271

Bioelectrochemistry and Bioenergetics, 24 (1990) 271-295

A section of J. Electroanal Chem., and constituting Vol . 299 (1990)
Elsevier Sequoia S .A ., Lausanne

Review

On electroporation of cell membranes and some related

phenomena

Tian Y . Tsong
Department of Biochemistry, University of Minnesota College of Biological Sciences, 1479 Gortner Avenue,
St. Paul, MN 55108 (U.S.A .)
(Received 25 June 1990)

ABSTRACT

Electroporation and other related cell membrane phenomena induced by an applied electric field,
either a direct (dc), an alternating (ac), or a dc shifted radio-frequency electric field, reflect the electrical
properties and hence the basic structure and molecular interactions of a cell membrane . The importance
of studying these phenomena, thus, goes beyond their practical application in genetic engineering,
agricultural research and biotechnology . One recent development in this area, namely, the electroactiva-
tion of membrane proteins by an ac field, poetises to yield crucial data on how a cell can process and
transmit electric signals for the regulation of its internal activities and communication with other cells
(T .Y . Tsong, Trends Biochem . Sci ., 14 (1990) 89) . Knowledge of cellular designs of these events would
have far reaching implications concerning our ability to understand cell membrane functions, especially
with respect to mechanisms of cellular energy and signal transduction (T .Y. Tsong, Annu . Rev . Biophys.
Chem ., 19 (1990) 83) .

(I) INTRODUCTION

Electricity and its effects on life have aroused intense curiosity in the inquiring
minds of men and women since ancient time . The development of the membrane
hypothesis of cells early this century has generated many studies on the electrical
properties of the cell membrane . Accounts for these early studies have been
documented in the monographs of Cole [1] and Tien [2] . Electrically induced
reversible changes in membrane conductance or permeability to small molecules,
e.g . water and organic solutes, are well known phenomena and a large body of
literature has developed since the 1940's [1] . Field effects on molecular and ionic
transport across the axonal membranes are the prime examples of these early works
[1] . Goldman [3] in 1943 used a current scanning technique to measure the
voltage-current (V-I) characteristics of the membrane of Chara australis and
found that when the membrane was hyperpolarized beyond a certain potential,

0302-4598/90/$03 .50 © 1990 - Elsevier Sequoia S .A .

272

there was a sudden rapid increase in current, similar to that of dielectric breakdown .
However, in this case the breakdown was completely reversible . The whole scan
could be repeated over and over again to give the same V-I curve. Coster called this
phenomenon the reversible electric punch-through [4] . By the 1960's the electric
breakdown potentials for the bilayers of many synthetic or natural lipids had been
measured and characterized [2] . Huang et al. in 1964 measured the dielectric
breakdown potential of a bilayer of purified egg phosphatidyl choline to be 200 mV
[5] . Of particular interest to investigators working in the area of electroporation, are
the publications of Sale and Hamilton in 1967 and 1968 [6-8] . In these papers, the
authors studied the effects of short (µs to ms) and intense (several kV/cm) electric
pulses on suspensions of cells and concluded that " . .the application of excessive
potential differences across the membrane may lead to irreversible changes in the
ordered structure to give rise to the observed forms of membrane breakdown" . "In
microorganisms the structural changes due to a potential difference of about 1 V
could give rise to irreversible loss of the membrane's function as the semipermeable
barrier between the cell and its environment . ." . Following these early works, there
was a gap of several years before the electric breakdown phenomenon caught the
attention of a few laboratories again . However, a great proliferation of literature has
been noted in recent years due largely to the successs of using electroporation
techniques for gene transfer into living cells [9,10] and the electrofusion methods for
preparing heterokaryons and hybridomas for genetic and biotechnological research
[11,12] . Electroactivation of membrane proteins [13-18], electrostimulation of cellu-
lar activities [19], electroinsertion of biologically active molecules into cell mem-
branes [20], and microsurgery or punctuation of tissues [21] using pulsed electric
fields are a few new developments which continue to broaden the basis of the field.
This article will summarize the results of our mechanistic studies on some of these
phenomena and what we have learned from thesis work . As suggested by the Editor,
it will be mainly a personal account. However, from time to time I will borrow from
the experiences of others to understand effects of pulsed electric fields on cell
membranes and also to plan for future studies . Several similar articles from other
laboratories have already appeared and many will soon appear in this journal . Thus
a survey of the literature will not be attempted .

(II) DIFFERENT EFFECTS OF PULSED ELECTRIC FIELDS

Work in my laboratory began by studying membrane lipid phase transitions

using the capacitor discharge technique [22,23] . Aqueous dispersions of single
component synthetic lipids of different preparations, multilamellar liposomes, uni-
lamellar small vesicles (25 ± 3 nm diameter) and large vesicles (95 ± 5 nm diameter)
were used. The crystalline-liquid crystalline phase transitions of the lipid bilayer
generally occur in an extremely narrow temperature range. Dimyristoylphosphatidyl
choline vesicles have a transition around 24'C and dipalmitoylphosphatidyl choline
vesicles at 42 ° C and the width of these transitions is less than 0 .5 ° C . Because of
this high sensitivity, most of the changes of the physical properties which we were


273

Some Molecular Processes In Membrane Dispersions

A . The crystalline-liquid crystalline phase transition

T4 0
B . Swelling rupture and annealing
T4 rt

C . Stripping rupture and fusion

Tl --

0 CO)i
FL
C 0
Tt

O
o o ~c t O
Fig. 1 . Different dynamic events of a lipid aqueous dispersion induced by a kV/cm, microsecond electric
pulse. An aqueous suspension of unilamellar small vesicles (25 ± 3 mm), large vesicles (95 ± 5 nm),
multilamellar liposomes of sysnthetic lipids, or B. subtilis was exposed to an exponential decaying electric
pulse (time constant 3 µs) of initial strength up to 35 kV/cm . Different molecular events are summarized .
This figure is an untouched reproduction of a slide the author presented at the 1974 Annual Meetings of
the Federation of American Societies of Experimental Biology . See Section (II) for an explanation of the
different molecular dynamics and conditions for inducing these events .

monitoring (turbidity, light scattering, fluorescent probes) were dominated by the

effect of Joule heating . However, it soon became clear to us that there were many
other chemical events which could not be explained by the effects of Joule heating
[22,23] . To illustrate this point, a slide which I presented in the 1974 Annual
Meetings of the Federation of American Societies for Experimental Biology [24] is
reproduced without modifications in Fig . 1 . This slide summarizes several processes
when a suspension of lipid vesicles or B. subtilis was exposed to an exponentially
decaying electric pulse of up to 35 kV/cm and with a decaying time constant of
approximately 3 µs . The first events belong to the pre-transition and the
crystalline-liquid crystalline phase transition (or S to F phase transition) of lipid
molecules in the bilayer structure. The pre-transition involves the reorientation of
lipid head groups, from a straight up to a tilted orientation, and the main transition
involves the formation of cis-isomeric C-C bonds in the hydrocarbon chains of
lipid molecules, generally called kinks, and the subsequent disordering of the bilayer
274

crystalline state. These events were thermally induced, and for the conditions used
in these experiments (approximately 0 .15 M NaCl, buffered), a pulsed electric field
of less than 5 kV/cm was sufficient to generate large signals, i .e . decreases in the
turbidity (indicated by T) of the suspension . The second class of reactions was
identified to be due to swellings, ruptures and annealing of vesicles, cells and
multilamellar lipid structures . The swelling was interpreted as resulting from either a
dielectric breakdown or an electro-osmosis effect which caused the permeation of
water into the vesicles . To induce these events required an electric pulse of
amplitudes greater than 15 kV/cm for the lipid vesicles . The turbidity changes
associated with these reactions are indicated by arrows . The third class of reactions
was identified to be due to a large scale disruption of the lipid bilayer structures .
After an electric field was turned off, annealing of the disrupted bilayer structures
and fusion of vesicles were observed . Fusion of lipid vesicles was mentioned in the
abstract [24] and reported in the 1974 meetings . These reactions were induced by
single electric pulses of 30 kV/cm or greater . The vesicles used were relatively small,
25 nm or 95 nm in diameter ; a 30 kV/cm electric field would induce transmem-

