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Electromediated permeabilization of frog skeletal muscle cell membrane: Effect of voltage-gated ion channels
Bioelectrochernistry
and Bioenergetics,
157
34 (1994) 157-167
Electromediated permeabilization of frog skeletal muscle cell membrane: effect of voltage-gated ion channels Wei Chen and Raphael C. Lee Department of Surgery, Section of Plastic and Reconstructive Chicago, IL 60637 bWA)
Surgery, The University of Chicago, 5841 South Maryland Avenue,
(Received 24 March 1994)
Abstract The asymmetrical electromediated permeabilization of cell membranes of frog skeletal muscle fibers with respect to the stimulation pulse polarity were studied using an improved double Vaseline gap voltage clamp. Channel-mediated currents in response to a variety of high voltage electrical pulses have been differentiated from the pulse-induced electroporated leakage currents induced in the cell membranes. Under voltage clamp conditions, electroporated current as a percentage of the total transmembrane currents and the ratios of the membrane conductances responding to positive electrical pulses over negative electrical pulses have been quantified. These results imply that protein channels have a protective effect, mediating some of the changes to the phospholipid membrane from electroporation by adjusting the membrane conductance. Our hypothesis states that, when skeletal muscle fibers are exposed to a high intensity electrical field in real life, the increment in membrane conductance due to ion channels opening on the depolarized side of the cell membrane initiates a redistribution of the membrane potential differences across the two opposed membranes. The field-induced voltage drop across the membrane hemisphere facing the positive electrode is much higher than that across the hemisphere facing the negative electrode, which results in unequal electromediated permeabilization of the two hemispheres..
1. Introduction The use of external electric fields to destabilize the plasma membrane, a process known as electroporation, has been widely studied. Electroporation has been used to open transient pores temporarily in specific regions of the cell membrane in order to transfect a variety of molecules such as DNA, proteins and antibodies [l-4]. Recently, electroporation has been postulated as a mechanism of electrical injury [5]. There is a growing interesting in the clinical diagnosis and treatment of electroporated cell membrane in electrically injured patients. Since the larger the cell, the higher the field-induced voltage drop across the cell membrane [61, electromediated permeabilization often appears on large excitable cells, such as skeletal muscles and nerve cells, as opposed to the thermal component of electrical injury. Previously, investigators assumed that during exposure to an external high intensity electrical field the field-induced potential differences across the two hemispherical cell membranes surrounding the cell were symmetric, but with opposite polarity. Recently, 0302-4598/94/$7.00 SSDZ 0302-4598(94)05011-I
however, several investigators have found that the electric-field-induced electroporation on these two opposite hemisphere membranes is asymmetric [7-111. With a few exceptions [12], these investigators reported that any membrane area of a cell which is closer to a positive electrode will show more evidence of electroporation than a membrane area closer to a negative electrode. Several hypotheses have been developed to explain this phenomenon. Since a large potential difference across a cell membrane is considered as the cause of electroporation, other investigators [lo] have suggested that the physiologic resting membrane potential, which is vectorially added to the external-field-induced membrane potential, results in asymmetrical electroporation on the spatially opposed membranes. Sowers [9] has postulated that the phenomenon of electro-osmosis may explain asymmetrical transport. It was also shown that it could be due to an artifactual process [9,12]. Most of these investigations involved optical dye measurements of non-excitable cells, such as nucleated plant cells, liposomes or erythrocyte ghosts. The phenomenon of asymmetrical membrane electroporation 0 1994 - Elsevier Science S.A. All rights reserved
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W. Chen, R.C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
was only observed after high voltage electrical stimulation. In other approaches, the voltage clamp technique has been used for decades to study the electrical mechanisms of cell membranes. Recently, it has also been employed to investigate electromediated permeabilization in cell membranes [6,13]. We introduced a modified double Vaseline gap voltage clamp to study the destructive effects on skeletal muscle fibers by an external high intensity electrical field [6]. The improved voltage clamp technique makes it possible to study changes in the transmembrane current both before and after electrical pulses, as well as to monitor transient changes in transmembrane current continuously during the pulses. The improved voltage clamp allows us to resolve the transmembrane current into its components and to differentiate membrane conductance changes attributed to ion channels opening from the electromediated permeabilization of cell membrane. In our laboratory we have utilized the improved double Vaseline gap voltage clamp to observe asymmetrical electromediated permeabilization in cell membranes of frog skeletal muscle fiber during exposure to high voltage pulses. The external electrical pulse that hyperpolarizes the cell membrane introduces much larger transmembrane leakage current than the pulse that depolarizes the membrane. Our hypothesis is that in the actual situation of muscle fiber exposure to a high intensity electrical field, the increment in membrane conductance due to ion channels opening on the depolarized side of the cell membrane initiates a redistribution of membrane potential differences across the two opposed membranes. The external-field-induced membrane potential on the depolarized membrane hemisphere facing the negative electrode is much lower than that of the hyperpolarized hemisphere facing the positive electrode. Therefore less electroporation occurs on the depolarized membrane hemisphere. The present study was designed to clarify the electromediated permeabilization of the asymmetrical membrane during high voltage electrical pulses, to differentiate the voltage-gated ionic channel currents from the membrane electroporated currents and to test whether opening of the voltage-gated ion channels in cytoplasmic membrane alters membrane conductance and reduces the external-field-induced membrane potential, thus protecting the cell membrane from electroporation. 2. Materials and methods 2.1. Cell preparation and equipment set-up Voltage clamp experiments using an improved double Vaseline gap chamber to study electroporation of skeletal muscle cells have been described previously
[6]. Cut fibers were dissected following the procedure used by Kovacs et al. [14], Irving et al. [15] and Hui et al. [16]. Briefly, single muscle fibers were hand-dissected in a high K+ relaxing solution from twitch skeletal muscle, the semitendinosus of the English frog Rana temperoria. The fiber was mounted in a custommade chamber filled with the relaxing solution and was isolated across three distinct pools by two Vaseline seals. Clips on the bottom of the chamber held the fiber in place. The cell membrane of the fiber segments in the two end-pools was chemically permeabilized by treatment with 0.01% saponin for 2 min in order to ensure that electrical and ionic transfer could take place between the interior and exterior of the cells. The solution in the two end-pools was then replaced by “internal solution” which mimics the composition of the cytoplasmic fluid [6,16]. The central pool was filled with either normal Ringer’s solution or an “external solution” which contained both Na+ and K+ channel blockers, tetrodotoxin (TTX) and tetraethanoammonia (TEA). Six agar bridges and six Ag-AgCl pellets were used to connect the solutions in the chamber to the electrical circuit. The following solutions were used. (1) The relaxing solution contained 120 mM potassium glutamate, 1 mM MgSO,, 0.1 mM K,EGTA and 5 mM K,PIPES (pH 7.0). (2) The internal solution contained 45.5 mM potassium glutamate, 20 mM Tris-creature phosphate, 20 mM EGTA, 5.5 mM Na,ATP, 5 mM glucose, 5 mM K,PIPES and 6.8 mM MgSO,. (3) The external solution contained 120 mM TEA Cl, 2.5 mM RbCl, 1.8 mM CaCI,, 2.15 mM Na,HPO,, 0.85 mM NaHPO, and 1 PM tetrodotoxin (pH 7.1). TEA- and Rb+ in the external solution were used to minimize Kf currents and TTX was used to block Na + channels. A Dagon Whole Clamp 8800 controlled by an American Wells 486 personal computer was used to generate and send pulse signals to the fiber in the chamber. Signals from the voltage clamp were digitized by a Tektronix 11401 digital oscilloscope and stored in the hard disk of the computer. A Data Translation ACIIC general purpose interface board (GPIB) was used as the computer interface. 2.2. Electrical measurements Cell membranes were stimulated by a series of square pulses of magnitude ranging from 120 to 600 mV and different polarities (top panels in Figs. l-7). Simultaneously, the corresponding transient transmembrane currents were recorded during each stimulation pulse. To save space, the term membrane potential difference is simplified to membrane potential in this
W! Chen, R C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
paper. It is necessary to emphasize the difference between the magnitude of the pulse applied to the cell membrane and the magnitude of the membrane potential which is the vectorial sum of the pulse magnitude and the membrane rest potential. There are several components in the transmembrane currents recorded in response to the high voltage electrical pulse: (1) leakage currents beneath the Vaseline seals, (2) the membrane capacitance current, (3) the normal membrane leakage current, (4) the pulseinduced electroporated leakage currents and (5) the ionic channel current. To distinguish the pulse-induced leakage current and the ionic channel currents from the total recorded current, a template current consisting of components (11, (2) and (3) was generated using the following procedure. Before exposure to each high voltage pulse, the cell membrane was held at - 110 mV for 20 ms and then a group of N prepulses was delivered for the same period as the subsequent high voltage pulse. The magnitude of each prepulse was l/N that of the subsequent pulse. The criterion for the choice of N is that the membrane potential produced by the prepulses is lower than the threshold of open ionic channels. Therefore the transmembrane current responding to the prepulse consists of only the membrane capacitance and normal resistance currents. The sum of these prepulse currents served as the template current for the membrane capacitance and normal resistance currents with respect to the subsequent high voltage pulse current. After subtracting the template current from each high voltage pulse current, a pulseinduced electroporated leakage current, an ion channel current or a combination of the two can be identified depending upon the experimental solution and the polarity of the high voltage pulses. In what follows the pulse-induced current is defined as the current remaining after subtraction of the template current from the total current response. To avoid any post-pulse sealing effect of the electromediated pores in the cell membrane, all current traces discussed in this study were recorded during the high voltage electrical pulse. 2.3. Membrane bilayer versus channel proteins In order to evaluate the effects of ion channels in protecting the cell membrane from electroporation, the pulse-induced electroporated leakage current must be differentiated from the ion channel currents. To do this, it is necessary to distinguish reference membrane voltages related to the membrane bilayer from those related to ion channels. The phospholipid membrane, which is the major component of the cell surface, is organized with the hydrophobic tails of the phospholipid structure on the inside of the membrane and the
