Download Voltage modulation of membrane permeability and energy utilization

Bioscience Reports 3, 487-505 (1983)
Printed in Great Britain
487
V o l t a g e m o d u l a t i o n of m e m b r a n e p e r m e a b i l i t y and e n e r g y
u t i l i z a t i o n in c e l l s
Review
Tian Yow TSONG
Department of Physiological Chemistry,
The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, U.S.A.
Cell m e m b r a n e s a r e i n h e r e n t l y e l e c t r i c a l in nature.
Charge
movements across, and e l e c t r i c potentials experienced by membranes
are used to process and transmit information, transduce energy, and
p e r f o r m o t h e r essential functions of a cell.
Thus, the el ect ri cal
p r o p e r t i e s of cell membranes are topics of study in nearly every
branch of the biological sciences (1-6).
This article will limit its
scope to two r e c e nt developments using e l e c t r i c pulses, namely, (1) to
implant pores of controlled size in cell membranes, and hence, to
modify the permeability of the membrane; (2) to a c t i v a t e membranebound ATPases, and hence, to study the energy utilization process in
cells. The use of e l e c t r i c pulses to induce cell alignments and fusions
will also be briefly mentioned.
It will focus on some basic findings
and how this newly acquired knowledge may be further developed into
useful tools for biological research.
As such, a large portion of the
material covered will be selected from our own experience.
Readers
i n t e r e s t e d in a b r o a d e r a s p e c t of t h e e l e c t r i c a l p r o p e r t i e s of
m e m b r a n e s , or in a survey of the l i t e r a t u r e in specific areas of
research, should consult regular and more com pl et e reviews (1-6).
When a cei l in suspension is exposed to a voltage pulse, it
experiences e f f e c t s of an e l e c t r i c field and 3oule heating (7; Table 1).
Electric field e f f e c t s , such as electrophoresis of charged molecules or
membrane proteins (8,9), orientation of ceils ( 1 0 - 1 2 ) , and dissociation
of molecules or complexes (13), are common to all chemical systems.
However, because o5 the specific a r c h i t e c t u r e of cells and membrane
vesicles, all of these e i f e c t s may be amplified. This occurs when a
s p h e r i c a l c e l l ( r a d i u s a), made of membrane material much less
conductive compared to the medium on both sides of the membrane, is
exposed to an e l e c t r i c field, inducing a transmembrane potential, AO.
A~p = 1.5 a E cos0 .
In the equation, A0 is expressed in volts; the radius of the cell a, in
cm; the e l e c t r i c field strength E, in V/cm; and 0, the angle between
tile field direction and points of interest on the membrane surface, in
degrees (7,1#-17).
The peak values of A0 occur at 0 equals 0 ~ and
180 ~ i.e. A0 = _+ 1.5 aE.
Thus, the potential experienced by a
molecule on a membrane surface is 1.5 a/d times great er than what
the molecule would experience in solution (d being the thickness of
01983
The Biochemical Society
488
TSONG
Table i.
Perturbations
of a cell suspension by a high electric pulse
Symbols used (please refer to references cited for details): A$, transmembrane
potential; a, radius of cell; E, electric field strength; d, thickness of cell
membrane; AT, temperature jump; i, current density; r, specific resistivity of
the suspension; At, electric pulse duration; O, density of the suspension; K,
chemical equilibrium constant; AK, shift in equilibrium constant; AH, heat of
reaction;
R, gas constant; AP, pressure generated by solvent expansion; ~,
expansion coefficient of the suspension; ~, compressibility of the suspension;
A~, osmotic pressure difference; c, concentration of solutes; Q, heat of
transfer of solvent across the membrane; V, partial molar volume of the solvent.
This table is adapted from Kinosita and Tsong (7).
I. Electric-field effects
A. Effects common to
all systems
B. Effects specific to
cell suspensions
Electrophoresis (8,9)
Field-induced
orientation (i0-12)
Field-induced transmembrane
potential (7,14-17)
A~ = 1.5 a E cos@
Field-induced
dissociation (13)
Amplification of common
effects within cell
membrane by 1.5 d/a times
Electrocompression
membrane (4)
Large transmembrane
2. Thermal effects (73,74)
(Current effects)
AT = i 2 r At/O C
Shift of chemical
equilibrium (73)
KAK = R~T 2 AT
Temperature
membrane
of
current
gradient across
Colligative effect
A~ = c R AT
Solvent expansion
(shock waves)
Thermal osmosis effect
(18,19)
(%
AP = ~ AT
AP = - Q AT
~T
membrane in cm).
