Characterization of the mobile charges in the membrane ofValonia utricularis

Summary An attempt for the characterization of the mobile charges has been made by investigation of the voltage relaxations following a charge pulse at various temperatures between 2 and 40°C. The translocation ratek, the membrane conductivity 1/R _m and the total concentration of mobile chargesN _t within the membrane were calculated from recently developed theory (U. Zimmermann. K.-H. Büchner and R. Benz,J. Membrane Biol. 67:183–197, 1982). Data from 21 cells show that the concentration of mobile charges does not change significantly over a temperature range of 5 to 34°C, whereas both the translocation rate and the membrane conductivity reveal a strong but reversible temperature dependence. In the temperature range of 18 to 5°C,k decreases by a factor of 10 to 20, while between 18 and 34°C the increase ink is only two- or threefold with a maximum around 25°C. In principle, the same temperature dependence was observed for the membrane conductivity. Hysteresis effects occurring in the low temperature range as well as at high temperatures indicate that a protein component is involved in the mobile charge system. Furthermore, addition of cycloheximide leads to a marked decrease in both the translocation rate and the membrane conductivity, however, leaving the concentration of mobile charges almost unchanged. Therefore, it is assumed that the mobile charges are coupled to, but not part of a carrier protein.     

Dependence of the electrical breakdown voltage on the charging time inValonia utricularis

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. For a charging time of 420 sec the breakdown voltage is about 750 mV (18°C), a value that is in close agreement with earlier results obtained with current pulses (Coster & Zimmermann, 1975;J. Membrane Biol. 22:73). If the membrane is charged to the breakdown voltage in a shorter time, the breakdown voltage is found to be a function of the duration of the charge pulses. Whereas towards smaller pulse lengths down to 10 sec only a small, but significant, increase in the breakdown voltage is observed (1.1 V at 10 sec pulse length and 18°C), a strong increase in the breakdown voltage is found for even shorter charging times. For a pulse length of 800 nsec the breakdown voltage has a value of about 2.4 V (18°C) and a plateau seems to be reached for a pulse duration of 500 nsec. The influence of temperature on the breakdown voltage as observed for short charging times is very similar to that reported earlier for current pulses of 500 sec duration. For charge pulses of 1 to 2 sec duration the breakdown voltage decreases from 3.6 V at 3°C to 1.6 V at 25°C by more than a factor of two.Voltage relaxation studies in the low-field range suggest that the time constants of the two membranes arranged in series, tonoplast and plasmalemma, are similar. From this, it is suggested that both membranes show electrical breakdown, whereby the breakdown voltage of a single membrane is probably half the value of the total breakdown voltage. Its dependence on pulse length is therefore considered to be an intrinsic property of one single membrane. The strong dependence of the breakdown voltage on the charging time of the membrane further supports the interpretation of the breakdown phenomenon on the basis of the electro-mechanical model proposed earlier. In this model it is assumed that the electrical and mechanical compressive forces are counter balanced by elastic restoring forces within the membrane. However, towards very short pulses (less than 800 nsec), where a plateau seems to be reached, other processes may be generated by the application of the electric field. We discuss whether one of these processes is the ion movement through the membranes induced by a high electric field (Born energy).     

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 μs. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1 V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$. Between charging times of 300 ns and 5 μs at 25°C and between 100 ns and 2 μs at 40°C, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$ showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25°C) and from 1 to 0.4 V (40°C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns.The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25°C) and 100 ns (40°C) other processes (such as the Born energy) become possible.     

The breakdown of cell membranes by electrical and mechanical stress.

We attempted to determine whether mechanical tension and electrical stress couple to cause membrane breakdown in cells. Using cell-attached patches from HEK293 cells, we estimated the mechanically produced tension from the applied pressure and geometry of the patch. Voltage pulses of increasing amplitude were applied until we observed a sudden increase in conductance and capacitance. For pulses of 50 micros duration, breakdown required >0.5 V and was dependent on the tension. For pulses of 50-100 ms duration, breakdown required 0.2-0.4 V and was independent of tension. Apparently two physically different processes can lead to membrane breakdown. We could explain the response to the short, high-voltage pulses if breakdown occurred in the lipid bilayer. The critical electromechanical energy per unit area for breakdown by short pulses was approximately 4 dyne/cm, in agreement with earlier results on bilayers. Our data suggest that, at least in a patch, the bilayer may hold a significant fraction (approximately 40%) of the mean tension. To be compatible with the large, nonlytic area changes of patches, the bilayer appears to be pulled toward the pipette tip, perhaps by hydrophobic forces wetting membrane proteins bound to the glass. Although breakdown voltages for long pulses were in agreement with earlier work on algae, the mechanism(s) for this breakdown remain unclear.     