Fig. 2. An electric pulse induced hemolysis of human erythrocytes . Erythrocytes in an isotonic saline were
exposed to a single pulse of an exponentially decaying electric field (time constant 3 µs, initial field
strength 15 kV/cm). Turbidity changes at 300 nm were followed as a function of time . In the lower
figure, small dots denote radioactive sucrose, small open circles denote hemoglobin or other cytoplasmic
macromolecules. Steps I to IV are, respectively, pore initiation, water influx, membrane rupture and
hemolysis, and annealing of a red cell. According to this early work, Step I occurred in less than 10 µs,
Step II in 5 ms, Step III in 100 ms and step IV in 500 ms . The initial temperature of the sample was
15 ° C and the final temperature was 17 ° C . After Step IV, the membrane stayed leaky to K + and Na +
for hours . In this work it was still not possible to distinguish effects of the electric field and the Joule
heating. (ref . 25) . These phenomena were shown later to be due to the effect of the electric field [26] .
 275

brane potentials in the range 45-200 mV . Repeated pulses greatly enhanced the
fusion yield in the suspension of lipid vesicles . The experimental evidence for
membrane fusion included formation of large vesicles or multilamellar vesicles when
the initial sample consisted of unilamellar vesicles . For dimyristoylphosphatidyl
choline, the small vesicles (25 nm) had a broader phase transition at 20-22 ° C
(transition width of about 2'Q . The same transition in the large unilamellar
vesicles (95 nm) was at 23'C with a width of 1 ° C and for the liposomes it was at
24 .1'C with a transition width smaller than 0.5'C .
The swelling and subsequent rupture of lipid bilayers and B. subtilis were of
particular interest to us, and we decided to examine events associated with the
tracer entrapment and the protein leakage induced by an electric pulse using human
erythrocytes as a model system . The results of these experiments are summarized in
Fig. 2, which is the reproduction of a figure from a 1975 paper [25] . In this figure, it
is shown that the initial event was the modification of the membrane permeability,
which resulted in the loading of a radioactive sucrose tracer into the red cells (steps
I and II) . Following the permeability changes were the swelling of cells, the rupture
of cell membranes, the leakage of the cytoplasmic contents, and finally the deflation
of the damaged cells and the annealing of the electrically perforated cell mem-
branes . In this study we also attempted to distinguish two different effects of a
pulsed electric field, namely effects of the electric field and Joule heating . Owing to
the short durations of the electric pulses used (decay constants 1-3 µs) the effect of
the electric field was difficult to separate from that of the Joule heating (see Fig . 5
of ref. 27). And our attempt was not successful .

(III) PROPOSITION, EXPERIMENTAL VERIFICATION, AND RESEALING OF PORES

After these early experiences, Kinosita and I decided to tackle the problem of the
electropermeabilization of cell membranes . It was during this time that we proposed
and provided experimental evidence for the "formation and resealing of pores of
controlled size " in human erythrocyte membranes by a transient electric field [28] .
The concept of aqueous pores was discussed [26-29] although it was slow to gain
acceptance because most investigators prior to 1980 used more familiar terms such
as dielectric breakdown and electric breakdown to describe the phenomenon [30,31] .
Nevertheless, when the highly graphic word electroporation was later adopted by
Sugar and Neumann to encompass both the phenomenon and the methodology [32],
it gained universal approval rather quickly . The work of Chang and Reese [33]
shows unequivocally that volcano-like pores are clearly visible in cell membranes
treated with an intense electric field .
The human erythrocyte was chosen for our study because of its availability and
also its fragility which would later prove to be essential for discriminating different
molecular events induced by a pulsed electric field . The results of these experiments
are summarized below.
(1) For human erythrocytes, the application of an electric pulse of 3-6 kV/cm
and of 2-200 µs duration to an aqueous solution, can generate many different


276

TABLE 1

Different effects of a pulsed electric field. See refs . 18 and 28 for details

(A) Effects common to all (B) Effects specific to cell

systems suspensions

(1) Electric field effects Electrophoresis Field induced transmembrane

potential
Field induced orientation Amplification of common ef-
fects within cell membrane by
1 .5Rcen/d, for a spherical cell
Wien effects, dissociation of
ion pairs
Electroconformational change
0K/K=t MtsE/RT
Electrocompression of cell
membrane
Large transmembrane current
Alternating field only Dielectrophoresis Amplification of common ef-
fects within cell membrane
Electroconformational oscilla-
tion energy and signal trans-
ductions

(2) Thermal effects (current ef- Enthalpy effects : shift of chem- Colligative effects, A77 = cR AT
fects), AT= i 2 rOt/4.18CP ical equilibrium AK/K =
(A H/RT 2 )OT
Trermal osmosis effects, L p =
- (Q/vT )OT
Solvent expansion ; shock
waves, sP = (a/K)L1T

Symbols used : R Ce,,, radius of cell ; AT, temperature change ; r, specific resistivity ; CP , specific heat
capacity ; c, concentration of solutes ; K, equilibrium constant ; OM, change in electric moment ; 0H,
enthalpy of reaction ; OP, pressure generated ; K, compressibility of solvent ; Q, heat of transfer across
membrane; d, membrane thickness (5 nm) ; i, current ; At, time of field exposure ; Air, change in osmotic
pressure ; R, gas constant ; AK, change in equilibrium ; DE, effective electric field ; T, Kelvin tempera-
ture ; a, expansion coefficient ; v, partial molar volume, solvent .

chemical reactions and physical effects, e .g . lipid thermal phase transitions, thermal
osmosis, electrophoretic movement of ions or charged molecules, electroconforma-
tional changes, etc . in the bulk solution and on the cell membrane (Table 1) .
Leakage of the cytoplasmic contents and lysis of cells, or hemolysis of erythrocytes,
are mainly due to the effect of the electric field [26] . This conclusion was drawn
from the fact that a correlation was found for the extent of hemolysis and the
applied field strength but not with the Joule heating . Although the conclusion was
explicitly stated and emphasized in several papers [26-29], it has often been
mis-quoted as that of a reviewer [30] .
(2) By measuring the kinetics of ion leakage, molecular permeation, and events
associated with hemolysis after the application of an electric pulse to a suspension
 277

of human erythrocytes, it was concluded that the primary effect of an electric pulse
was to implant aqueous pores of limited sizes into the cell membranes . Under most
of the conditions used by Kinosita and Tsong (a single square wave dc pulsee of
approximately 4 kV/cm, 5-120 µs), the electropores would allow permeation of
ions and small molecules (molar mass less than 2000 g), but would not allow the
passage of hemoglobin or enzymes [26-29] . By measuring the rate of diffusion of a
K + tracer, the number of pores was estimated to be small, approximately 200 per
red cells, and the average size of the stable pores was of the order of 1-3 nm [27,28] .
The membrane surface occupied by these stable pores is 0 .01 to 0 .1% [27,28,34] .
Here, "stable pores" simply mean those pores existing between 1 s and a few min
after the electric pulse is turned off . The results of Sowers and Lieber [35] and
Chang and Reese [33] are in basic agreement with our results .
(3) Once the membranes have lost their function as a semipermeable barrier, the
term "isotonic" loses its meaning for a solution of saline, sucrose, or a mixture of
saline and sucrose . However, the membranes are still impermeable to the cyto-
plasmic macromolecules . The cells begin to swell in such an "isotonic" suspension
in the time scale of seconds to min owing to the osmotic pressure of these
cytoplasmic macromolecular contents (which have an equivalent pressure of 20-30
mOs) . Paralleling the cell swelling, there is also a gradual shrinking of the diameter
of the electropores . The colloid osmotic swelling eventually leads to the rupture of
the cell membranes and hence the lysis of the cells when the cell volume approaches
155% of the normal volume [26,29] . The phenomenon, known as colloidal osmotic
hemolysis, is similar to that of the cell lysis caused by the complement fixation of
red cells . In this case, complement forms membrane pores in the cell membrane,
causing the colloid osmotic swelling and hemolysis of the cells .
(4) The swelling and the consequent hemolysis of the field treated red cells could
be prevented by adding molecules larger than the sizes of the electropores to the
external medium to balance the colloidal osmotic pressure of the cytoplasmic fluid .
Oligosaccharides and small proteins have been used [28,29] .
(5) Under osmotically balanced conditions, the permeability of the perforated
cell membranes would return to its normal level . The rate of recovery depends on
the severity of electroporation, or size of the pores, and on temperature . At 4 0 C,
resealing is slow and takes hours or days . At 37 ° C, the resealing is complete within
minutes to hours . For pores which permit permeation of Rb + but not sucrose,
resealing takes about 20 min . But for pores that admitted sucrose, initially, it takes
about 20 h to reseal completely, i .e . to the normal permeability for Rb +. Other cells
with cell walls or a stronger cytoskeleton than erythrocytes, may not swell as readily
and may reseal much faster . In some cells, resealing takes place naturally and does
not require osmotic balancing or any special resealing procedure . However, for
those cells with fragile membranes, the use of different compositions of pulsing
medium and resealing medium has been recommended [28,29,36] . A resealing
medium should contain 20-35 mM of a compound of molecular size larger than the
average size of electropores .
(6) The size of the pores could be controlled . A higher intensity, a longer pulse