For erythrocytes, this value is around 500-1000.
As will be discussed in later sections, cells are capable of utilizing
this enormous electric field to activate their transport apparatus or to
capture the energy for ATP synthesis.
Likewise, the effects of 3oule heating on a ceil are also very
different compared to the effects on a molecule in solution (Table i ) .
In addition to a perturbation of chemical equilibria and a shock wave
generated by a voltage pulse, a cell also experiences osmotic pressure
due to a difference in the a c t i v i t y of solvent molecules across the
membrane, e.g. the thermal osmosis effect (18,19). All these effects
should be considered in the design of an experiment or for the
interpretation of results.
1. V o l t a g e M o d i f i c a t i o n of M e m b r a n e P e r m e a b i l i t y
Electric-field-induced
lysis of cells
I n t e r e s t in e l e c t r i c a l p r o p e r t i e s of cell m e m b r a n e s is n e a r l y as old
as i n t e r e s t in the m e m b r a n e itself.
S t u w a r t ' s m e a s u r e m e n t s of the
c o n d u c t a n c e of blood suspensions (20) r e s e m b l e m o d e r n e x p e r i m e n t s
w h e r e b y cells, in suspension, a r e exposed to an e l e c t r i c field. The use
ELECTRIC FIELD ON CELLS
489
of an electric field for sterilization purposes has also been practiced
for some time (21,22). However, systematic studies of the effects of
an intense electric field on cell suspensions did not start until many
years later. In 1967 and 1968, Sale and Hamilton (1%23,2tt) reported
that electric pulses of a few kV/cm in the microsecond time range
caused hemolysis of red blood cells, lysis of yeasts, destruction of
b a c t e r i a , and release of DNA bundles from viral particles.
By
analysing the extent of cell killing and hemolysis of erythrocytes as
functions of either the field strength/duration or the power input into
the suspensions, it was concluded that the cell killing was the result
of the field-induced transmembrane potential. They also observed that
membrane ruptures occurred with electric pulses that produced a
transmembrane p o t e n t i a l greater than I V.
Later, Neuman and
Rosenheck (25) reported that chromaffin granules, when exposed to
e~ectric pulses, released catecholamine and ATP.
By light-scattering
measurements of the chromaffin-granule suspensions, following the
a p p l i c a t i o n of a voltage pulse, they concluded that the electric
breakdown of the membranes took place in the microsecond time range
and that the ruptured membranes were resealed within 200 ms. They
attributed this phenomenon to a reversible dielectric breakdown of the
membranes.
Zimmermann and coworkers observed that cells exposed
to a high electric field in a Coulter Counter lysed and released their
cytoplasmic contents (16,26).
The transmembrane potential required
for breakdown was roughly i V although the time for which the cells
were exposed to the electric field was less well defined in the Coulter
Counter.
They also a t t r i b u t e d the cell lysis to the dielectric
breakdown of the cell membranes.
Other studies (27-29) have considered the effects of rapid 3oule
heating on proteins or iipids of cell membranes.
Thermally induced
lipid phase transitions (30,31) and protein conformational changes
(32,33) are well documented, and these transitions could accompany a
change in the membrane permeability. Although the available evidence
strongly favored an electric field as the cause of the voltage-induced
cell lysis (34)9 at the time, no compelling evidence had been
presented to rule out the various e f f e c t s of rapid temperature
perturbation (Table I ) .
To settle the question9 Kinosita, then working in my laboratory,
designed the f o l l o w i n g e x p e r i m e n t ( 7 ) .
The e x p e r i m e n t took
advantage of the fact that erythrocytes are stable in an isotonic
medium containing either NaCl or sucrose. By varying the composition
of the suspending medium with NaCJ and sucrose, maintaining the
isotonicity but changing the ionic strength of the medium, it was
possible to apply a constant electric-field perturbation with different
degrees of the temperature jump.
Voltage-induced hemolysis of red
cells could then be related either to the electric-field perturbation or
to the temperature jump.
Four isotonic media of different NaCI/
sucrose ratios were used, and the hemolysis data, plotted against the
temperature jump and the electric-field strength of voltage pulses, are
shown in Figs. I A and IB, respectively.