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hydrophilic heads facing outward. The amphipathic structure of the phospholipid membrane maintains the integrity of a hydrophobic permeability barrier in close proximity to an aqueous medium. Unlike ion channels, which have a high ionic selectivity and a clear rectification effect on channel-based currents, the phospholipid-bilayer-based membrane current is neither ion selective nor current-direction specified. The externalfield-induced phospholipid-bilayer-based currents should not depend on the polarity of the applied electric field, i.e. the reference voltage of the phospholipid membrane with respect to a pair of symmetrical external electrical fields should be zero. In contrast, the asymmetric structures of the voltage-gated ion channels in the cell membrane result from the pattern in which proteins are embedded in the phospholipid membrane. Most types of voltagegated ion channels show ionic selectivity and rectifier channel currents. Because of the various ion selectivities of different kinds of channels and the electrochemical force due to different ion concentrations across the cell membrane generated by Na+-K+ ATPase pumps, the equilibrium membrane potential in the resting condition is -90 mV for natural intact fibers of frog skeletal muscle. To mimic the real situation, in our experiments cell membranes were always held at -90 mV except during the stimulation period. Any positive change in membrane potential from -90 mV can depolarize the cell membrane. If the cell membrane is depolarized at a higher potential than the threshold value, the voltage-gated channels will open. Any negative changes in membrane potential from - 90 mV that hyperpolarize the cell membrane will not open the voltage-gated ion channels, such as the Na and the delayed rectifier K channels. Therefore in order to study the effects of pulse polarity on the voltage-gated ion channels, the reference voltage must be at the rest potential or the holding potential (- 90 mV). In the present study, the cell membrane potential was held at - 90 mV and was then exposed to one of two groups of pulses: (1) pulses of the same magnitude and opposite polarities with respect to zero, and (2) pulses of the same magnitude and opposite polarities with respect to the membrane rest potential (- 90 mV>. The transmembrane current responses were simultaneously monitored for all these pulses. This technique makes it possible to separate pulse-induced transmembrane currents, caused by electroporation of the phospholipid membrane, from the opening of the voltage-gated ion channels. 2.4. Experiment protocol In most experiments, the external environment in the central pool was normal Ringer’s solution. High
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voltage pulses of various magnitudes were delivered across the cell membrane using the voltage clamp. The time interval between two pulses varied from 3 min, when the pulse magnitude was below the threshold of membrane electroporation, to 5 min when it was above the threshold, and to 10 min when it was much higher than the threshold. The criterion for choosing the time interval was that the post-pulse holding current, which is defined as the transmembrane current when the membrane is held at the holding potential (-90 mV), fell to the original value before further high voltage pulses were applied. In some experiments, high voltage pulses were applied to cell membranes when the external solution in the central pool contained both the Na+ and K+ channel blockers TEA and TTX. All high voltage pulses had a duration of 4 ms in order to mimic the effects of a half-cycle of the commercial powerline. A pulse of duration 4 ms at the RMS value and a half-cycle sinusoid current at commercial power frequency (60 Hz) have the same energy. To reduce the number of stimulation pulses and maintain a healthy fiber, the pulse magnitudes were carefully chosen as follows. (1) High voltage pulses of four different magnitudes were used: 120 mV, which is below the threshold voltage for membrane electroporation; 300 mV, which is slightly above the electroporation threshold [17]; 480 mV, which is near the damage threshold for the voltage-gated K+ channel [17]; 600 mV, which is higher than both the membrane electroporation and the ion channel damage thresholds [17], but not high enough to induce permanent change in either the phospholipid membrane or the protein channels. Each of the four groups consisted of two pulses of the same magnitude but opposite polarities. (2) Except for the last group, the differences between the pulse magnitudes of two sequential groups were twice the rest potential. The advantage of this is that while the pair of pulses in each group have the same magnitude with opposite polarities, the pair of pulses in sequential groups retain the same membrane potential with opposite polarities. In other words, the first pulse pair holds the membrane potential at opposite polarities with respect to the reference voltage at the rest potential and the second pulse pair with holds the membrane potential at opposite polarities with respect to the reference voltage at zero. 3. Results and discussion Comparison of the stable holding current following a positive pulse with the significantly increased holding current following a negative pulse immediately shows
that cell membranes can tolerate a higher positive membrane potential than a negative potential without evidence of electroporation. Because of this phenomenon, a positive pulse was always applied to the cell membrane prior to a negative pulse in each pulse group in order to maximize the life of each fiber. 3.1. Electroporation of the membrane bilayer When normal Ringer’s solution in the central pool was replaced by an external solution containing 120 mM TEA *Cl and 1 PM TI’X, most of the voltage-gated Na+ and K+ channels were blocked so that it was possible to concentrate on the electroporation of the membrane bilayer. Under these conditions, the reference voltage with respect to the symmetrical pulses in all four pulse groups should be zero. The first pulse was applied to the cell membrane to hold the membrane potential at +510 mV. The recorded total transmembrane current is shown in Fig. l(b). After 5 min, the current response to a negative pulse holding the membrane potential at -510 mV was also recorded and is shown in Fig. 2. Since the voltage-gated ion channels were blocked, these current traces consisted of the membrane capacitance current, the normal leakage current and the pulse-induced electroporation current. The directions of the two total current responses were opposite. Because the capacitance and normal resistance currents were linearly dependent on the pulse magnitude and independent of the pulse polarity, the difference in the pulse magnitudes (600 mV versus 420 mV) resulted in both the shapes and the magnitudes of the two response current traces being different (Figs. l(b) and 2(b)). After removing the membrane capacitance and resistance currents by subtracting the template currents, the remaining current can be attributed to the electromediated pores on the cell membrane. These currents are shown in the lower panels of Figs. l(c) and 2(c). Interestingly, the pulse-induced leakage currents responding to the two pulses that held the cell membrane at a potential of equal value (-600 mV), but opposite polarity, were very similar. This similarity is important evidence that the two sides of the phospholipid bilayer are electrically and statistically symmetrical. Identical stimulation pulses with opposite polarities cause almost equal amounts of electroporation on the membrane bilayer. The negligible difference occurred because not every channel was blocked. It should be noted that, under the voltage clamp conditions, the membrane potential was held according to the command pulse regardless of any change in cell membrane, such as ion channel opening. In other words, because ion channel opening does not influence membrane potential, electroporation is not affected
WOChen, R. C. Lee / Electromediated
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either. Thus the electromediated pore currents responding to a pair of pulses will be equal if the membrane potential is clamped at the same value but opposite polarities by the two pulses. This result will be used in the rest of the paper to isolate the electroporation current from the recorded total transmembrane current responding to a positive high voltage pulse when no channel blockers are used to block the voltage-gated channels. 3.2. Response of transmembrane currents to symmetrical pulses with different reference voltages The external solution used for the remaining experiments was normal Ringer’s solution without any channel blockers. Fig. 3(b) shows the recorded total transmembrane current trace responding to a pulse of - 120 mV with
-2000 Fig. 2. As Fig. 1 except that the stimulation pulse is negative. (a) A negative stimulation pulse of -420 mV. Since the intrinsic rest potential was -90 mV, the actual membrane potential across the cell membrane was - 510 mV during the stimulation pulse. (b) The transmembrane current in response to the stimulation pulse. The external solution in the central pool is the same as that used in Fig. 1, containing TTX and TEA. (c) the isolated electrically mediated leakage current induced by the stimulation pulse. Capacitance and resistance currents have been subtracted using a template current obtained from a group of prestimulation pulses. Since the stimulation pulse hyperpolarized the cell membrane, no voltage-gated ion channels were open.