278

width, or a lower ionic strength of the suspending medium all lead to formation of
larger pore sizes . For a super-threshold electric pulse, the ionic strength of the
medium seems the most effective for controlling pore size . It is believed that at the
initial millisecond, the pores can be very large if the induced membrane potential
D Pinduc >- 1 V . However, the lifetime of large pores is short (a few ms) . These pores
shrank to 1-3 nm in ms [27,29,36] . To entrap large molecules, e.g. DNA and
proteins, these molecules must first be bound to the cell surface [37] .
(7) Drugs, which were impermeant to cell membranes and had a small molecular
weight, e .g . less than 1000, could be readily loaded into human erythrocytes . And if
the protocol was followed properly, loss of hemoglobin content would be negligible .
Millimolar concentrations of radioactive sucrose and drugs were loaded into mouse
erythrocytes and the sucrose tracer loaded erythrocytes were shown to survive in the
circulation of mice with a normal lifetime of 20 days . Without going through the
resealing step, the sucrose tracer was eliminated from the circulation in 2 h
[36,38,39] .

Electroporation Osmotic imbalance Swelling Membrane rupture

Loading of macromolecules
into a cell envelope or ghost

o • o o 0 0
o, •
o
0

Osmotically balanced No swelling Drug loaded/healed

Fig. 3 . Mechanisms of electric field induced hemolysis of erythrocytes, and loading of enzymes or
antibodies into ghosts, or drugs into 100% hemoglobin retained erythrocytes . In the figure, small dots
denote drugs, small dark circles, cytoplasmic macromolecules ; small open circles, inulin or oligosac-
charide ; Y-shaped molecules, antibodies . In the upper path, an electroporated erythrocyte swells due to
the colloidal osmotic pressure of hemoglobin . When the membrane is ruptured hemoglobin leaks out and
in the process of membrane resealing, antibodies are loaded into the ghost . In the lower path, the
colloidal osmotic pressure of hemoglobin is counterbalanced by inulin or oligosaccharide added to the
external medium. Swelling of the electroporated cell is prevented . A drug of small molar mass (smaller
than 2000 g) can be loaded, and the cell membrane resealed completely . The final product is the drug
loaded intact erythrocytes . See refs . 28, 29 and 38 for details .


279

(8) For the loading of large molecules, such as enzymes or antibodies, rather
drastic conditions had to be used (e .g . a square wave of 10 kV/cm, 20 µs, in a low
ionic strength medium) . In this case, fractions of erythrocytes would be destroyed
beyong repair . Most other red cells would be hemolyzed . The end product would be
ghosts loaded with these macromolecules . Figure 3, which is essentially identical to
that which we proposed in 1977 [26-28], summarizes different steps of pore
formation, loading of molecules of different sizes, and resealing [27,36] . A kinetic
analysis of these events is discussed below .

(IV) KINETICS AND MECHANISMS OF ELECTROPORATION

Electroporation of lipid vesicles

Lipid bilayers are dynamic structures . For a pure synthetic lipid such as dimyri-
stoylphosphatidyl choline (DMPC) or dipalmitoylphosphatidyl choline (DPPC),
analysis of the phase properties has suggested that there are lattice defects in the
bilayer even at temperatures 10 or 15 ° C below their respective crystalline-liquid
crystalline phase transition temperatures [23,40,41] . As the temperature approaches
the phase transition temperature, Thaase (24 .2 ° C for DMPC and 42.2 ° C for DPPC)
the concentration of these defects increases. These defects are composed of mixtures
of lipid molecules with their hydrocarbon chains either in all-trans or in mixed
trans/cis configurations. According to the analysis of Kanehisa and Tsong [40], the
permeability of a lipid vesicle depends on the state of the lipid bilayer and on the
quantity and the size distribution of these defects . They used the following relation-
ship to express the permeability of a lipid bilayer .
P = Psa e-E,/RT + Pf (1 - a) e - Ef/RT + Pbr) e - Eb/RT (1)
Equation (1) relates the total permeability of the bilayer to an ion or a molecule, P,
to its permeability through the solid-like state, Ps, to the fluid-like state, P f , and to
the boundary region between these two states, P b . a, E, Ef , E b, and j are,
respectively, the fraction of lipid in the solid-like (S) state, the activation energy of
the permeation process in the solid-like state, in the fluid-like (F) state, in the
boundary region of the two states, and the quantity of the boundary state . For an
ion, PS and Pf are negligible compared to P b, and its permeability is dominated by
the last term of eqn . (1), i.e . the permeation through the lattice defects . At room
temperature, as much as 0 .01 to 1% of lipid bilayer surface is composed of these
defects . These defects could be similar to the hydrophobic pores proposed by Abidor
et al . [42] although the defects were considered dynamic, flickering structures and
they had limited life times ranging from us to ms [22,23,43] . Once a 4membr is
imposed, a current will flow through these defects and as much as i 2R At of heat
will be generated in the time span of At . This heat can induce the S to F phase
transition and also greatly increases the value of q [40] . If 01P ;n d Ue exceeds D'~ CC1,,
i.e . the dielectric strength of the bilayer, there will be an irreversible expansion of
pores until the field is turned off . In extreme cases, single pores as large as 1-2 µm
in diameter could be generated in a large lipid vesicle (several µm in diameter)


2 80

[34,44] . When the electric field is turned off, these pores reseal in µs to ms. Equation
(2) sumarizes these kinetic steps.

(2)

B' C'
The first step (A = B) is the pore initiation step and the second step (B -* C) is the
pore expansion step . After the field is turn off, the system will return to its initial
state by a different path (C --> C' - B' -A) simply because the conditions for
electroporation and for annealing are different, the former being under the influence
of a strong electric field and the latter being under zero field .
If an applied field is much higher than the critical field, i .e . E appl > E,,a ,, a large
vesicle will be fragmented and small vesicles will form from these membrane
fragments when the field is turned off. A simple kinetic scheme describing these
events is
C Ai

A B _ D _ AZ (3)

E ' A3
In this case, the system starts with A and ends up in A, and is thus irreversible .
In Schemes (2) and (3), the A to B transition includes the formation of new kinks
(or trans to cis isomerization of CH2-CH2 bonds) in lipid molecules, which is
known to occur in the 10 ns time range [45] . In Scheme (2), the B to C transition
involves conformational changes and dipole re-orientation of the lipid molecule and
should occur in the µs to ms time range, and the C to C' transition may involve the
formation of hydrophilic pores, or aqueous pores, in the ms range. The C' to B' to A
transitions are the relaxation reactions which return the system to the zero field
equilibrium . These processes occur in the ms to s time range . In Scheme (3), the B to
C, D and E transitions involve charge repulsions and the breakdown of hydrophobic
interactions resulting in fragmentation of the bilayers . The transitions from B, C,
and D to A, are the reformation of stable small vesicles from these fragments . These
reactions should occur in the µs to s time range . Time constants for some of these
dynamic events in the lipid bilayer have not been measured directly, but one would
expect them to be similar to the time constants of corresponding molecular events
induced by the pulsed laser temperature jump [43,45] .