In a low-ionic-strength
medium (396 isotonic NaCI, 9796 isotonic sucrose), I00% hemolysis
was achieved w i t h Jess than a 0. i ~
temperature jump.
Total
hemolysis of the sample occurred only if the temperature jump was
greater than 3~
in isotonic NaCI.
However, when these data are
t~90
TSONG
100| .
}
/
" / ; ; ?/
.
.
.
,
,
I,
' o]
/
2
!
4
0l/ : .....
0.03
0.1
0.3
1
3
Temperatureincrement(~
J
0
2
4
Electricfield(kV/cm)
Fig. i. (A) Hemolysis at different ionic strengths
of h u m a n e r t h r o c y t e s is plotted against the
magnitude of temperature jump. Washed erythrocytes
were suspended in i00 vol. of isotonic NaCl/sucrose
solutions, the relative NaCI content being: O, 100%;
O, 30%, A, 10%; 9
3%. Aliquots of the suspensions
were subject to a single electric pulse of various
intensities.
After the pulsation, the cells were
rapidly diluted into 30 vol. of isotonic NaCI to
provide an identical environment~ in which hemolysis
took place. After 15 h, the erythrocytes were spun
down at i0 000 g for i0 min, and the extent of
hemolysis was determined from the absorption of
hemoglobin in the supernatant.
The value for 100%
hemolysis was obtained by hypotonic hemolysis.
Temperature was 25 • 2~
and the pulse duration was
20 ps.
(B) The same set of data in A is plotted against
field strength of the electric pulse.
The result
indicates that the voltage-induced hemolysis was due
to the effect of the electric field, not Joule
heating. (After Kinosita and Tsong (7)).
plotted against the electric-field strength, they all fall on the same
hemolysis curve.
This result unequivocally ruled out thermal osmosis
as the cause of the cell ]ysis. The data in Fig. IB also suggest that
there is a threshold field strength for red-cell hemolysis, although this
value strongly depends on the duration of voltage pulses employed
(35).
With a 20-ps pulse, the threshold field strength was roughly 2
kV/cm, which can induce a transmembrane potential of I V in human
erythrocytes, a value similar to that found in earlier observations of
the rupture of cell membranes by microsecond voltage pulses (1#,23).
Electric breakdown of unilamellar vesicles or planar bilayers, made of
purified lipid or a mixture of lipids, occurs at much lower potentials,
usually between tOO and 300 mV (2,17,36).
ELECTRIC FIELD ON CELLS
491
Implantation of pores in cell membranes
Dielectric breakdown of membranes has been used to describe the
v o l t a g e - t r i g g e r e d release of the c y t o p l a s m i c c o n t e n t s of ceils.
Cytoplasmic contents, regardless of their molecular weight and size,
were thought to leak out at the instant the membrane was ruptured.
If this was the case, the term 'dielectric breakdown' would be an
adequate description of the observed phenomena, and the process could
serve as a convenient way of preparing erythrocyte ghosts in isotonic
solvents (16).
Indeed, the advantages of preparing erythrocyte ghosts
in isotonic solvents, and the use of these ghosts as carriers of drugs
and enzymes to specific ceil types, have been emphasized (26). Since
biological membranes are very complex, containing functional proteins
and s t r u c t u r a l c l u s t e r s ~ f these proteins within the bilayers, we
reasoned that if dielectric breakdown took place, it might occur at
certain specific locations, such as ion-transport sites. Once these sites
were open, the membrane potential could dissipate (15) and the
membrane rupture would be limited to local structures instead of
extending over a large area, as in a 'global breakdown'. This turned
out to be the case.
The e x p e r i m e n t a l strategy was to measure the kinetics of the
release of d i f f e r e n t classes of m o l e c u l e s from v o l t a g e - t r e a t e d
erythrocytes (7).
If the breakdown was global, all the cytoplasmic
contents should leak out at the same rate. If, on the other hand, the
breakdown was local, then different classes of molecules might be
released at different rates. The results are shown in Fig. 2. With a
3.7-kV/cm 20-ps pulse, in which 100% hemolysis was expected, not
o n l y did h e m o g l o b i n not leak out, but, if the experiment was
performed in isotonic NaCI, even a sucrose tracer could not permeate
the voltage-treated erythrocytes (Fig. 2A).