Fig. 1. Transmembrane current under a voltage clamp. (a) A stimulation pulse of magnitude +600 mV and duration 4 ms. (b) Total transmembrane currents recorded during the stimulation pulse. The cell membrane was held at a potential of +510 mV (initial rest potential, -90 mV). The solution in the central pool is an external solution containing the Na+ and K+ channel blockers TTX (0.1 PM) and TEA.Cl (120 mM). (c) Isolated pulse-induced membrane electroporated leakage current obtained after removal of the membrane capacitance current and the normal membrane leakage current. The narrow spikes (also visible in some later figures) along the two edges of the stimulation pulse are due to experimental variability in the alignment of the total and the template currents during subtraction.
the membrane potential held at - 210 mV. This potential is lower than the threshold of formation of electromediated pores on cell membranes [6]. This can be demonstrated by removing both the membrane capacitive and normal resistive currents by subtracting the template current from the total current response. Because of hyperpolarizing cell membranes, most of the Na+ and K+ channels are closed. The pulse-induced electroporated leakage current is almost zero, and is shown as the remaining current in Fig. 3(c). The next pulse was +300 mV. Thus the cell membrane was held at + 210 mV. The total transmembrane current responding to this depolarizing pulse is shown in Fig. 4(b). The spike at the rising edge with an exponential decay represents the membrane capacitance current. After the spike, the transmembrane current increases until the end of the pulse. The current remaining after subtracting the membrane capacitive and normal resistive currents is shown in Fig. 4(c).
W. Chen, R C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
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This is the sum of the voltage-gated ion channel currents and the electromediated membrane leakage currents, if any exist. It is not certain how much of this current can be attributed to the pulse-induced membrane leakage current. Fortunately, as mentioned previously and supported by the results shown in the Fig. 1, the two sides of the phospholipid bilayer are electrically and statistically symmetrical. Thus identical stimulation pulses with opposite polarities cause almost equal electroporation on the membrane bilayer. In other words, a pair of symmetrical pulses with a reference voltage of zero should introduce the same amount of electromediated leakage currents. Therefore the contribution of membrane electroporated currents in Fig. 4(c) should be equal to those responding to a pulse holding the membrane potential at -210 mV, which was recorded and is shown in Fig. 3(c). The negligible current in Fig. 3(c) indicates that the contribution of the electroporated current to the trace in Fig. 4(b) is almost zero. Therefore all currents in Fig. 4(c) can be attributed to the channel-mediated currents. These results imply that a pulse of magnitude of +300 mV
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Fig. 3. A pulse with a magnitude of - 120 mV: (a) cell membrane held at -210 mV, (b) capacitance and normal leakage currents subtracted from the total recorded transmembrane currents showing that this membrane potential is still below the electroporation threshold; (c) the remaining current is almost zero which demonstrates that no electroporation occurred.
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Fig. 4. (a) A positive pulse of 300 mV, which held the cell membrane at a potential of +210 mV, (b) the responding transmembrane current recorded during the stimulation; (c) remaining current after subtracting the capacitance current and normal leakage current. These currents are channel mediated currents, because there was no pulse-induced leakage current responding to a pulse holding the membrane potential at - 210 mV (see text).
cannot destabilize the integrity of the membrane structure. The total transmembrane current responding to a stimulation pulse of -300 mV was shown in Fig. 5(b). Because of the intrinsic membrane rest potential of -90 mV, the cell membrane was actually held at a potential of - 390 mV, which is higher than the threshold of electroporation of cell membranes [17]. Then Fig. 5(c), which was obtained by subtracting the membrane capacitance and normal resistance currents, should be the sum of the pulse-induced electroporated leakage currents and the ionic channel current, if it exists. Since the cell membrane was hyperpolarized, most of the voltage-gated Na+ and K+ channels did not open, and hence the current trace in Fig. 5(c) represents primarily the pulse-induced electroporated leakage current across the cytoplasmic membrane. It is now possible to discuss the pulse-induced membrane conductance with respect to the same pulse magnitude and opposite polarity. Because the pulse magnitudes are the same, the pulse-induced membrane conductances are proportional to the pulse-induced
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currents shown in Figs. 4(c) and 5(c). Since we are concerned here with a comparison of the effects of pulse polarity, we only need to consider the ratio of the pulse-induced conductance of the depolarized membrane to that of the hyperpolarized membrane. This can easily be calculated using the largest electromediated currents in Figs. 4(c) and 5(c) (an average of the last 20 data points at the end of the pulse). When the pulse magnitude is 300 mV, the membrane conductance ratio is 1.8: 1. Using a pair of pulses of magnitude 480 mV and opposite polarity, the cell membrane can be held at either +390 mV or -570 mV, as shown in Figs. 6(a) and 7(a) respectively. Both these membrane potentials are high enough to electroporate cell membranes. The total current responses to these opposite pulses were recorded and are shown in Figs. 6(b) and 7(b). The isolated pulse-induced transmembrane current of the depolarized cell membranes (membrane potential at +390 mV>, obtained by subtracting the membrane capacitance and normal leakage currents, is shown in Fig. 6(c). It is a combination of the channel currents
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Fig. 6. (a) Positive simulation pulse of 480 mV, (b) total transmembrane current recorded during the stimulation in which the membrane was held at a potential of +390 mV; (c) remaining current after subtracting the capacitance and resistance currents. The current in (c) represents a combination of ion channel currents and electroporated membrane current.
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Fig. 5. (a) A negative stimulation pulse of - 300 mV. The membrane potential was actually - 390 mV because of the vector summation of the stimulation pulse and an intrinsic membrane rest potential of -90 mV. (b) The total transmembrane current: since the pulse hyperpolarized the cell membrane, no voltage-gated ion channels were open. (c) The current obtained by subtracting the template currents, which is mainly electroporated membrane current.
and the electromediated electropore currents. With the same token, the pulse-induced electroporation current corresponding to a membrane potential of +390 mV shown in Fig. 6(c) is the same as the pulse-induced leakage current corresponding to - 390 mV (Fig. 5(c)). The channel-mediated currents in Fig. 6(c) can be determined by subtracting the pulse-induced leakage currents shown in Fig. 5(c). Comparison of Figs. 5(c) and tic> shows that about 40% of the pulse-induced current can be attributed to the electroporated leakage current and 60% to the channel-mediated current. In contrast, the current shown in Fig. 7(c) represents only the electromediated permeabilization current of cell membrane, since most voltage-gated ion channels are not opened by the hyperpolarizing pulse. Comparison of Figs. 5(c) and 7(c) shows that the pulse-induced leakage current corresponding to a pulse of +480 mV is only about 30% of that corresponding to a pulse of -480 mV. Again, using the method described in the previous paragraph, the ratio of the pulse-induced membrane conductance of the depolarized membrane to that of the hyperpolarized membrane for a pulse of magnitude 480 mV is about 3.4 : 4.7.
W. Chen, R.C. Lee / Electrornediated permeabilization of frog skeletal muscle cell membrane
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Fig. 7. (a) Negative stimulation pulse of -480 mV that held the membrane at a potential of 570 mV, (b) total transmembrane current is shown; Cc) the remaining current obtained by subtracting capacitance and normal leakage currents which, since the pulse hyperpolarized the cell membrane, was primarily membrane electroporation current.
3.3. Pulse-dependent percentages of the electroporated currents among the total transmembrane currents responding to different positive pulse magnitudes Fig. 8 was drawn by combining all the percentages of the pulse-induced leakage current with respect to the total electromediated currents corresponding to positive pulses of different magnitudes. Each point represents a percentage corresponding to a positive pulse. This percentage starts from zero when the stimulation pulse is below the threshold of membrane electroporation. It then increases with increasing pulse magnitude, but does not reach 100% except when all protein channels are damaged. It is important to note that these results were obtained using a voltage clamp, where the membrane potential was constrained regardless of changes in the membrane characteristics. This result shows that the voltage-gated ion channels tend to prevent the formation of electromediated pores in the phospholipid membrane bilayer. 3.4. Electroporation on cell membranes in a real situation of electrical injury In the previous section we discussed the percentages of the electromediated leakage current with respect to
the total transmembrane currents corresponding to different magnitudes of positive pulses. It is worth noting that the conditions of our experiment are different from those which exist during an actual electrical injury. Here, a voltage clamp was used to constrain the membrane potential to a prescribed value. No matter what the status of the ion channels, the membrane potential is a constant. In other words, the opening or closing of ion channels does not affect the formation of electromediated pores on the phospholipid membrane bilayer. In practice, the transmembrane potential across the cytoplasmic membranes of a skeletal muscle fiber induced by an external high intensity electrical field depends upon the membrane conductances. If no voltage-gated ion channels were present in cell membrane, the conductances of the two membrane hemispheres would be identical. Therefore the field-induced membrane potentials on the two hemispheres would also be the same. In fact, the presence of the voltage-gated ionic channels and their high opening conductance plays a significant role in the distribution of membrane potentials. We assume that when muscle fiber is initially exposed to an applied external electric field, not all voltage-gated ionic channels are activated. The rela-
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Fig. 8. The relationship between the magnitude of positive stimulation pulses and the percentage of the pulse-induced electroporated currents with respect to the total transmembrane current response. The absolute value of the stimulation pulses is plotted on the abscissa, and the percentage of isolated pulse-induced leakage currents with respect to the total transmembrane current (after subtracting capacitance and normal resistive currents) is plotted on the ordinate. The points represent data obtained in the experiments shown in the previous figures; the curve is hand drawn. All experiments were performed using normal Ringer’s solution in the central pool. The percentage increased with the magnitude of stimulation pulses, but did not reach 100% until all ion channels were damaged. Five fibers were used.