Electroporation of cell membranes

The key structure of a cell membrane is similar to that of a lipid bilayer, i .e . a
hydrophobic central layer, about 2-4 nm thick, sandwiched between the bitract
hydrophilic layers (each 2-4 nm thick), even though the composition of a cell
 28 1

membrane is much more complex, being composed of lipids, proteins, carbohydrates

and their derivatives . One would expect that in a first approximation, their electric
and dielectric properties would be similar . This is borne out by the fact that many
theories developed for lipid vesicles seem equally applicable to cell membranes, at
least when details are not crucial [46] . However, the biological function of a cell
membrane starts from where the property of the membrane diverts from that of the
lipid bilayer and this fact would undoubtedly be reflected in its response to an
applied electric field . In a cell membrane, there are many specific channels for
cations, H+, K +, Na +, Ca", etc . and anions, Cl -, P043-, etc . There are pumps and
other types of membrane transport systems, as well . Many of these channels or
pumps are voltage sensitive . The gating voltage is in the range 10 to 150 mV .
Therefore, a change in the membrane potential much smaller than the dielectric
breakdown potential of a lipid bilayer, which is in the range 150 to 350 mV [2], may
open or close some membrane transport systems . Single channel measurements
indicate that the transit time for the channel opening and closing is typically in the
µs or faster time scales . Once such a channel is forced to open by an excessive
potential induced by the applied field, it will experience an enormous local heating
due to the high current density passing through the channel and as a consequence it
may be thermally denatured . When the field is turned off, a denatured membrane
protein may renature slowly or be unable to restore its initial conformational state .
If the duration of an electric pulse is prolonged to ms or longer, these heat
denatured pores will expand even further due to the mechanical effect of the
hydrodynamic flow induced by the electroosmosis of ions [47] .
Experiments were designed to test these ideas . The electric conductance arising
from membrane pores may be measured if one uses a dense suspension of cells .
Kinosita and Tsong [48] investigated the kinetics of pore formation by monitoring
the time dependent evolution of membrane conductance. It was found that pore
initiation took place in less than 1 µs for the human erythrocyte membrane and pore
expansion occurred in the 50 µs to ms time range when a field of 3-5 kV/cm was
used . Once the electric field was turned off, pores began to shrink in ms and this
process continued for min . However, these electropores were never resealed
completely. For complete resealing, the procedures outlined earlier had to be
performed. Later, Serpersu et al . [49] used short rectangular electric pulses (3-5
kV/cm) for their experiments and found that no change in the membrane permea-
bility was detectable for the erythrocytes treated with a pulse of duration less than
0.2 µs (comparable to the membrane relaxation time of an erythrocyte) . However,
these red cells became permeable to Rb+ if the pulse width was increased to 0 .5 µs
or longer . For the membranes to become permeable to sucrose required a pulse with
a duration longer than several µs. These results confirmed the results of the
conductivity measurements and dismissed some suggestions that the conductance
measured by Kinosita and Tsong [48] reflected mainly the relaxation of the surface
charges [30].
Based on the above data, I propose that there are two different pathways for the
electroporation of erythrocyte membranes ; one occurring in lipid domains or lipid


2 82

patches and the other occurring in protein channels, transport systems or pumps .
The simplest, yet most consistent mechanisms would be,

A B-->C- *C' >B' 'A

- >
Pclose ~- Popen Pdenatr

[O]

Equation (4 .1) describes events that occur in the lipid domains and eqn . (4.2)
describes events that occur in protein components of the membrane . Equation (4 .1)
is identical to eqn. (2). In eqn . (4 .2), Pclose, Popen' Pdenatr' Pirrev and [O] are,
respectively, a membrane channel or pore in its closed conformation, in its open
conformation, in a denatured (possibly due to heat) state, in an irreversibly
denatured state, and in an excised state (e.g . removed from cell membrane by
exocytosis) . Both pathways compete in parallel . In principle, the Pclose to Pope
transition should occur when the D 'membr reaches the gating voltage of a channel .
However, opening of such channels may not be sufficient to dissipate the AY'induc
rapidly and therefore pathway (4 .1) would take place irrespective of pathway (4 .2) .
Equations (4 .1) and (4.2) are considered the primary events in the electroporation
of a cell membrane . There are other secondary effects which may have more direct
consequences than the primary effects on the destiny of the treated cells . A few of
the possible secondary events are discussed . First, once artificial pores are intro-
duced, the semipermeable barrier of the membrane is destroyed. This would lead to
colloidal swelling and all of the succeeding events outlined in Fig. 3 [26,29] . Second,
loss of the cell's natural membrane potential may lead to a disintegration of the
cytoskeletal network or the disruption of other membrane properties and cellular
processes, e.g . lipid asymmetry . Third, electric fields may modify and change the
chemistry of lipids, proteins, or carbohydrates, especially of those functional groups
involved in redox reactions . One such modification could be the phosphorylation or
dephosphorylation of membrane proteins . These modifications could lead to a state
of the membrane described by Sowers as fusogenic [50] . Loss of lipid asymmetry, if
it occurs, must be confined to a limited area . A global loss of lipid asymmetry in a
cell is known to cause cell death .
Evidence for "semi-permanent" modification of membrane proteins has been
accumulating [51,53] . Teissie and I [53,54] showed that the field induced membrane
conductance described by Kinosita and Tsong [48] contained a component (ap-
proximately 30%) which could be inhibited quantitatively by ouabain, a specific
inhibitor of Na, K-ATPase . They then showed that if the applied field induced
transmembrane potential was comparable to the physiological potential of the cell,
e.g . by using a low amplitude (16 V/cm) ac field, this field induced membrane
conductance would be completely reversible [54] . Cells treated with such ac fields
were normal with respect to their survivability and ion transport activity
[14,15,17,54] . Apparently, only the reversible step in eqn . (4 .2) was induced by the
low amplitude ac field . This concept captivated our attention for the next few years .


283

Structures of pores
The structure of an electropore remains illusive despite intense interest among
investigators and many attempts to image it . This is not surprising as these aqueous
pores are dynamic structures and they are constantly changing, in tts to s in lipid
domains and in µs to hours in protein meshwork . To take a snapshot of the
structure in the .ts to ms time scale is possible by newly developed pulsed laser
fluorescence microscopy [34,44] and by rapid-freezing electron microscopy [33,55,56],
but these techniques, so far, have achieved limited spatial resolution . However, in a
lipid bilayer, there are few ways lipid molecules can be arranged to form a stable
pore, and many investigators agree on some structures which would be consistent
with the physical chemistry of the amphipathic interactions of lipid molecules at the
lipid-water interface . Figure 4 gives an example commonly seen in the literature
[32,42] . Similar pores may also occur in limited regions of a cell membrane where
small lipid patches are permissible . However, in a cell membrane other factors must
be taken into consideration .
Electropores of erythrocyte membranes imaged by rapid-freezing electron mi-
croscopy are particularly informative [33] . These pores have been induced by a dc
shifted radio-frequency (RF) field of 4-5 kV/cm with a duration of 0 .3 ms (100
kHz) and show volcano-like structures with diameters of 25 to 125 nm approxi-
mately 40 ms after the electric treatment. The composition of the surrounding
materials and the detailed arrangement of molecules in these volcano-like structures
cannot be determined . They are probably made of lipids and cytoskeletal protein
meshwork. The actual opening of a volcano could be much smaller than the
diameter of the volcano's aperture . A thin layer of lipids may permeate across the
orifice. Figure 5 gives some images of electropores obtained by Chang and Reese
[33] . It is understood that if an electric pulse is much stronger than the critical
strength, E,,,.; t (approximately 3 kV/cm for human erythrocyte), and longer than 10
ms, membrane cracks may develop [55,56] . A large piece of membrane may also be
ripped away from a cell [55,56] .

,
ffy
till
A B
Fig . 4 . Hypothetical pores of a lipid bilayer . (A) a lattice defect, or a hydrophobic pore ; (B) a hydrophilic
pore . A hydrophobic pore is small and unstable but a hydrophilic pore is relatively large and stable . The
pore initiation step induced by an electric pulse is to enlarge a lattice defect to form a hydrophobic pore
and the pore expansion step is to expand a hydrophobic pore further, leading to rearrangement of the
lipid molecules to form a larger hydrophilic pore . See text for discussion .
284

Fig. 5 . Experimental visualization of electropores on membranes of human erythrocyte . Chang and Reese
[33] used rapid freezing electron microscopy to investigate the kinetics of membrane structural changes
induced by a direct current shifted radio-frequency electric field (100 kHz, 4-5 kV/cm, 0.3 ms) in the
time range of a few ms to many min . Freezing of a cell was accomplished in 0.1 ms. The left photo is an
E-face replica (the inwardly directed outer half-membrane) of a red cell frozen at 40 ms following the
electroporation . The right photo is a P-face replia (the outwardly directed inner half-membrane) 220 ms
after the electroporation . 40 ms after the electroporation, pores were clearly visible but 220 ms later, these
pores seemed to be covered by a layer of membrane . The bar at the lower left represents a length of 0 .2
µm. See ref. 33 for details. These photos were kindly provided by Dr . D . Chang .

Electroporation by an oscillating electric field

Most electroporation experiments use square wave dc pulses or exponentially
decaying dc pulses . To calculate the transmembrane electric potential induced by a
dc pulse, A `Y induc, the Maxwell relationship has been used .