However, K + ion leaked
out within seconds, and, Na + ion entered just as fast because of large
initial concentration gradients. Interestingly, if the same voltage pulse
was applied to erythrocytes in a lower-ionic-strength medium (NaCI/
sucrose = ?,0%/70%), then the sucrose tracer permeated within 10 min
(Fig. 2B).
Release of hemoglobin, in both cases, occurred only after
several hours of i n c u b a t i o n at 25~
Hematocrit measurements
indicated that before hemolysis, there was a swelling of ceils due to
the colloid osmotic pressure of hemoglobin.
This process, which is
much slower than the i n i t i a l movement of ions, continued for several
hours, and when the cell volume reached 155% of the initial volume,
the cell membranes were ruptured and the cytoplasmic contents of
c e l l s were released.
These observations indicate that the voltage
pulses implanted pores of limited size in the cell membranes, and the
hemolysis was due to a secondary effect, namely the colloid osmotic
s w e l l i n g of the c e i l s ( 7 ) .
Once the membranes were rendered
permeable to ions or to small molecules, these ions reached Donnan
equilibrium rapidly.
However, because cytoplasmic proteins could not
permeate these pores, they exerted an excess osmotic pressure on the
ceil membranes.
As a result, the cells swelled, and, eventually, t h e
cell membranes were punctured because of the oversweJling.
The
c o n c e p t of ' p o r e s ' , as i n t r o d u c e d here, is valid as long as the
membranes become semipermeable, and the solutes exhibit colligative
properties. This will become clear following the next section.
#92
TSONG
A
150
150
AA
:~ ~00 ........................................... :~- 100
}
D
50
~0
9
D
C
,
...............
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
o ._-_-__:__:
.......
?
~..........
--:-o
o
20
40
,20
Time (mJn)
o
2'o
20
u ,~o
Time (min)
Fig. 2. (A) Permeation of ions and sucrose and the
swelling of erythrocytes prior to the voltageinduced hemolysis.
Erythrocytes suspended in
isotonic NaCI were subject to a single electric
pulse at 3.7 kV/cm 20 ~s.
At various intervals
after the pulsation 9 samples were taken and their
ionic
contents
and 1 4 C - s u c r o s e
entrapment
determined: Curve A 9 the volume of the unlysed cell
relative to the untreated; B 9 sodium ion penetration; C, sucrose penetration~ D~ extent of hemolysis.
(B) A similar experiment to that shown in A~
except that the erythrocytes were suspended in
isotonic NaCl/sucrose (30%/70% mixture).
In this
case, sucrose permeated into the voltage-treated
erythrocytes. (After Kinosita and Tsong (7)).
Control of pore size and resealing
In Fig. 2, it is shown that in an isotonic saline, a 3.7-kV/cm
electric pulse for 20 ps rendered erythrocyte membranes peremeable to
Na + (and K +, data not shown) ions9 but not to sucrose. On the other
hand9 a similar voltage t r e a t m e n t for erythrocytes suspended in a
3096/70% i s o t o n i c mixture of NaCl/sucrose rendered the red cells
permeable to radioactive sucrose. It would appear that by a suitable
choice of experimental conditions, one should be able to control the
size of pores induced by a voltage pulse.
Variables such as pulse
length, field strength, and solvent ionic strength have been examined
(35,37).
Fig. 3 shows an experiment monitoring pore size by probe
permeation rate (see reference 37 for the method). In the examples
given here, permeation of the probes was measured before hemolysis
of the red cells, and, in fact, these pores could be properly resealed
without the loss of cellular hemoglobin. The result indicates that it is
ELECTRIC FIELD ON CELLS
~-, > 2
.
t~93
0
~
_h
-,o[
.E
=
~
e
;
i
5
6
Radius (A)
Fig. 3.
Permeability of the pulse-treated erythrocyte m e m b r a n e
to various carbohydrate molecules.
Curve a t untreated cells; b~ cells treated with a
3.7-kV/cm 20-~s pulse in isotonic NaCI; c 9 treated
with a 5.3-kV/cm 20-~s pulse in isotonic NaCI; d t
treated with a 3.7-kV/cm 80-~s pulse in isotonic
NaCI; e t treated with a 3.7-kV/cm 20-~s pulse in 3:7
mixture of isotonic NaCI and isotonic sucrose.