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tive symmetrical conductance of the two opposite membranes results in the field-induced membrane potentials with the same magnitudes but opposite polarities. The field-induced membrane potential at the membrane facing the negative electrode depolarizes the cell membrane and thus activates the voltage-gated ion channels. The opening of these channels significantly increases the conductance of the depolarized membrane, while the hyperpolarized membrane facing the positive electrode maintains the same conductance. The different membrane conductances lead to a redistribution of the field-induced membrane potentials across the two membrane hemispheres. The transmembrane potential at the membrane facing the positive electrode, which hyperpolarizes the cell membrane, is higher than that at the membrane facing the negative electrode, which depolarizes the cell membrane. If the depolarizing membrane potential is higher than the threshold for opening protein channels, the redistribution procedure will continue and the membrane potential will be further adjusted to a lower value until all protein channels are open. In consequence, the hyperpolarizing membrane potential at the membrane facing the positive electrode has been adjusted to an even higher value. One of the characteristics of the electrical circuit is the continuity of the field-induced electrical current. This means that the field-induced transmembrane current crossing the two membrane hemispheres should be the same, since there is no source of electrical force inside the cell. The result of the membrane potential adjustment is that the higher hyperpolarizing membrane potential at the membrane facing the positive electrode produces the same influx current as the efflux current driven by the lower depolarizing membrane potential at the membrane facing the negative electrode. For the membrane facing the positive electrode, all the field-induced transmembrane currents have to pass though the electromediated pores because there is no open channel. For the membrane facing the negative electrode, these currents can first be attributed to the ion channel currents. The extra currents beyond the capability of the protein channels are the electroporated currents. If the field-induced transmembrane current is in the range of the current capability of ion channels, no electroporation occurs on this membrane hemisphere. Therefore the voltage-gated ion channels fully protect phospholipid membranes from electroporation, which is the situation shown in Fig. 8 when the magnitude of the pulse is below 300 mV. When the electric field is higher than 300 mV, where the field-induced transmembrane current is beyond the channel current capability, then the electropore-based current is observed. The protective effect
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Fig. 9. The ratio of the membrane conductance in response to positive stimulation pulses relative to that in response to negative pulses as a function of pulse magnitude. The pulse magnitude was the same for each data point and the membrane conductances were monitored during the stimulation pulses. For the low voltage stimulation pulses, the membrane conductance of a depolarized cell membrane could be several times higher than that of a hyperpolarized membrane. The ratio decreased as the magnitude of the stimulation pulses was increased. Five fibers were used.
on the phospholipid membrane bilayer produced by opening the voltage-gated ion channels depends upon the total current driven by the applied external field. Fig. 9 shows the ratio of the conductance of the depolarized membrane to that of the hyperpolarized membrane as a function of the magnitude of the electrical pulse. At a low pulse magnitude, the conductance of the depolarized membrane is two to three times greater than that of the hyperpolarized membrane. This implies that when muscle fibers are exposed to a high intensity electrical field in real life, the field-induced membrane potential across the membrane facing the positive electrode could be two or three times higher that across the membrane facing the negative electrode. Direct evidence of this may need further investigation using a fluorescent potentiometric dye or a current clamp to monitor the membrane potential directly. 4. Conclusions Channel-mediated transmembrane currents produced in response to a variety of high voltage electrical pulses have been distinguished from pulse-induced electroporated leakage currents on the membrane of skeletal muscle fibers. The electropore-based current has been quantified as a percentage of the total transmembrane current under voltage clamp conditions. This percentage starts from zero at low membrane poten-
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tials and then increases with the membrane potential. At low membrane potentials, the ratio of the membrane conductance in response to a positive pulse relative to that in response to a negative pulse of the same magnitude can be as high as 3 or 4, and gradually decreases as the electrical pulse increases. These results imply that the voltage-gated ion channels have a protective effect, mediating some of the damage to the phospholipid membrane from electroporation by adjusting the membrane conductance. Under voltage clamp conditions the transmembrane potential is held constant even when the ion channels are open. Opening the ion channels can only lead to a decrease in membrane conductance and has no effect on the formation of electropores. In real life, the two membrane hemispheres surrounding the cell are electrically connected in series with respect to the external electrical field. The field-induced total potential difference across the whole cell is constant as long as the size and shape of the cell remain the same. The distribution of the membrane potentials across the two opposed membrane hemispheres depends upon their conductances. The opening of the voltage-gated ion channels in the membrane facing an anode causes an increase in membrane conductance and consequently a reduction in the transmembrane potential. The membrane potential across the membrane facing the cathode will spontaneously increase as a consequence. 4.1. The effects of the membrane rest potential on the protection of phospholipid membrane The membrane rest potential, as a constant, is vectorially added to the external-field-induced membrane potential, which also results in unequal membrane potentials across the two opposite membrane hemispheres. The polarity of the field-induced membrane potentials across the two membrane hemispheres is always opposite; however, the magnitude is not necessarily the same. Distribution of the field-induced membrane potential depends on the distribution of membrane conductance of the two hemispheres. The higher the membrane conductance, the lower is the field-induced membrane potential. In fact, distribution of the membrane potential across the two membrane hemispheres involves a dynamic procedure of potential redistribution depending upon changes in the membrane conductance. This redistribution of membrane potential does not involve the membrane rest potential. If it were assumed that the membrane rest potential is zero, the membrane hemisphere with open voltage-gated ion channels would have a larger membrane conductance than the hemisphere with fewer open channels regardless of the membrane resting potential. The difference between the membrane conductances results in a dif-
ference in the field-induced voltage drop across the two membrane hemispheres, which promotes a dynamic redistribution of membrane potentials. The consequence is that the hyperpolarizing membrane potential is much higher than the depolarizing potential. Meanwhile, because of the continuity of electrical current, the current crossing the two membrane hemispheres must be the same. The influx current passing the hyperpolarized membrane hemisphere, where no channel is open, must be equal to the efflux current passing the depolarized hemisphere, where many channels are open. Consequently, the higher membrane potential on the hyperpolarized membrane hemisphere with fewer open ion channels results in the formation of electrically mediated pores. Acknowledgments
We wish to thank Diane Rudall and Philip River for editorial assistance. This research is supported by grants from the Electric Power Research Institute and the Empire State Electric Energy Research Corporation. Reference 1 U. Zimmermann, J. Vienken and G. Pilwat, Development of drug carrier systems: electric field induced effects in cell membranes. J. Electroanal. Chem., 116 (19801553. 2 T.K. Wong and E. Neumann, Electric field mediated gene transfer. Biochem. Biophys. Res. Commun., 107 (19821584. 3 R.A.Winegar, J.W. Phillips, J.H. Yongblom and W.F. Morgan, Cell electroporation is a highly efficient method for introducing restriction endonuclease into cells. Mutat. Res., 225 (1989) 49. 4 DC. Chang, P.Q. Gao and B.L. Maxwell, High efficiency gene transfection by electroporation using a radio-frequency electric field. Biochim. Biophys. Acta., 1992, p. 153. 5 R.C. Lee and MS. Kolodney, Electrical injury mechanisms: electrical breakdown of cell membrane. Plast. Reconstr. Surg., 80 (19871672. 6 W. Chen and R.C. Lee, An improved double Vaseline voltage clamp to study electroporated skeletal muscle fibers. Biophys. J., 66(31(19931700. 7 D.P. Rossignol, G.L. Decker, W.J. Lennarz, R.Y. Tsong and J. Teissie, Introduction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation, Biochim. Biophys. Acta, 148 (1983) 781. 8 W. Mehrle, U. Zimmermann and R. Hampp, Evidence for asymmetrical uptake of fluorescent dyes through electro-permeabilized membranes of Auena mesophyll portoplasts, FEBS Lett., 185 (1985) 89. 9 A.E. Sowers, Fusion events and nonfusion contents mixing events induced in erythrocyte ghosts by an electric pulse. Biophys. J., 54 (1988) 619. 10 E. Tekle, R.D. Astumian and P.B. Chock, Electro-permeabilization of cell membranes: effect of the resting membrane potential. Biochem. Biophy. Res. Commun., 172(l) (1990) 282. 11 M. Hibino, M. Shigemori, H. Itoh, K. Nagayama and K. Kinosita Jr., Membrane conductance of an electroporated cell analyzed by
W. Chen, R. C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
submicrosecond imaging of transmembrane potential, Biophys. J., 59 (19911 209. 12 D.S. Dimitrov and A.E. Sowers, B&him. Biophys. Acta, 1022 (1990) 381. 13 R.J. O’Neill and L. Tung, Cell-attached patch clamp study of the electropermeabilization of amphibian cardiac cells, Biophys. J., 59 (1991) 1028. 14 L. Kovacs, E. Rios and M.F. Schneider, Measurement and modification of free calcium transients in frog skeletal muscle fibers by a metallochromic indicator dye. J. Physiol., 343 (1983) 161.