A`Pinduc - 1 .SR Ceii Eappi cos 0 (5)

Equation (5) is applicable to cells of spherical shape with radius R Ceii

[18,27,31,44,57,58] . The angular distribution of 4
induc was recorded using a poten-
tial sensitive fluorescence dye [34,44,58] . The relationship has also been used to
calculate a correct breakdown potential O~,,.; t for lipid vesicles of well defined size
distribution, 95 ± 5 nm, to be 210 mV [59,60] . For a cell of non-spherical shape,
eqn. (5) must be modified [27,57,58] . However, AY'induc calculated from eqn . (5) is a
good approximation for most cells .
When an ac field is used, the actual induced membrane potential is smaller than
the AY'induc calculated from eqn. (5) when the frequency of the ac field is close to the
 2 85

membrane relaxation time, Tmembr • Schwan [61] has derived a relationship from the
basic electromagnetic theory for calculating an ac induced transmembrane potential,
0 0 induc,ac •
1/2
1
0 `Yinduc,ac - 1 .SRceI,Eappl Cos 6/[1 + (WTmembr) Z (6)
where

Tmembr = RcellCmembr( Pint + Pext/ 2 ) (7)

Here W, Cmembr, Pint and peat are, respectively, the angular frequency of the ac field,
the membrane capacitance, the resistivity of the internal fluid, and the resistivity of

MOU
Fig . 6. Electroporation of myeloma by an alternating electric field . Myeloma cell line Tib 9 was used in
the experiment . Electroporation was monitored by the permeation of the probe, propidium iodide (upper
formula), which became strongly fluorescent on binding to DNA . When the ac field reached Ecr; t (in this
case 1 kHz, 550 V/cm, 200 ms), the membrane was perforated at the two poles facing the electrodes, and
the dye permeated into the cell rapidly . The center figure shows a cell in bright field and its relative
orientation to the electrodes . The lower figure gives photos of the cell 3 s, 20 s and 3 min after the
electroporation (from left to right) . The dye permeated from the edge to the center in a few min . The
radius of the cell was 6 .5 µm. See Marszalek et al . [62] for details of the experiment and the analysis .
286

the external medium. Marszalek et al. [62] have used ac fields of different frequen-
cies to perform electroporation on a line of myeloma cells Tib9 . Membrane
permeabilization was monitored by the entry of a fluorescent probe, propidium
iodide, which has a high affinity for DNA . This dye is weakly fluorescent in solution
but becomes strongly fluorescent when bound to DNA . The Eapp, to generate Olf/cr;,
was strongly dependent on the frequency of the ac field . The Ecri, were fitted to
eqns . (6) and (7) by regression analysis . The analysis also allowed these authors to
obtain Cmembr and p in ,. A good fit of the data to eqns. (6) and (7) indicates that the
Schwan equation is appropriate for calculating 0 `Y induc,ac of a cell of spherical shape .
Figure 6 gives an example of the dye permeating from the two poles of a myeloma
cell.

Loading of exogenous molecules or ions into living cells

Since electropores are stable at temperatures below 15 ° C and can be resealed at
temperatures near the growth temperature of a cell, generally at 37 ° C, the electro-
poration method may be used to load exogenous molecules or ions into living cells .
Loading of drugs and ions into cell envelopes or erythrocyte ghosts had been
conceived early . An advantage of the electroporation method is that ghosts may be
obtained in phosphate buffer saline instead of low ionic strength medium [30,46] .
Having worked out the colloidal osmotic hemolysis mechanism of erythrocytes,
Kinosita and Tsong attempted to load drugs into erythrocytes by electroporation
[36,38,39] . Loading of radioactive sucrose was successful and it was shown that
sucrose loaded mouse erythrocytes exhibited a normal life time in the mouse
circulation. A relatively constant level of the model drug was obtained in the mouse
circulation for 15-20 days [38] . If the sucrose was injected directly by venipuncture,
or if the sucrose loaded erythrocytes were not resealed according to the protocol
described earlier, the loaded sucrose leaked out and dissipated from the circulation
within 2-3 h [38] . These results demonstrate that after proper resealing, the drug
loaded erythrocytes were fully compatible with the normal erythrocytes . Erythro-
cytes with leaky membranes or lost lipid asymmetry are known to be rejected from
the circulation rapidly . In this respect, it is interesting to point out that Chalmer has
used 20 kV cm - ' ms - ' electric pulses to prepare membrane debris and broken
ghosts of erythrocytes and has used this debris as drug carrier. He claimed that this
debris is superior as a drug carrier to intact erythrocytes or living cells [63] . His
results contrast a general consensus in the field that any damaged erythrocytes
cannot survive in the circulation . Many investigators have attempted to use
erythrocyte ghosts as drug or enzyme carriers . To my knowledge, none have
reported positive results .
Kaibara and Tsong [64] loaded antisickling reagents into heterozygous sickled
erythrocytes and showed that these drugs could prevent sickling of the red cells
under deoxygenated conditions . However, the method was abandoned because these
drugs are mostly membrane permeant and would leak out within a few hours at
37'C . Modification of drugs to reduce their permeability would be a logical
approach .
 2 87

Loading of Ca 21 into sea urchin eggs has been done by Rossignol et al . [65] .
They have shown that Ca 2+ can trigger development of partial fertilization en-
velopes. These electric field treated eggs were viable and could later develop
normally when sperm was added . Knox et al . loaded radioactive ATP into dic-
tyostelium and followed its metabolisms in the cells [66] . These experiments demon-
strated that exogenous molecules could be loaded into living cells, not just cell
envelopes or ghosts, and set the stage for the loading of genetic material into living
cells .

Mechanisms of DNA transfection by electroporation

The molar mass of plasmid DNA is generally in the 2 X 106 g range . How such a
large molecule enters a cell by electroporation is of considerable interest [9,10] . One
theory is that a DNA molecule, which is highly charged, will orient in a strong
electric field . An electrophoretic force will then drive the molecule across the cell
membrane, either through an electropore or simply by "piercing through" the lipid
bilayer . Xie et al . [37] have investigated transfection of E . coli by plasmid pBR322
at a low DNA/cell ratio (< 0 .1) . When DNA was mainly in the solution, the
probability of a DNA molecule being close to an E . coli cell was small . Since the
area of electropores on the membrane surface has been estimated to be about 0 .01%
[27,34,35], the chance of a direct hit of the pores by a DNA molecule would be
negligibly small . In their experiments, approximately 0 .7% of cells were transfected .
Thus, direct electrophoresis of DNA in solution across membrane pores is unlikely .
DNA entry by the second mechanism has a much larger probability . However, Xie
et al. [37] showed that transfection as high as 2 X 104/µg DNA was obtained when
DNA was added 10 min after the electric field was turned off . In other words, entry
of the DNA did not require the presence of an applied field . The transfection
efficiency of 2 x 104/µg DNA was much smaller than the 2 X 108/µg DNA
obtained if DNA was added at least 30 s before electroporation . However, it was
still approximately 1000-10000 times greater than the control in which the cells
were not exposed to an electric pulse . The greatly reduced efficiency in the first case
was due to shrinking of the pores, which is known to occur during the 10 min
incubation time. Other experiments indicate that if DNA is bound to the cell
surface before the electroporation, the transfection efficiency is increased dramati-
cally. DNA binding is promoted by cations, especially divalent cations, Ca 2+ or
Mg t+ . Figure 7 compares several mechanisms for DNA entry into a cell by
electroporation. Our present understanding favors mechanisms B and C . It remains
unclear how a molecule the size of DNA can enter a cell by surface diffusion
through an electropore . Chizmadzhev and co-workers did find that an electro-
phoretic force enhances the transfection efficiency 2 .5-3-fold [67] . Chernomordik et
al . [68] have shown that DNA taken up by large lipid vesicles is inaccessible to
binding by ethydium bromide and have suggested that DNA uptake is by endo-
some-like lipid vesicles . Further studies should resolve these questions .
Another exploratory experiment of particular interest to us is the use of low
amplitude, low frequency ac fields for DNA transfection . Xie and Tsong [69] have


288

shown that a 200 V/cm (peak-to-peak) ac field, which can induce a transmembrane
potential in E. coli of the order of 20-30 mV, can induce transfection of E. coli by
plasmid DNA, with a transfection efficiency as high as 1 .2 X 10 5/µg DNA. Since
A ' induc is much smaller than A`Ycrit, it is unliekly that the ac field caused
electroporation. In these experiments, an ac field as low as 50 V/cm was shown to
cause transfection of E. coli, although in this case the efficiency was low. To
interpret these results, we have proposed that the low amplitude low frequency ac
field could enhance conformational oscillation of certain transport or carrier pro-
teins, or DNA binding sites, and in doing so facilitate the transfer of DNA into the
cells . This would be similar to the electroconformational coupling model for the
enzyme activation discussed below [18,70] .