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.     

An investigation of electrical breakdown of bimolecular lipid membranes

The investigation is concerned with the irreversible electrical breakdown of bimolecular lipid membranes, depending on the velocity of linear voltage scanning. It was found that the membrane breakdown potential depended on the velocity of electric field variation. For instance, at voltage scanning velocities of up to 0.1 V/s, the rupture of membrane from glycerol monooleate occurs at 0.20–0.25 V and, at velocities higher than 1 V/s, at 0.5–0.6 V. Then the film breakdown depending on lipid phase transition was studied. At high velocities of imposed voltage scanning, the disruption of the bimolecular lipid membranes was shown not to depend on their phase states; at the same time, at low velocities, one could note a slight difference in the stability of the films at temperatures higher and lower than those of the phase transition. Whereas transition from gel to liquid-crystalline state involves transition from an ordered to a less ordered membrane structure with a sharp increase in the number of defects in the membrane, the authors, conclude that the film breakdown in the second case occurs by the ‘defect’ mechanism suggested earlier. It was also assumed that, in certain cases involving low velocities of voltage scanning, membrane breakdown may occur because of variation in the interfacial tension and in the contact angle between the film and torus. Possible mechanisms of the membrane irreversible electrical breakdown at high velocities of voltage variation are discussed. It was shown that breakdown should occur as a result of membrane compression in an electric field by a mechanism previously examined. The elastic moduli of a number of membranes were calculated by the breakdown criterion suggested earlier. They were found to coincide with the results of other investigators and, depending on the type of lipid, to equal 10⁵–10⁶ Pa.     

Electrical injury mechanisms: electrical breakdown of cell membranes

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.     

The effect of pressure on the electrical breakdown in the membranes of Valonia utricularis

The interpretation of electrical breakdown in terms of electro-mechanical instabilities, predicts that the breakdown potential should decrease with increasing cell turgor pressure.Experiments were conducted to test this hypothesis on cells of Valonia utricularis over a turgor pressure range of 0.5 · 10⁵–5.0 · 10⁵ N/m². Electrical breakdown was measured using intracellular electrodes and 500 μs current pulses. The pressure was monitored by an intracellular micropipette pressure transducer. The results obtained show a linear decrease in the critical breakdown potential with pressure. The effective compressive modulus of the cell membrane, γ, is calculated from the slope of this line to 69 ± 10 · 10⁵N/m² (average value of seven measurements). This is consistent with the theoretical prediction of the electromechanical model using our previously determined values of the elastic modulus of the membrane.A theoretical analysis is given of the effects of pressure on the breakdown. This includes also considerations of the indirect effect of pressure on the membrane via stretching of the cell wall with a possible coupling of such strains to the cell membrane. The results and analysis presented allow us to conclude on the basis of the experimentally determined breakdown P.D. of 959 mV that the region of membrane where electrical breakdown occurs is a dielectric with one of the following combinations of parameters: (A) a thickness δ = 7–9 nm with a dielectric constant ? = >10, e.g. a hydrated protein spanning the whole membrane. (B) δ = 4–5 nm with ? = 3–8, e.g. a lipoprotein of lipid bilayer dimensions. (C) δ ≈ 2 nm with ? = 2–3, e.g. a half lipid bilayer.If we assume that the breakdown P.D. of the tonoplast and plasmalemma are identical, that is 480 mV, then there is only one reasonable choice for the membrane thickness and the dielectric constant: δ = 2nm, ? = 3–8, e.g. a (lipo-)proteinaceous module facing a half lipid bilayer.     

On the effect of prestin on the electrical breakdown of cell membranes

The voltage-dependent activity of prestin, the outer hair cell (OHC) motor protein essential for its electromotility, enhances the mammalian inner ear's auditory sensitivity. We investigated the effect of prestin's activity on the plasma membrane's (PM) susceptibility to electroporation (EP) via cell-attached patch-clamping. Guinea pig OHCs, TSA201 cells, and prestin-transfected TSA cells were subjected to incremental 50 mus and/or 50 ms voltage pulse trains, or ramps, at rates from 10 V/s to 1 kV/s, to a maximum transmembrane potential of +/-1000 mV. EP was determined by an increase in capacitance to whole-cell levels. OHCs were probed at the prestin-rich lateral PM or prestin-devoid basal portion; TSA cells were patched at random points. OHCs were consistently electroporated with 50 ms pulses, with significant resistance to depolarizing pulses. Although EP rarely occurred with 50 mus pulses, prior stimulation with this protocol had a significant effect on the sensitivity to EP with 50 ms pulses, regardless of polarity or PM domain. Consistent with these results, resistance to EP with depolarizing 10-V/s ramps was also found. Our findings with TSA cells were comparable, showing resistance to EP with both depolarizing 50-ms pulses and 10 V/s ramps. We conclude prestin significantly affects susceptibility to EP, possibly via known biophysical influences on specific membrane capacitance and/or membrane stiffness.     