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15 M. Irving, J. Maylie, N.L. Sizto and W.K. Chandler, Intrinsic optical and passive electrical properties of cut frog twitch fibers. J. Gen. Physiol., 89 (1987) 1. 16 C.S. Hui and W. Chen, Separation of QP and Q-y charge components in frog cut twitch fibers with tetracaine, J. Gen. Physiol., 99 (19921 985. 17 W. Chen and R. Lee, The effects of high voltage pulsed electric field on voltage-gated ionic channels on frog skeletal muscle membrane, Biophys. J. in press.
and Bioenergetics,
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34 (1994) 157-167
Electromediated permeabilization of frog skeletal muscle cell membrane: effect of voltage-gated ion channels Wei Chen and Raphael C. Lee Department of Surgery, Section of Plastic and Reconstructive Chicago, IL 60637 bWA)
Surgery, The University of Chicago, 5841 South Maryland Avenue,
(Received 24 March 1994)
Abstract The asymmetrical electromediated permeabilization of cell membranes of frog skeletal muscle fibers with respect to the stimulation pulse polarity were studied using an improved double Vaseline gap voltage clamp. Channel-mediated currents in response to a variety of high voltage electrical pulses have been differentiated from the pulse-induced electroporated leakage currents induced in the cell membranes. Under voltage clamp conditions, electroporated current as a percentage of the total transmembrane currents and the ratios of the membrane conductances responding to positive electrical pulses over negative electrical pulses have been quantified. These results imply that protein channels have a protective effect, mediating some of the changes to the phospholipid membrane from electroporation by adjusting the membrane conductance. Our hypothesis states that, when skeletal muscle fibers are exposed to a high intensity electrical field in real life, the increment in membrane conductance due to ion channels opening on the depolarized side of the cell membrane initiates a redistribution of the membrane potential differences across the two opposed membranes. The field-induced voltage drop across the membrane hemisphere facing the positive electrode is much higher than that across the hemisphere facing the negative electrode, which results in unequal electromediated permeabilization of the two hemispheres..
1. Introduction The use of external electric fields to destabilize the plasma membrane, a process known as electroporation, has been widely studied. Electroporation has been used to open transient pores temporarily in specific regions of the cell membrane in order to transfect a variety of molecules such as DNA, proteins and antibodies [l-4]. Recently, electroporation has been postulated as a mechanism of electrical injury [5]. There is a growing interesting in the clinical diagnosis and treatment of electroporated cell membrane in electrically injured patients. Since the larger the cell, the higher the field-induced voltage drop across the cell membrane [61, electromediated permeabilization often appears on large excitable cells, such as skeletal muscles and nerve cells, as opposed to the thermal component of electrical injury. Previously, investigators assumed that during exposure to an external high intensity electrical field the field-induced potential differences across the two hemispherical cell membranes surrounding the cell were symmetric, but with opposite polarity. Recently, 0302-4598/94/$7.00 SSDZ 0302-4598(94)05011-I
however, several investigators have found that the electric-field-induced electroporation on these two opposite hemisphere membranes is asymmetric [7-111. With a few exceptions [12], these investigators reported that any membrane area of a cell which is closer to a positive electrode will show more evidence of electroporation than a membrane area closer to a negative electrode. Several hypotheses have been developed to explain this phenomenon. Since a large potential difference across a cell membrane is considered as the cause of electroporation, other investigators [lo] have suggested that the physiologic resting membrane potential, which is vectorially added to the external-field-induced membrane potential, results in asymmetrical electroporation on the spatially opposed membranes. Sowers [9] has postulated that the phenomenon of electro-osmosis may explain asymmetrical transport. It was also shown that it could be due to an artifactual process [9,12]. Most of these investigations involved optical dye measurements of non-excitable cells, such as nucleated plant cells, liposomes or erythrocyte ghosts. The phenomenon of asymmetrical membrane electroporation 0 1994 - Elsevier Science S.A. All rights reserved
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was only observed after high voltage electrical stimulation. In other approaches, the voltage clamp technique has been used for decades to study the electrical mechanisms of cell membranes. Recently, it has also been employed to investigate electromediated permeabilization in cell membranes [6,13]. We introduced a modified double Vaseline gap voltage clamp to study the destructive effects on skeletal muscle fibers by an external high intensity electrical field [6]. The improved voltage clamp technique makes it possible to study changes in the transmembrane current both before and after electrical pulses, as well as to monitor transient changes in transmembrane current continuously during the pulses. The improved voltage clamp allows us to resolve the transmembrane current into its components and to differentiate membrane conductance changes attributed to ion channels opening from the electromediated permeabilization of cell membrane. In our laboratory we have utilized the improved double Vaseline gap voltage clamp to observe asymmetrical electromediated permeabilization in cell membranes of frog skeletal muscle fiber during exposure to high voltage pulses. The external electrical pulse that hyperpolarizes the cell membrane introduces much larger transmembrane leakage current than the pulse that depolarizes the membrane. Our hypothesis is that in the actual situation of muscle fiber exposure to a high intensity electrical field, the increment in membrane conductance due to ion channels opening on the depolarized side of the cell membrane initiates a redistribution of membrane potential differences across the two opposed membranes. The external-field-induced membrane potential on the depolarized membrane hemisphere facing the negative electrode is much lower than that of the hyperpolarized hemisphere facing the positive electrode. Therefore less electroporation occurs on the depolarized membrane hemisphere. The present study was designed to clarify the electromediated permeabilization of the asymmetrical membrane during high voltage electrical pulses, to differentiate the voltage-gated ionic channel currents from the membrane electroporated currents and to test whether opening of the voltage-gated ion channels in cytoplasmic membrane alters membrane conductance and reduces the external-field-induced membrane potential, thus protecting the cell membrane from electroporation. 2. Materials and methods 2.1. Cell preparation and equipment set-up Voltage clamp experiments using an improved double Vaseline gap chamber to study electroporation of skeletal muscle cells have been described previously
[6]. Cut fibers were dissected following the procedure used by Kovacs et al. [14], Irving et al. [15] and Hui et al. [16]. Briefly, single muscle fibers were hand-dissected in a high K+ relaxing solution from twitch skeletal muscle, the semitendinosus of the English frog Rana temperoria. The fiber was mounted in a custommade chamber filled with the relaxing solution and was isolated across three distinct pools by two Vaseline seals. Clips on the bottom of the chamber held the fiber in place. The cell membrane of the fiber segments in the two end-pools was chemically permeabilized by treatment with 0.01% saponin for 2 min in order to ensure that electrical and ionic transfer could take place between the interior and exterior of the cells. The solution in the two end-pools was then replaced by “internal solution” which mimics the composition of the cytoplasmic fluid [6,16]. The central pool was filled with either normal Ringer’s solution or an “external solution” which contained both Na+ and K+ channel blockers, tetrodotoxin (TTX) and tetraethanoammonia (TEA). Six agar bridges and six Ag-AgCl pellets were used to connect the solutions in the chamber to the electrical circuit. The following solutions were used. (1) The relaxing solution contained 120 mM potassium glutamate, 1 mM MgSO,, 0.1 mM K,EGTA and 5 mM K,PIPES (pH 7.0). (2) The internal solution contained 45.5 mM potassium glutamate, 20 mM Tris-creature phosphate, 20 mM EGTA, 5.5 mM Na,ATP, 5 mM glucose, 5 mM K,PIPES and 6.8 mM MgSO,. (3) The external solution contained 120 mM TEA Cl, 2.5 mM RbCl, 1.8 mM CaCI,, 2.15 mM Na,HPO,, 0.85 mM NaHPO, and 1 PM tetrodotoxin (pH 7.1). TEA- and Rb+ in the external solution were used to minimize Kf currents and TTX was used to block Na + channels. A Dagon Whole Clamp 8800 controlled by an American Wells 486 personal computer was used to generate and send pulse signals to the fiber in the chamber. Signals from the voltage clamp were digitized by a Tektronix 11401 digital oscilloscope and stored in the hard disk of the computer. A Data Translation ACIIC general purpose interface board (GPIB) was used as the computer interface. 2.2. Electrical measurements Cell membranes were stimulated by a series of square pulses of magnitude ranging from 120 to 600 mV and different polarities (top panels in Figs. l-7). Simultaneously, the corresponding transient transmembrane currents were recorded during each stimulation pulse. To save space, the term membrane potential difference is simplified to membrane potential in this
W! Chen, R C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
