdown voltage of a single membrane for very short and long pulse lengths would be 0.9 V and 0.3 V, respectively. Thus, the extreme values of the breakdown voltage of a single m e m b r a n e of cells of H. parvula agree well with the corresponding values of the breakdown voltage of artificial lipid bilayer membranes (Benz and Z i m m e r m a n n 1980). These values were determined to be 1 V (ns-range) and 0.4 V (10 las-range). The value of the breakdown voltage at long pulse lengths also agrees well with that determined for Fucus serratus (Gauger and Ben trup 1979). On the other hand, the pulse length dependence of the breakdown voltage of cells of H. parvula shows a quite different behavior from that of V. utricularis cells and of planar bilayer membranes made up of oxidized cholesterol. With these membranes, the dependence of the breakdown voltage on the charging time of the m e m b r a n e only occurs in a narrow pulse length range which is roughly between 0.8 gs and 10 gs, whereas the pulse length dependence of the breakdown voltage of the membranes of H. parvula cells is observed between 10 gs and 100 gs. There is a further remarkable difference between the breakdown behavior of membranes of V. utricularis cells and that of H. parvula cells. !In contrast to V. utricularis cells the membrane breakdown in H. parvula cells is much more clear-cut and rather resembles, in this respect, the breakdown behavior of planar lipid bilayer membranes. Comparison of the results obtained with bilayer membranes (ref. Benz and Z i m m e r m a n n 1980, Fig. 13) with those in Fig. 2 A and B clearly demonstrates that the change in resistance at the breakdown voltage is much more dramatic in H. parvula than in V. utricularis (ref. Z i m m e r m a n n and Benz 1980, Fig. 10). Thus, we can postulate that the breakdown in the membrane of H. parvula may predominantly occur in the lipid domain rather than at the lipid protein junctions, as was indicated for cell membranes of V. utricularis ( Z i m m e r m a n n et al. 1977). If this were true the mem-

317 brane structure or composition of these two species should differ considerably. On the other hand, it cannot be excluded at present that turgor pressure, which is different for both alga (normally 0.5 bar in H. parvula and 1.5 bar in V. utricularis), may have a direct influence on the electrical breakdown behavior of cell membranes. Finally we would like to point out that cell-to-cell fusion was found to occur only in certain narrow pulse-length ranges (at slightly higher electric field strengths than required for breakdown). The optim u m pulse-length range for the fusion of erythrocytes to form giant cells was 3 gs (Scheurich and Zimmermann 1981), whereas the optimum pulse length for fusion of plant protoplast cells was between 20 to 50 gs (Zimmermann and Scheurich 1981). The different o p t i m u m pulse-length ranges for fusion found so far may be partly explained by the fact that the pulse-length dependence of the breakdown voltage is different for these species. To predict the optimum conditions for fusion as well as for entrapment of impermeable substances, techniques must be developed to measure the pulse-length dependence of the breakdown voltage in smaller cells and protoplasts. The authors are very grateful to Dr. P. Lfiuger for many helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft(Sonderforschungsbereiche 138 (R.B.) and 160 (U.Z.). References

Benz, R., Beckers, F. Zimmermann, U. (1979) Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study. J. Membr. Biol. 48, 181-204 Benz, R., Lfiuger, P. (1976) Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique. J. Membr. Biol. 27, 171-191 Benz, R., Lfiuger, P., Janko, K. (1976) Transport kinetics of hydrophobic ions in lipid bilayer membranes. Charge pulse relaxation studies. Biochim. Biophys. Acta 455, 701 720 Benz, R., Zimmermann, U. (1980) Pulse-length dependence of the electrical breakdown in lipid bilayer membranes. Biochim. Biophys. Acta 597, 637 642 Gauger, B., Bentrup, F.W. (1979). A study of dielectric membrane breakdown in the Fucus egg. J. Membr. Biol. 48, 249-264 Gimmler, H., Schirling, R., ToNer, U. (1977) Cation permeability of the plasmalemma of the halotolerant alga Dunaliella parva Z. Pflanzenphysiol. 83, 145-148 Graves, J., Gutknecht, J. (1976) Ion transport studies and determination of the cell wall elasticity in the marine algae Halicystis parvula. J. Gen. Physiol. 67, 579-597 Gutknecht, J., Hastings, D.F., Bisson, M.A. (1978) Ion transport and turgor pressure regulation in giant algal ceils. In: Membrane transport in biology, pp. 125 174, Giebisch, G., Tosteson, D.C., Ussing, H.H., eds. Springer Verlag, Berlin Heidelberg New York Kauss, H. (1978) Osmotic regulation in algae. In: Progress in phytochemistry, Vol. 5, pp. 1 27, Reinhold, L., Harborn, J.B., Swain, T., eds. Pergamon Press, Elmsford, N.Y.

318 Scheurich, P., Zimmermann, U. (1981) Giant human erythrocytes by electric field induced cell-to-cell fusion. Naturwissenschaften 68, 45-46 Zimmermann, U. (1981) Cellular drug-carrier systems and their possible targeting. In: Targeted drugs, Goldberg, E., Donaruma, L., Vogl, O., eds. John Wiley and Sons, New York, in press Zimmermann, U., Beckers, F., Coster, H.G.L. (1977) The effect of pressure on the electrical breakdown in the membranes of Valonia utricularis. Biochim. Biophys. Acta 464, 399M16 Zimmermann, U,, Benz, R. (1980) Dependence of the electrical breakdown voltage on the charging time in Valonia utricularis. J. Membr. Biol. 53, 33~43 Zimmermann, U., Schulz, J., Pilwat, G. (1973) Transcellular ion flow in Escherichia coli B and electrical sizing of bacteria. Biophys. J. 13, 1005-1013

R. Benz and U. Zimmermann: Field effects in Halieystis Zimmermann and Stendle (1978) Physical aspects of water relations of plant cells'. Adv. Bot. Res. 6, 45-117 Zimmermann, U., Hf~sken, D. (1980) Turgot pressure and cell volume relaxation in Halicystis parvula. J. Membr. Biol. 56, 55-64 Zimmermann, U. Pilwat, G., Beckers, F., Riemann, F. (1976) Effects of external electrical fields on cell membranes. Bioelectrochem. Bioenerg. 3, 58-83 Zimmermann, U., Scheurich, P. (1981) High frequency fusion of plant protoplasts by electric fields. Planta 151, 26-32

Received 20 December 1980; accepted 23 February 1981

High electric field effects on the cell membranes of Halicystis parvula : A charge pulse study.

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically i...
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