Dielectric breakdowm in the membranes of itValonia utricularis. The role of energy dissipation

The electrical properties of the membranes of Valoniautricularis were investigated using intracellular electrodes. Using short (0.5–1.0 ms) current pulses it was found that at a critical membrane potential difference of 0.85 V there was a large and discontinuous decrease in the membrane impedance and the slope resistance beyond this potential was virtually zero.The electrical breakdown of the membranes did not lead to global damage of the cells and after a resealing time of approx. 5 s could be repeated with identical results.Experiments with long current pulses and long bursts of pulses repeated at 1 kHz are described which show that the electrical breakdown is not due to thermal damage arising from localized heating in the membrane. Thus a dissipation of some 10³–10⁵ times the energy normally dissipated during the onset of breakdown did not lead to breakdown itself unless the critical membrane potential was exceeded.The results also show that punch-through and avalanche ionization are not likely to be important in the breakdown mechanism. The results are consitent, however, with there being a critical instability in the electro-mechanical stresses set up in the membrane at large electric field strengths.     

Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study

Summary Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50–200 sec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10⁸ to 0.1 cm²), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential differenceV _c. For 1m alkali ion chloridesV _c was about 1 V. The temperature dependence of the electrical breakdown voltageV _c is comparable to that being observed with cell membranes.V _c decreases between 2 and 48°C from 1.5 to 0.6 V in the presence of 1m KCl.Breakdown experiments were also performed with lipid bilayer membranes composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability.The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).     

Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes.

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.     

The breakdown of bilayer lipid membranes by dendrimers

The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.     

The electrical properties of synthetic membranes

By the addition of n-butyl bromide to a 1:19 copolymer of 4-vinylpyridine and styrene, water-insoluble, strong polyelectrolytes can be prepared. The addition of a hydrocarbon plasticizer permits the casting of flexible films in which large polycations are immobilized but in which bromide ions (or other small anions) are free to move. Electrical measurements on these membranes showed that they could be represented by a complex admittance: an electrolytic conductance in parallel with a pure A. C. impedance. The latter gives a circular arc when real component is plotted against imaginary. These synthetic membranes thus resemble in their electrical behavior that found by Cole for a variety of biological membranes.     

Contribution of electrogenic ion transport to impedance of the algae Valonia utricularis and artificial membranes.

The cell membrane of Valonia utricularis contains an electrogenic carrier system for chloride (Wang et al., Biophys J. 59:235-248 (1991)). The electrical impedance of V. utricularis was measured in the frequency range between 1 Hz and 50 kHz. The analysis of the impedance spectra from V. utricularis and its comparison with equivalent circuit models showed that the transport system created a characteristic contribution to the impedance in the frequency range between 10 Hz and 5 kHz. The fit of the impedance spectra with the formalism derived from the theory of carrier-mediated transport allowed the determination of the kinetic parameters of chloride transport through the cell membrane of V. utricularis, and its passive electrical properties. Simultaneous measurements of the kinetic parameters with the charge pulse method demonstrated the equivalence of both experimental approaches with respect to the evaluation of the translocation rate constants of the free and the charged carriers and the total density of carriers within the membrane. Moreover, the impedance spectra of the protonophor-mediated proton transport by FCCP (carbonylcyanide p-trifluoromethoxyphenyl-hydrazone) were measured in model membranes. The carrier system made a substantial contribution to the impedance of the artificial membranes. The analysis of the spectra in terms of a simple carrier system (Benz and McLaughlin, 1983, Biophys. J. 41:381-398) allowed the evaluation of the kinetic and equilibrium parameters of the FCCP-mediated proton transport. The possible application of the measurement of impedance spectra for the study of biological transport systems is discussed.     

Plasma membrane regeneration in the myxomyceteBadhamia utricularis

Summary Plasmodial strands of the myxomyceteBadhamia utricularis were injured and, after fixation and sectioning, examined using light and transmission electron microscopy. Injury results in the formation of a cytoplasmic droplet that rapidly regenerates a plasma membrane by the apparent fusion of vesicles at its surface. During the same time several structurally distinct layers form within the droplet. Some of these internal layers, which persist for several minutes after injury, may also arise from vesicle fusion. These results have differences from those reported for the relatedPhysarum polycephalum.