paper. It is necessary to emphasize the difference between the magnitude of the pulse applied to the cell membrane and the magnitude of the membrane potential which is the vectorial sum of the pulse magnitude and the membrane rest potential. There are several components in the transmembrane currents recorded in response to the high voltage electrical pulse: (1) leakage currents beneath the Vaseline seals, (2) the membrane capacitance current, (3) the normal membrane leakage current, (4) the pulseinduced electroporated leakage currents and (5) the ionic channel current. To distinguish the pulse-induced leakage current and the ionic channel currents from the total recorded current, a template current consisting of components (11, (2) and (3) was generated using the following procedure. Before exposure to each high voltage pulse, the cell membrane was held at - 110 mV for 20 ms and then a group of N prepulses was delivered for the same period as the subsequent high voltage pulse. The magnitude of each prepulse was l/N that of the subsequent pulse. The criterion for the choice of N is that the membrane potential produced by the prepulses is lower than the threshold of open ionic channels. Therefore the transmembrane current responding to the prepulse consists of only the membrane capacitance and normal resistance currents. The sum of these prepulse currents served as the template current for the membrane capacitance and normal resistance currents with respect to the subsequent high voltage pulse current. After subtracting the template current from each high voltage pulse current, a pulseinduced electroporated leakage current, an ion channel current or a combination of the two can be identified depending upon the experimental solution and the polarity of the high voltage pulses. In what follows the pulse-induced current is defined as the current remaining after subtraction of the template current from the total current response. To avoid any post-pulse sealing effect of the electromediated pores in the cell membrane, all current traces discussed in this study were recorded during the high voltage electrical pulse. 2.3. Membrane bilayer versus channel proteins In order to evaluate the effects of ion channels in protecting the cell membrane from electroporation, the pulse-induced electroporated leakage current must be differentiated from the ion channel currents. To do this, it is necessary to distinguish reference membrane voltages related to the membrane bilayer from those related to ion channels. The phospholipid membrane, which is the major component of the cell surface, is organized with the hydrophobic tails of the phospholipid structure on the inside of the membrane and the
159
hydrophilic heads facing outward. The amphipathic structure of the phospholipid membrane maintains the integrity of a hydrophobic permeability barrier in close proximity to an aqueous medium. Unlike ion channels, which have a high ionic selectivity and a clear rectification effect on channel-based currents, the phospholipid-bilayer-based membrane current is neither ion selective nor current-direction specified. The externalfield-induced phospholipid-bilayer-based currents should not depend on the polarity of the applied electric field, i.e. the reference voltage of the phospholipid membrane with respect to a pair of symmetrical external electrical fields should be zero. In contrast, the asymmetric structures of the voltage-gated ion channels in the cell membrane result from the pattern in which proteins are embedded in the phospholipid membrane. Most types of voltagegated ion channels show ionic selectivity and rectifier channel currents. Because of the various ion selectivities of different kinds of channels and the electrochemical force due to different ion concentrations across the cell membrane generated by Na+-K+ ATPase pumps, the equilibrium membrane potential in the resting condition is -90 mV for natural intact fibers of frog skeletal muscle. To mimic the real situation, in our experiments cell membranes were always held at -90 mV except during the stimulation period. Any positive change in membrane potential from -90 mV can depolarize the cell membrane. If the cell membrane is depolarized at a higher potential than the threshold value, the voltage-gated channels will open. Any negative changes in membrane potential from - 90 mV that hyperpolarize the cell membrane will not open the voltage-gated ion channels, such as the Na and the delayed rectifier K channels. Therefore in order to study the effects of pulse polarity on the voltage-gated ion channels, the reference voltage must be at the rest potential or the holding potential (- 90 mV). In the present study, the cell membrane potential was held at - 90 mV and was then exposed to one of two groups of pulses: (1) pulses of the same magnitude and opposite polarities with respect to zero, and (2) pulses of the same magnitude and opposite polarities with respect to the membrane rest potential (- 90 mV>. The transmembrane current responses were simultaneously monitored for all these pulses. This technique makes it possible to separate pulse-induced transmembrane currents, caused by electroporation of the phospholipid membrane, from the opening of the voltage-gated ion channels. 2.4. Experiment protocol In most experiments, the external environment in the central pool was normal Ringer’s solution. High
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voltage pulses of various magnitudes were delivered across the cell membrane using the voltage clamp. The time interval between two pulses varied from 3 min, when the pulse magnitude was below the threshold of membrane electroporation, to 5 min when it was above the threshold, and to 10 min when it was much higher than the threshold. The criterion for choosing the time interval was that the post-pulse holding current, which is defined as the transmembrane current when the membrane is held at the holding potential (-90 mV), fell to the original value before further high voltage pulses were applied. In some experiments, high voltage pulses were applied to cell membranes when the external solution in the central pool contained both the Na+ and K+ channel blockers TEA and TTX. All high voltage pulses had a duration of 4 ms in order to mimic the effects of a half-cycle of the commercial powerline. A pulse of duration 4 ms at the RMS value and a half-cycle sinusoid current at commercial power frequency (60 Hz) have the same energy. To reduce the number of stimulation pulses and maintain a healthy fiber, the pulse magnitudes were carefully chosen as follows. (1) High voltage pulses of four different magnitudes were used: 120 mV, which is below the threshold voltage for membrane electroporation; 300 mV, which is slightly above the electroporation threshold [17]; 480 mV, which is near the damage threshold for the voltage-gated K+ channel [17]; 600 mV, which is higher than both the membrane electroporation and the ion channel damage thresholds [17], but not high enough to induce permanent change in either the phospholipid membrane or the protein channels. Each of the four groups consisted of two pulses of the same magnitude but opposite polarities. (2) Except for the last group, the differences between the pulse magnitudes of two sequential groups were twice the rest potential. The advantage of this is that while the pair of pulses in each group have the same magnitude with opposite polarities, the pair of pulses in sequential groups retain the same membrane potential with opposite polarities. In other words, the first pulse pair holds the membrane potential at opposite polarities with respect to the reference voltage at the rest potential and the second pulse pair with holds the membrane potential at opposite polarities with respect to the reference voltage at zero. 3. Results and discussion Comparison of the stable holding current following a positive pulse with the significantly increased holding current following a negative pulse immediately shows
that cell membranes can tolerate a higher positive membrane potential than a negative potential without evidence of electroporation. Because of this phenomenon, a positive pulse was always applied to the cell membrane prior to a negative pulse in each pulse group in order to maximize the life of each fiber. 3.1. Electroporation of the membrane bilayer When normal Ringer’s solution in the central pool was replaced by an external solution containing 120 mM TEA *Cl and 1 PM TI’X, most of the voltage-gated Na+ and K+ channels were blocked so that it was possible to concentrate on the electroporation of the membrane bilayer. Under these conditions, the reference voltage with respect to the symmetrical pulses in all four pulse groups should be zero. The first pulse was applied to the cell membrane to hold the membrane potential at +510 mV. The recorded total transmembrane current is shown in Fig. l(b). After 5 min, the current response to a negative pulse holding the membrane potential at -510 mV was also recorded and is shown in Fig. 2. Since the voltage-gated ion channels were blocked, these current traces consisted of the membrane capacitance current, the normal leakage current and the pulse-induced electroporation current. The directions of the two total current responses were opposite. Because the capacitance and normal resistance currents were linearly dependent on the pulse magnitude and independent of the pulse polarity, the difference in the pulse magnitudes (600 mV versus 420 mV) resulted in both the shapes and the magnitudes of the two response current traces being different (Figs. l(b) and 2(b)). After removing the membrane capacitance and resistance currents by subtracting the template currents, the remaining current can be attributed to the electromediated pores on the cell membrane. These currents are shown in the lower panels of Figs. l(c) and 2(c). Interestingly, the pulse-induced leakage currents responding to the two pulses that held the cell membrane at a potential of equal value (-600 mV), but opposite polarity, were very similar. This similarity is important evidence that the two sides of the phospholipid bilayer are electrically and statistically symmetrical. Identical stimulation pulses with opposite polarities cause almost equal amounts of electroporation on the membrane bilayer. The negligible difference occurred because not every channel was blocked. It should be noted that, under the voltage clamp conditions, the membrane potential was held according to the command pulse regardless of any change in cell membrane, such as ion channel opening. In other words, because ion channel opening does not influence membrane potential, electroporation is not affected
WOChen, R. C. Lee / Electromediated
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161
either. Thus the electromediated pore currents responding to a pair of pulses will be equal if the membrane potential is clamped at the same value but opposite polarities by the two pulses. This result will be used in the rest of the paper to isolate the electroporation current from the recorded total transmembrane current responding to a positive high voltage pulse when no channel blockers are used to block the voltage-gated channels. 3.2. Response of transmembrane currents to symmetrical pulses with different reference voltages The external solution used for the remaining experiments was normal Ringer’s solution without any channel blockers. Fig. 3(b) shows the recorded total transmembrane current trace responding to a pulse of - 120 mV with
-2000 Fig. 2. As Fig. 1 except that the stimulation pulse is negative. (a) A negative stimulation pulse of -420 mV. Since the intrinsic rest potential was -90 mV, the actual membrane potential across the cell membrane was - 510 mV during the stimulation pulse. (b) The transmembrane current in response to the stimulation pulse. The external solution in the central pool is the same as that used in Fig. 1, containing TTX and TEA. (c) the isolated electrically mediated leakage current induced by the stimulation pulse. Capacitance and resistance currents have been subtracted using a template current obtained from a group of prestimulation pulses. Since the stimulation pulse hyperpolarized the cell membrane, no voltage-gated ion channels were open.