I II III

E
B

CO \ rfnl
i \\
C '- l 8

D
O
Fig . 7 . Mechanisms of DNA transfection by the electroporation method . In Model A, a DNA in solution
diffuses into a cell through the electropore . In Model B, a DNA would enter a cell only if it first binds to
the cell surface . In Model C, DNA was added after the electroporation was done . DNA entrance in this
case is accomplished in the absence of a field and cannot be driven by any electrophoretic forces . In
Model D, a DNA is electrophoretically driven into a cell and in the process carries a piece of lipid
membrane with it . See text for comparisons and discussion of evidence in favor of Models B and C over
the other models.
 289

(V) SOME RELATED MEMBRANE PHENOMENA

Membrane fusion
Membrane fusion apparently is closely associated with the electroporation phe-
nomenon . Fusion can occur when an electroporation pulse is applied while two cells
are in contact [30,71] or when cells pretreated with electroporation pulses are
brought into contact [50] . Fusion occurs because of modification of the membrane
surface by the pulsed electric field . As was discussed above, the "fusogenic" state of
a cell membrane could mean : loss of lipid asymmetry, existence of pores, modifica-
tion of lipid compositions or protein meshwork, and other unspecified changes .
Until now, no convincing evidence favors one explanation over the others . Many
molecular models seen in the literature are reasonable and are supported mainly by
theories rather than by experimental evidence . However, studies by Hermann et al .
[72] provide strong evidence that fusion of vesicular stomatitis virus to erythrocyte
ghosts occurs more rapidly when the lipid asymmetry in the ghost is lost . Modifica-
tion of surface charges and the head group composition did not render the ghost
more fusogenic to the virus . If the loss of lipid asymmetry would extend over the
whole cell, the cell would have a shortened life time and can no longer be considered
normal . Whether a cell can restore the lost lipid asymmetry is not yet known . If a
cell cannot restore its lipid asymmetry, then electrofusion of cell membranes cannot
be due to a loss of the lipid asymmetry because most of the fused cells will survive .
Most investigators use a high frequency (100 kHz to 10 MHz) low amplitude
(about 5 V/cm) ac field to elicit cell contact for electrofusion [30,46] . Dielectro-
phoresis is convenient for a direct visual observation . The phenomenon itself has a
certain theoretical appeal and allows some cell parameters, e .g . membrane conduc-
tance (Gmembr ), capacitance (Cmembr) and resistivity of the cytoplasmic fluid (Pin,) ,
to be determined [73] . However, for application to the preparation of hetrokaryons
and hybridomas, dielectrophoresis is no more convenient than simply pelleting a cell
mixture by a centrifugation followed by an incomplete dispersion of the pellet . We
have tested another approach, namely the surface receptor mediated selection of
cells for electrofusion [11,74] . The basic design of the method, using hybridoma
preparations as an example, is to use an antigen to select B-lymphocytes for fusing
with myeloma cells . An antigen is chemically cross-linked to avidin and the
myeloma is coated with biotin . Avidin has a high affinity for biotin . Thus,
B-lymphocytes recognized by, and binding to the antigen/avidin complex will be
directed to biotin coated myeloma cells . Most cell doublets thus formed are
composed of one myeloma cell and one appropriate B-lymphocyte . B-lymphocytes
which are not destined to secrete antibodies against the antigen will not conjugate
with the myeloma cells and thus cannot fuse with them . This procedure, or any
procedure of similar design, will have the advantage of selecting the right partners
for fusions rather than fusing with random partners .
Membrane bleb formation
One interesting phenomenon associated with electroporation of cells concerns
membrane bleb formation (see ref . 75 and references therein) . Gass and
290

Chernomordik [75] have investigated these membrane blebs induced by pulsed

electric fields in fetal bovine trachea, mouse embryo fibroblasts, rat fibloblasts and
L-cells . Their results further strengthen our interpretation that pores induced in the
lipid domains of a cell membrane are rapidly reversible, in contrast to those induced
in the protein domains . Consider a volcano-like membrane pore such as shown by
Chang and Reese [33] . After a short period, the volcano aperture is permeated with
a bilayer of membrane lipids . The permeability of the lipid layer is small. However,
since there are still irreversibly denatured small protein pores which continue to be
leaky to ions, there would be an influx of water due to the colloidal osmotic pressure
of the cytoplasmic content and the cell will swell [26,29], as mentioned earlier . If a
cell has a strong cytoskeletal network or cell wall, its envelope volume cannot
expand readily . This will result in the formation of membrane blebs, composed
mainly of lipids, to accommodate the excess volume of fluid . If this interpretation is
correct, a counterbalancing of the cytoplasmic osmolality in the external medium
(by molecules larger than the pore size, such as inulin) should prevent bleb
formation. If the osmolality of the external medium exceeds that of the internal
medium, the membrane blebs already formed should shrink . Obviously, the number
of blebs a cell can form is limited unless lipids are supplied from the internal stores
of the cell . A similar phenomenon, field induced membrane protrusion, is said to be
due to electro-mechanical effects [76] . Its mechanism of formation is likely different
from that of bleb formation .

Electroinsertion
An electric pulse can induce translocation of a glycoprotein across a lipid bilayer
[77] . If the field strength is chosen to be slightly lower than the critical field, Ecdl ,
which induces a breakdown potential, A~ c ;t , glycoproteins may be inserted into the
membrane [20] . This type of technique has been applied to the insertion of CD4 (an
AIDS virus receptor protein of lymphocyte) into erythrocyte membranes and
activity of the inserted form has been demonstrated [78] . Besides its potential
clinical applications, electroinsertion combined with DNA cloning techniques will
provide an important tool for studies of the reconstitution of cell membranes .

Electroactivation
Electroporation and electrofusion of cell membranes require an electric field
which induces an unnaturally high transmembrane potential, i .e . when Atpinduc >
A`Ycrit, where 4crit is the breakdown potential (O grit/d being the dielectric
strength and d the thickness of the membrane) . These phenomena are thus often
considered purely artificial and have little relevance to the normal function of cells .
This may not be a proper assessment of the phenomenon . A membrane potential
can influence ionic and molecular transport, protein trafficking, protein phosphory-
lation, cell-to-cell communication, and energy transduction . Pathological cells are
often found to have weaker dielectric strength than normal cells [78,79] and are
easily "electroporated" by a normal physiological membrane potential . From the
 291

above experiments, we concluded that if we could apply a proper field strength to

induce the PclOSe ;'~ Popen transition without producing the irreversible steps in eqns .
(4)-(2), we might be able to control reversibly the activity of an enzyme by applied
fields . The physiological membrane potential, '64physiol, of human erythrocyte is
about 10 mV. To generate this magnitude of transmembrane potential would require
an applied field of about 20 V/cm . The low amplitude ac stimulation experiments
mentioned above [54] represent our first attempt .
A convincing observation was made by Serpersu and Tsong with the ac activation
of Na,K-ATPase in human erythrocytes [14,15] . These authors used an ac field of
approximately 20 V/cm and a frequency around 1 kHz to activate the K + pumping
mode of the Na,K-ATPase . The electric field induced activity was completely
reversible . Red cells treated with such a field for 2 h were found to exhibit the
normal biconcave shape and normal ion transport activities when the field was
turned off . No hemolysis beyond the control samples was detected in the ac treated
samples . The ac stimulated activity was assayed by the influx of radioactive tracers,
42 K + or 86 Rb' and the evidence that the influx of these ions was catalyzed by
Na,K-ATPase was obtained by its inhibition by ouabain, a specific inhibitor of the
enzyme, with a Km of 0.15 µM . Most interestingly, this ac stimulated activity
showed a voltage optimum at 20 V/cm and a frequency optimum at 1 kHz [14] . Liu
et al. continued this investigation and were able to activate the Na' pumping mode
of the enzyme at 20 V/cm, and at 1 MHz [17] . These ac activated transport
activities were pumping ions up their respective concentration gradients without
requiring the splitting of ATP . The energy required must have been derived from the
applied ac fields . Na,K-ATPase is not the only enzyme shown to absorb energy and
couple it to drive an endergonic reaction . ATP synthases from different sources have
also been shown to capture energy from a dc field to fuel ATP synthesis [13,16] . A
recent study by Chauvin et al . [80] indicates that beef heart mitochondrial ATPase
can also be induced to synthesize ATP with a low amplitude ac field (60 V/cm)
when newly formed ATP is rapidly converted to glucose-6-phosphate by hexokinase .
This greatly reduces the phosphorylation potential, and the rate of ATP synthesis is
solely determined by catalytic rate constants . An ac field enhances the rate and
there is a frequency optimum of 10 Hz.
Electroactivation has also been adopted to interpret transfection of E. coli by
plasmid DNA PCU18 using low amplitude (50-200 V/cm) ac fields [69] . The
A Pinduc was in the range of 30 mV and was much lower than D on, for electropora-
tion. The authors of ref. 69 suggested that DNA entry into E. coli could be by a
similar mechanism as that which activates a membrane transport system by an
oscillating electric field, i .e. by electroconformational coupling of a membrane
transport system or protein .

Electroconformational coupling
Another development in our study of effects of electric fields on membrane
functions is the theory of electroconformational coupling . This concept has been
used to guide the design of many electroactivation experiments since the early years,
2 92

especially for those experiments concerning the activation of ATPases [18,29,70] .

Initially, dc pulses were used to induce ATP synthesis in mitochondrial ATPase
[16,81]. But it soon became clear to us that it would be difficult to achieve enzyme
turnover with a dc pulse by electroconformational change unless there existed some
internal mechanism for modulating a dc field to become an oscillating field . Only an
ac field can turn the catalytic wheel (cycle) of an enzyme or a membrane trans-
porter . Later, the thermodynamic basis of electroconformational changes and cou-
pling of these changes to a ligand binding reaction for energy transduction was laid
out explicitly [18,70] . A simple four-state facilitated membrane transport model was
shown to interact with an oscillating electric field, capture energy and transduce it
to the chemical potential energy of a ligand, or the y-phosphodiester bond energy of
ATP [70] . Many properties of the electroconformational coupling model have been
examined [82-91] and it is now clear that such a mechanism, or mechanisms similar
to it, are implicated in biological energy and signal transduction [18,92] . Until now,
detailed analyses have been done only for the case of an electric field ; however, any
other oscillating fields which can effectively interact with a protein and enforce
conformational oscillations of the protein should have the same effect as an electric
field [18,92] . Markin and Tsong [93] have formulated a piezo-electric coupling
model based on the same principle . A membrane transport protein which has
several conformational states of different molar volumes or of different areas in the
cell membrane is shown to absorb energy from an acoustic wave to pump an ion
across the membrane . This process converts acoustic energy into the electrochemical
potential energy of the ion. Any enzyme which exhibits Michaelis-Menten kinetics
has the ability to interact with an oscillating potential, and through the enforced
conformational oscillations of the enzyme, energy or signals may be converted into
other forms [18,87] .

Perspective
Electric modula membrane permeability is a natural process of a cell .
When an irreversible modification occurs because of an excess transmembrane
electric potential imposed externally, the bilayer structure and protein constituents
of a cell membrane are modified . Some processes are reversible and some are
irreversible. One manifestation in a cell membrane is the implantation of aqueous
pores. Invisible pores are easily modeled and analyzed by theories and consequently
they have received disproportionately greater attention . Visible, volcano-like pores
are much more difficult to analyze but they are the genuine imprints of the applied
electric field . Other molecular events, such as the disorganization of lipid arrange-
ment, denaturation of membrane proteins, chemical modification of molecules, etc .,
may be responsible for cell membranes becoming fusogenic . If an applied field is
controlled carefully, it may activate a membrane enzyme, a transporter, a receptor,
an energy transducer, a signal transducer, or a gene regulator . Electroactivation
promises to bridge the gap between the physiology and the biochemistry of the cell
membrane . Studies of the molecular basis for signal processing and energy transduc-
tion would further our knowledge for the fabrication of microelectronics [94] .
 293

NOTE ADDED IN PROOF

I am delighted to obtain two pieces of information before this paper goes to

print . Pliquett reported effects of ac fields on single Oxytrichia cells in 1968 [95] . He
observed, what appeared to be membrane ruptures and formation of membrane
blebs . A threshold frequency of 100 Hz was detected when critical breakdown
potential was plotted vs . the frequency of the ac field . This work was further
discussed in a later paper [96] . Matzke and Matzke [97] proposed, in 1985, that
electric potential across the nuclear membrane could be involved in the regulation
of gene expression . Matzke et al . [98] then used a potential sensitive fluorescence
probe, DiOC6, to probe the nuclear membrane potential in various stages of
differentiation in Acetabularia cells [98]. They observed dramatic changes in the
nuclear membrane in potential during cell differentiations .

ACKNOWLEDGEMENTS

I wish to thank my past and present colleagues who have participated in this
work for their dedication and contributions . Dr. Donald Chang of Baylor College of
Medicine kindly provided images of electropores (Fig . 5) for this article. Thanks are
also due to C.J. Gross for help with the manuscript . This work was supported by a
grant from the United States Office of Naval Research .

REFERENCES

1 K.S . Cole, Membranes, Ions and Impulses,, University of California Press, Berkeley, 1972.
2 H.T. Tien, Bilayer Lipid Membranes (BLM), Marcel Dekker, New York, 1974 .
3 D.E. Goldman, J. Gen . Physiol ., 27 (1943) 37 .
4 H.G .L . Coster, Biophys . J ., 5 (1965) 669 .
5 C. Huang, L . Wheeldon and T.E. Thompson, J . Mol. Biol., 8 (1964) 148 .
6 A .J.H. Sale and W .A . Hamilton, Biochim. Biophys. Acta, 148 (1967) 781 .
7 W.A. Hamilton and A .J .H . Sale, Biochim. Biophys. Acta, 148 (1967) 789.
8 A .J.H. Sale and W .A . Hamilton, Biochim. Biophys. Acta, 163 (1968) 73 .
9 T: K. Wong and E. Neumann, Biochem . Biophys . Res . Commun., 107 (1982) 584 .
10 E. Neumann, M. Schaefer-Ridder, Y . Wang and P.H . Hofschneider, EMBO J., 1 (1982) 841 .
11 M.M .S. Lo, T.Y . Tsong, M.K. Conrad, S .M. Strittmatter, L .D. Hester and S. Snyder, Nature
(London), 310 (1984) 792 .
12 U . Zimmermann, J. Vienken, J. Halfmann and C .C . Emeis, Adv . Biotechnol. Proc . 4(1985) 79 .
13 H .T . Witt, E. Schlodder and P . Graber, FEBS Lett ., 69 (1976) 272 .
14 E.H . Serpersu and T .Y . Tsong, J. Membr . Biol., 74 (1983) 191 .
15 E.H . Serpersu and T .Y . Tsong, J. Biol . Chem., 259 (1984) 7155 .
16 J. Teissie, B .E. Knox, T.Y. Tsong and J. Wherle, Proc . Natl. Acad. Sci . USA, 78 (1981) 7473.
17 D .S . Liu, R .D. Astumian and T.Y . Tsong, J . Biol. Chem ., 265 (1990) 7260.
18 T.Y . Tsong, Annu. Rev. Biophys. Biophys. Chem., 19 (1990) 83 .
19 M . Blank and E. Findl, Mechanistic Approaches to Interactions of Electric and and Electromagnetic
Fields with Living Systems, Plenum Press, New York, 1987 .
20 Y . Mouneimne, P : F . Tosi, Y . Gazitt and C . Nicolau, Biochem. Biophys . Res . Commun ., 159 (1989)
34 .
21 K.T. Powell, A.W . Morgenthaler and J .C . Weaver, Biophys . J., 56 (1989) 1163 .
294