Fig. 1. Transmembrane current under a voltage clamp. (a) A stimulation pulse of magnitude +600 mV and duration 4 ms. (b) Total transmembrane currents recorded during the stimulation pulse. The cell membrane was held at a potential of +510 mV (initial rest potential, -90 mV). The solution in the central pool is an external solution containing the Na+ and K+ channel blockers TTX (0.1 PM) and TEA.Cl (120 mM). (c) Isolated pulse-induced membrane electroporated leakage current obtained after removal of the membrane capacitance current and the normal membrane leakage current. The narrow spikes (also visible in some later figures) along the two edges of the stimulation pulse are due to experimental variability in the alignment of the total and the template currents during subtraction.
the membrane potential held at - 210 mV. This potential is lower than the threshold of formation of electromediated pores on cell membranes [6]. This can be demonstrated by removing both the membrane capacitive and normal resistive currents by subtracting the template current from the total current response. Because of hyperpolarizing cell membranes, most of the Na+ and K+ channels are closed. The pulse-induced electroporated leakage current is almost zero, and is shown as the remaining current in Fig. 3(c). The next pulse was +300 mV. Thus the cell membrane was held at + 210 mV. The total transmembrane current responding to this depolarizing pulse is shown in Fig. 4(b). The spike at the rising edge with an exponential decay represents the membrane capacitance current. After the spike, the transmembrane current increases until the end of the pulse. The current remaining after subtracting the membrane capacitive and normal resistive currents is shown in Fig. 4(c).
W. Chen, R C. Lee / Electromediated permeabilization of frog skeletal muscle cell membrane
162
This is the sum of the voltage-gated ion channel currents and the electromediated membrane leakage currents, if any exist. It is not certain how much of this current can be attributed to the pulse-induced membrane leakage current. Fortunately, as mentioned previously and supported by the results shown in the Fig. 1, the two sides of the phospholipid bilayer are electrically and statistically symmetrical. Thus identical stimulation pulses with opposite polarities cause almost equal electroporation on the membrane bilayer. In other words, a pair of symmetrical pulses with a reference voltage of zero should introduce the same amount of electromediated leakage currents. Therefore the contribution of membrane electroporated currents in Fig. 4(c) should be equal to those responding to a pulse holding the membrane potential at -210 mV, which was recorded and is shown in Fig. 3(c). The negligible current in Fig. 3(c) indicates that the contribution of the electroporated current to the trace in Fig. 4(b) is almost zero. Therefore all currents in Fig. 4(c) can be attributed to the channel-mediated currents. These results imply that a pulse of magnitude of +300 mV
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Fig. 3. A pulse with a magnitude of - 120 mV: (a) cell membrane held at -210 mV, (b) capacitance and normal leakage currents subtracted from the total recorded transmembrane currents showing that this membrane potential is still below the electroporation threshold; (c) the remaining current is almost zero which demonstrates that no electroporation occurred.
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Fig. 4. (a) A positive pulse of 300 mV, which held the cell membrane at a potential of +210 mV, (b) the responding transmembrane current recorded during the stimulation; (c) remaining current after subtracting the capacitance current and normal leakage current. These currents are channel mediated currents, because there was no pulse-induced leakage current responding to a pulse holding the membrane potential at - 210 mV (see text).
cannot destabilize the integrity of the membrane structure. The total transmembrane current responding to a stimulation pulse of -300 mV was shown in Fig. 5(b). Because of the intrinsic membrane rest potential of -90 mV, the cell membrane was actually held at a potential of - 390 mV, which is higher than the threshold of electroporation of cell membranes [17]. Then Fig. 5(c), which was obtained by subtracting the membrane capacitance and normal resistance currents, should be the sum of the pulse-induced electroporated leakage currents and the ionic channel current, if it exists. Since the cell membrane was hyperpolarized, most of the voltage-gated Na+ and K+ channels did not open, and hence the current trace in Fig. 5(c) represents primarily the pulse-induced electroporated leakage current across the cytoplasmic membrane. It is now possible to discuss the pulse-induced membrane conductance with respect to the same pulse magnitude and opposite polarity. Because the pulse magnitudes are the same, the pulse-induced membrane conductances are proportional to the pulse-induced
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W. Chen, R.C. Lee / Electromediated permeabilization of frog skeletnl muscle cell membrane
currents shown in Figs. 4(c) and 5(c). Since we are concerned here with a comparison of the effects of pulse polarity, we only need to consider the ratio of the pulse-induced conductance of the depolarized membrane to that of the hyperpolarized membrane. This can easily be calculated using the largest electromediated currents in Figs. 4(c) and 5(c) (an average of the last 20 data points at the end of the pulse). When the pulse magnitude is 300 mV, the membrane conductance ratio is 1.8: 1. Using a pair of pulses of magnitude 480 mV and opposite polarity, the cell membrane can be held at either +390 mV or -570 mV, as shown in Figs. 6(a) and 7(a) respectively. Both these membrane potentials are high enough to electroporate cell membranes. The total current responses to these opposite pulses were recorded and are shown in Figs. 6(b) and 7(b). The isolated pulse-induced transmembrane current of the depolarized cell membranes (membrane potential at +390 mV>, obtained by subtracting the membrane capacitance and normal leakage currents, is shown in Fig. 6(c). It is a combination of the channel currents
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Fig. 6. (a) Positive simulation pulse of 480 mV, (b) total transmembrane current recorded during the stimulation in which the membrane was held at a potential of +390 mV; (c) remaining current after subtracting the capacitance and resistance currents. The current in (c) represents a combination of ion channel currents and electroporated membrane current.
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Fig. 5. (a) A negative stimulation pulse of - 300 mV. The membrane potential was actually - 390 mV because of the vector summation of the stimulation pulse and an intrinsic membrane rest potential of -90 mV. (b) The total transmembrane current: since the pulse hyperpolarized the cell membrane, no voltage-gated ion channels were open. (c) The current obtained by subtracting the template currents, which is mainly electroporated membrane current.
and the electromediated electropore currents. With the same token, the pulse-induced electroporation current corresponding to a membrane potential of +390 mV shown in Fig. 6(c) is the same as the pulse-induced leakage current corresponding to - 390 mV (Fig. 5(c)). The channel-mediated currents in Fig. 6(c) can be determined by subtracting the pulse-induced leakage currents shown in Fig. 5(c). Comparison of Figs. 5(c) and tic> shows that about 40% of the pulse-induced current can be attributed to the electroporated leakage current and 60% to the channel-mediated current. In contrast, the current shown in Fig. 7(c) represents only the electromediated permeabilization current of cell membrane, since most voltage-gated ion channels are not opened by the hyperpolarizing pulse. Comparison of Figs. 5(c) and 7(c) shows that the pulse-induced leakage current corresponding to a pulse of +480 mV is only about 30% of that corresponding to a pulse of -480 mV. Again, using the method described in the previous paragraph, the ratio of the pulse-induced membrane conductance of the depolarized membrane to that of the hyperpolarized membrane for a pulse of magnitude 480 mV is about 3.4 : 4.7.