22 T.Y . Tsong, Proc . Natl . Acad. Sci . USA, 71 (1974) 2684 .

23 T.Y. Tsong and M.I. Kanehisa, Biochemistry, 16 (1977) 2674 .
24 T.Y . Tsong, FASEB J., Fed . Proc., 33 (1974) 1342 .
25 T.Y. Tsong and E. Kingsley, J . Biol . Chem ., 250 (1975) 786.
26 K. Kinosita and T.Y. Tsong, Proc . Natl . Acad . Sci . USA, 74 (1977) 1923 .
27 K . Kinosita and T.Y . Tsong, Biochim . Biophys. Acta, 471 (1977) 438 .
28 K. Kinosita and T.Y . Tsong, Nature (London), 268 (1977) 438 .
29 T.Y. Tsong, Biosci . Rep ., 3 (1983) 487 .
30 U . Zimmermann, Biochim . Biophys. Acta, 694 (1982) 227 .
31 E. Neumann and K. Rosenheck, J . Membr . Biol., 14 (1973) 194 .
32 I .P . Sugar and E. Neumann, Biophys . Chen ., 26 (1984) 211 .
33 D .C. Chang and T.S . Reese, Biophys. J., 58 (1990) 1 .
34 M . Hibino, M . Shigemori, H . Itch, K . Nagayama and K. Kinosita, Biophys . J ., in press.
35 A.E. Sowers and M.L. Lieber, FEBS Lett., 205 (1986) 179 .
36 T .Y . Tsong, Meth . Enzymol ., 149 (1987) 248 .
37 T.-D . Xie, L . Sun and T .Y . Tsong, Biophys . J., 58 (1990) 13 .
38 K . Kinosita and T .Y Tsong, Nature (London), 272 (1978) 258 .
39 T .Y . Tsong and K . Kinosita, Bibl . Haematol . (Basel), 51 (1985) 108 .
40 M .I . Kanehisa and T .Y. Tsong, J. Am. Chem . Soc ., 100 (1978) 424.
41 T .Y . Tsong, M . Greenberg and M .I. Kanehisa, Biochemistry, 16 (1977) 2674.
42 1G . Abidor, V.B . Arakelyan, L.V . Chernomordik, Y .A . Chizmadzhev, V .F . Pastushenko and M .R .
Tarasevich, Bioelectrochem. Bioenerg., 6 (1979) 37 .
43 A . Genz, J .F. Holzwarth and T .Y . Tsong, Biophys. J ., 50 (1986) 1043 .
44 K . Kinosita, I . Ashikawa, N . Saita, H . Yoshimura, H . Itch, K. Nagayama and A. Ikegami, Biophys.
J ., 53 (1988) 1015 .
45 B . Gruenewald, W . Frisch and J.F . Holzwarth, Biochim . Biophys. Acta, 641 (1981) 311 .
46 E . Neumann, A.E . Sowers and C .A . Jordan, Electroporation and Electrofusion in Cell Biology,
Plenum Press, New York, 1989 .
47 S .D . Dimitrov and A .E. Sowers, Biochim . Biophys. Acta, 1022 (1990) 381 .
48 K . Kinosita and T .Y . Tsong, Biochim . Biophys . Acta, 554 (1979) 479 .
49 E.H . Serpersu, K . Kinosita and T .Y . Tsong, Biochim . Biophys . Acta, 812 (1985) 779 .
50 A.E . Sowers, J . Cell Biol ., 103 (1986) 1358 .
51 B . Deuticke, P . Lutkemeier and M . Sistemich, Biochim . Biophys . Acta, 775 (1984) 150 .
52 K . Schwister and B . Deuticke, Biochim . Biophys . Acta, 816 (1985) 332 .
53 J . Teissie and T.Y. Tsong, J . Membr . Biol ., 55 (1980) 133 .
54 J . Teissie and T .Y. Tsong, J . Physiol. (Paris), 77 (1981) 1043 .
55 A.E . Sowers and C.R. Hackenbrock, Proc . Natl . Acad . Sci . USA, 78 (1981) 6246 .
56 D .A . Stenger and S .W . Hui, J . Membr. Biol ., 93 (1986) 43 .
57 D .L . Farkas, R. Korenstein and S .Malkin, Biophys . J ., 45 (1984) 363 .
58 D . Gross, L.M . Loew and W .W . Webb, Biophys. J ., 50 (1986) 339 .
59 J . Teissie and T.Y. Tsong, Biochemistry, 20 (1981) 1548 .
60 M .E. El-Mashak and T.Y . Tsong, Biochemistry, 24 (1985) 2884 .
61 H .P . Schwan In M . Grandolfo, S .M. Michaelson and A . Rindi (Eds.), Biological Effects and
Dosimetry of Nonionizing Radiation, Plenum Press, New York, 1983, pp . 213-231 .
62 P . Marszalek, T.-S . Liu and T .Y . Tsong, Biophys. J., in press .
63 R.A . Charlmers, Bibl . Haematol. (Basel), 51 (1985) 15 .
64 M . Kaibara and T.Y. Tsong, unpublished results, 1980.
65 D .P. Rossignol, G .L . Decker, W .J . Lennarz, T .Y . Tsong and J. Teissie, Biochim . Biophys . Acta, 736
(1983) 346 .
66 B . Knox, P. Derroetes and T.Y . Tsong, unpublished results, 1980 .
67 Yu.A. Chizmaedhzer and co-workers, to be published .
68 L .V . Chernomordik, A .V . Sokolov and V .G. Budker, Biochim . Biophys . Acta, in press.
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69 T .-D . Xie and T.Y . Tsong, Biophys . J ., in press.

70 T .Y . Tsong and R .D . Astumian, Bioelectrochem. Bioenerg., 211 (1986) 457 .
71 J . Teissie, V .P . Knutson, T.Y . Tsong and M .D. Lane, Science, 216 (1982) 537 .
72 A . Hermann, M .J . Clague, A . Puri, S .J. Morris, R. Blumenthal and S. Grimaldi, Biochemistry, 29
(1990) 4054 .
73 P . Marszalek, J .J. Zielinski and M . Fikus, Bioelectrochem . Bioenerg . 22 (1989) 289.
74 T.Y . Tsong and M . Tomita, Meth . Enzymol ., in press .
75 G.V . Gass and L.V. Chernomordik, Biochim . Biophys. Acta, 1023 (1990) 1 .
76 S.V . Popov and L .B . Margolis, J . Cell. Sci., 90 (1988) 379 .
77 J.N . Weinstein, R . Blumenthal, J . van Renswoude, C . Kempf and R.D . Klausner, J .Membr . Biol., 66
(1982) 203 .
78 M. Kaibara and T.Y . Tsong, Biochim. Biophys. Acta, 595 (1980) 146 .
78 C . Nicolau et al., to be published.
79 M . Kaibara and T.Y . Tsong, unpublished results, 1980 .
80 F. Chauvin, R .D. Astumian and T .Y. Tsong, submitted .
81 B.E. Knox and T.Y Tsong, J . Biol . Chem ., 259 (1984) 4757 .
82 H.V . Westerhoff, T.Y . Tsong, P.B. Chock, Y : D . Chen . and R.D . Astumian, Proc . Natl . Acad . Sci.
USA, 83 (1986) 4734 .
83 R.D. Astumian, P.B . Chock, T.Y . Tsong and H.V . Westerhoff, Phys. Rev. A, 39 (1989) 6416 .
84 T.Y . Tsong, R.D . Astumian, Prog. Biophys . Mol . Biol., 50 (1987) 1 .
85 R.D . Astumian, P .B . Chock, T.Y . Tsong, Y: D. Chen and H .V. Westerhoff, Proc . Natl. Acad . Sci .
USA, 84 (1987) 434 .
86 T.Y . Tsong and R .D. Astumian, Annu . Rev . Physiol., 50 (1988) 273 .
87 T.Y. Tsong, D .-S . Liu, F . Chauvin, A. Gaigalas and R .D. Astumian, Bioelectrochem . Bioenerg., 21
(1989) 319 .
88 T .Y. Tsong, D .-S . Liu, F . Chauvin and R .D . Astumian, Biosci . Rep ., 9 (1989) 13 .
89 J .C. Weaver and R.D . Astumian, Science, 247 (1990) 459 .
90 B. Robertson and R.D . Astumian, Biophys . J., 57 (1990) 689.
91 V .S. Markin, T .Y . Tsong, B. Robertson and R.D . Astumian, J . Chem . Phys ., press.
92 T.Y. Tsong, Trends Biochem . Sci ., 14 (1989) 89 .
93 V .S. Markin, T.Y . Tsong, submitted .
94 T.Y . Tsong, in H. Hong (Ed .), Molecular Electronics, Plenum Press, New York, 1989, pp . 83-95 .
95 F . Pliquett, Z . Biol., 116 (1968) 10 .
96 F. Pliquett, Studia Biophys., 94 (1983) 85 .
97 M .A . Matzke and A .J .M . Matzke, J . Bioelectricity, 4 (1985) 461 .
98 M .A . Matzke, A.J .M. Matzke and G. Neuhaus, Pant Cell Environ ., 11 (1988) 157.