W. Chen, R.C. Lee / Electrornediated permeabilization of frog skeletal muscle cell membrane
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Fig. 7. (a) Negative stimulation pulse of -480 mV that held the membrane at a potential of 570 mV, (b) total transmembrane current is shown; Cc) the remaining current obtained by subtracting capacitance and normal leakage currents which, since the pulse hyperpolarized the cell membrane, was primarily membrane electroporation current.
3.3. Pulse-dependent percentages of the electroporated currents among the total transmembrane currents responding to different positive pulse magnitudes Fig. 8 was drawn by combining all the percentages of the pulse-induced leakage current with respect to the total electromediated currents corresponding to positive pulses of different magnitudes. Each point represents a percentage corresponding to a positive pulse. This percentage starts from zero when the stimulation pulse is below the threshold of membrane electroporation. It then increases with increasing pulse magnitude, but does not reach 100% except when all protein channels are damaged. It is important to note that these results were obtained using a voltage clamp, where the membrane potential was constrained regardless of changes in the membrane characteristics. This result shows that the voltage-gated ion channels tend to prevent the formation of electromediated pores in the phospholipid membrane bilayer. 3.4. Electroporation on cell membranes in a real situation of electrical injury In the previous section we discussed the percentages of the electromediated leakage current with respect to
the total transmembrane currents corresponding to different magnitudes of positive pulses. It is worth noting that the conditions of our experiment are different from those which exist during an actual electrical injury. Here, a voltage clamp was used to constrain the membrane potential to a prescribed value. No matter what the status of the ion channels, the membrane potential is a constant. In other words, the opening or closing of ion channels does not affect the formation of electromediated pores on the phospholipid membrane bilayer. In practice, the transmembrane potential across the cytoplasmic membranes of a skeletal muscle fiber induced by an external high intensity electrical field depends upon the membrane conductances. If no voltage-gated ion channels were present in cell membrane, the conductances of the two membrane hemispheres would be identical. Therefore the field-induced membrane potentials on the two hemispheres would also be the same. In fact, the presence of the voltage-gated ionic channels and their high opening conductance plays a significant role in the distribution of membrane potentials. We assume that when muscle fiber is initially exposed to an applied external electric field, not all voltage-gated ionic channels are activated. The rela-
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Fig. 8. The relationship between the magnitude of positive stimulation pulses and the percentage of the pulse-induced electroporated currents with respect to the total transmembrane current response. The absolute value of the stimulation pulses is plotted on the abscissa, and the percentage of isolated pulse-induced leakage currents with respect to the total transmembrane current (after subtracting capacitance and normal resistive currents) is plotted on the ordinate. The points represent data obtained in the experiments shown in the previous figures; the curve is hand drawn. All experiments were performed using normal Ringer’s solution in the central pool. The percentage increased with the magnitude of stimulation pulses, but did not reach 100% until all ion channels were damaged. Five fibers were used.
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tive symmetrical conductance of the two opposite membranes results in the field-induced membrane potentials with the same magnitudes but opposite polarities. The field-induced membrane potential at the membrane facing the negative electrode depolarizes the cell membrane and thus activates the voltage-gated ion channels. The opening of these channels significantly increases the conductance of the depolarized membrane, while the hyperpolarized membrane facing the positive electrode maintains the same conductance. The different membrane conductances lead to a redistribution of the field-induced membrane potentials across the two membrane hemispheres. The transmembrane potential at the membrane facing the positive electrode, which hyperpolarizes the cell membrane, is higher than that at the membrane facing the negative electrode, which depolarizes the cell membrane. If the depolarizing membrane potential is higher than the threshold for opening protein channels, the redistribution procedure will continue and the membrane potential will be further adjusted to a lower value until all protein channels are open. In consequence, the hyperpolarizing membrane potential at the membrane facing the positive electrode has been adjusted to an even higher value. One of the characteristics of the electrical circuit is the continuity of the field-induced electrical current. This means that the field-induced transmembrane current crossing the two membrane hemispheres should be the same, since there is no source of electrical force inside the cell. The result of the membrane potential adjustment is that the higher hyperpolarizing membrane potential at the membrane facing the positive electrode produces the same influx current as the efflux current driven by the lower depolarizing membrane potential at the membrane facing the negative electrode. For the membrane facing the positive electrode, all the field-induced transmembrane currents have to pass though the electromediated pores because there is no open channel. For the membrane facing the negative electrode, these currents can first be attributed to the ion channel currents. The extra currents beyond the capability of the protein channels are the electroporated currents. If the field-induced transmembrane current is in the range of the current capability of ion channels, no electroporation occurs on this membrane hemisphere. Therefore the voltage-gated ion channels fully protect phospholipid membranes from electroporation, which is the situation shown in Fig. 8 when the magnitude of the pulse is below 300 mV. When the electric field is higher than 300 mV, where the field-induced transmembrane current is beyond the channel current capability, then the electropore-based current is observed. The protective effect
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Fig. 9. The ratio of the membrane conductance in response to positive stimulation pulses relative to that in response to negative pulses as a function of pulse magnitude. The pulse magnitude was the same for each data point and the membrane conductances were monitored during the stimulation pulses. For the low voltage stimulation pulses, the membrane conductance of a depolarized cell membrane could be several times higher than that of a hyperpolarized membrane. The ratio decreased as the magnitude of the stimulation pulses was increased. Five fibers were used.
on the phospholipid membrane bilayer produced by opening the voltage-gated ion channels depends upon the total current driven by the applied external field. Fig. 9 shows the ratio of the conductance of the depolarized membrane to that of the hyperpolarized membrane as a function of the magnitude of the electrical pulse. At a low pulse magnitude, the conductance of the depolarized membrane is two to three times greater than that of the hyperpolarized membrane. This implies that when muscle fibers are exposed to a high intensity electrical field in real life, the field-induced membrane potential across the membrane facing the positive electrode could be two or three times higher that across the membrane facing the negative electrode. Direct evidence of this may need further investigation using a fluorescent potentiometric dye or a current clamp to monitor the membrane potential directly. 4. Conclusions Channel-mediated transmembrane currents produced in response to a variety of high voltage electrical pulses have been distinguished from pulse-induced electroporated leakage currents on the membrane of skeletal muscle fibers. The electropore-based current has been quantified as a percentage of the total transmembrane current under voltage clamp conditions. This percentage starts from zero at low membrane poten-
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tials and then increases with the membrane potential. At low membrane potentials, the ratio of the membrane conductance in response to a positive pulse relative to that in response to a negative pulse of the same magnitude can be as high as 3 or 4, and gradually decreases as the electrical pulse increases. These results imply that the voltage-gated ion channels have a protective effect, mediating some of the damage to the phospholipid membrane from electroporation by adjusting the membrane conductance. Under voltage clamp conditions the transmembrane potential is held constant even when the ion channels are open. Opening the ion channels can only lead to a decrease in membrane conductance and has no effect on the formation of electropores. In real life, the two membrane hemispheres surrounding the cell are electrically connected in series with respect to the external electrical field. The field-induced total potential difference across the whole cell is constant as long as the size and shape of the cell remain the same. The distribution of the membrane potentials across the two opposed membrane hemispheres depends upon their conductances. The opening of the voltage-gated ion channels in the membrane facing an anode causes an increase in membrane conductance and consequently a reduction in the transmembrane potential. The membrane potential across the membrane facing the cathode will spontaneously increase as a consequence. 4.1. The effects of the membrane rest potential on the protection of phospholipid membrane The membrane rest potential, as a constant, is vectorially added to the external-field-induced membrane potential, which also results in unequal membrane potentials across the two opposite membrane hemispheres. The polarity of the field-induced membrane potentials across the two membrane hemispheres is always opposite; however, the magnitude is not necessarily the same. Distribution of the field-induced membrane potential depends on the distribution of membrane conductance of the two hemispheres. The higher the membrane conductance, the lower is the field-induced membrane potential. In fact, distribution of the membrane potential across the two membrane hemispheres involves a dynamic procedure of potential redistribution depending upon changes in the membrane conductance. This redistribution of membrane potential does not involve the membrane rest potential. If it were assumed that the membrane rest potential is zero, the membrane hemisphere with open voltage-gated ion channels would have a larger membrane conductance than the hemisphere with fewer open channels regardless of the membrane resting potential. The difference between the membrane conductances results in a dif-
ference in the field-induced voltage drop across the two membrane hemispheres, which promotes a dynamic redistribution of membrane potentials. The consequence is that the hyperpolarizing membrane potential is much higher than the depolarizing potential. Meanwhile, because of the continuity of electrical current, the current crossing the two membrane hemispheres must be the same. The influx current passing the hyperpolarized membrane hemisphere, where no channel is open, must be equal to the efflux current passing the depolarized hemisphere, where many channels are open. Consequently, the higher membrane potential on the hyperpolarized membrane hemisphere with fewer open ion channels results in the formation of electrically mediated pores. Acknowledgments
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