The
rate of permeation and the radius of permeants are
obtained
as e x p l a i n e d
elsewhere (37).
D means
D-glucose;
Lt L-glucose;
E~ meso-erythritol;
x9
sylitol; A t D-arabitol; G t glycerol; M~ D-mannitol;
I t myo-inositol;
S~ sucrose; Z~ melezitose; and T~
stachyose.
(After Kinosita and Tsong (37)).
possible t o implant pores of controlled size with the voltage pulse
method by varying experimental parameters such as pulse width, field
strength, and ionic strength of the medium.
The next question asked was whether the voltage perforation of the
cell membranes was a reversible process. Light-scattering of the cell
suspension decreased upon application of a voltage pulse in microseconds, but was quickly restored to a range within milliseconds to
seconds (25,29).
It would seem logical to interpret the signals as
being due to, f i r s t , m e m b r a n e pore f o r m a t i o n , and second, the
resealing of pores. This was clearly not the case. If one incubated a
s a m p l e , t r e a t e d with a v o l t a g e pulse greater than the threshold
potential, for a longer period of time (e.g. a few hours), all cells
were e v e n t u a l l y lysed.
This means that the pores generated by
voltage pulses were irreversible, contrary to the view that these pores
494
TSONG
are rapidly reversible.
The distinction of reversibility and irreversib i l i t y is essential if one is to develop the method for biological
applications. Indeed, irreversibly damaged membranes can be resealed
if suitable conditions can be found.
A misjudgement of the pore
r e v e r s i b i l i t y usually leads to a doomed experiment because of the
destruction of the cells.
As mentioned, the hemolysis of voltagep e r f o r a t e d e r y t h r o c y t e s is due to the colloid osmotic pressure of
cytoplasmic hemoglobin.
The pressure amounts to roughly 25 m i l l i osmolal (mOsm).
If one adds to the suspending medium 25 mOsm of
molecules larger than the pore size, the colloid osmotic pressure of
hemoglobin can be balanced, and, under such conditions, ceils should
not swell. Hence, the hemolysis would be prevented indefinitely. We
have used the tetrasaccharide, stachiose, or small proteins, such as
r i b o n u c l e a s e or c y t o c h r o m e c, to compensate for the hemoglobin
pressure.
Voltage-perforated red cells were stable in the osmotically
balanced medium. No swelling, and hence no hemolysis, was detected.
The pores were found to be stable for several days if the temperature
was kept low, e.g. at 4~
Small molecules or drugs could be loaded
into the erythrocytes, which retained their full hemoglobin content. In
an osmotically balanced medium, and at higher temperature, e.g. 37~
these pores spontaneously resealed within 2 h (37), again, without
losing their cytoplasmic content.
Loading of
contents
molecules
into
cells
without
loss
of cytoplasmic
Because electrically perforated cell membranes can be resealed, it
will be possible to load molecules into a cell without killing the ceil.
Radioactive oligosaccharides have been loaded into voltage-perforated
erythrocytes in our laboratory.
In the case of sucrose loading, the
resealed erythrocytes (that retained fuji hemoglobin content) were
then injected back into the mouse circulation to demonstrate that
these ceils were indistinguishable from normal cells in both l i f e t i m e
and function (3g).
The dissipation of radioactive tracer from the
circulation had a halflife of approx. 20 days, compared to less than I
h if the tracer is directly administered.
Radioactive-tracer-loaded
erythrocytes, without going through a normal resealing procedure, were
unable to retain the tracer molecules, indicating that pores induced by
the voltage pulse were indeed not reversible (Fig. 4). While in other
cell types, because of stronger membrane structures or the presence of
a cell wail, no Iysis would accompany voltage perforation of the
membranes, i t is i m p o r t a n t to r e c o g n i z e t h a t these pores are
irreversible, and the resealing of these pores requires special attention.
Usually, this does not require more than balancing the osmolality of
solvent to that of the cytoplasmic fluid, and incubating voltage-treated
cells at the growth temperature for a few hours. Ceils are able to
repair the perforated membranes. Once the perforated membranes are
resealed, these cells behave like ordinary ceils.
The above experiment immediately suggested to us that erythrocytes could be used as carriers for drug delivery.
The use of
liposomes or erythrocyte ghosts as drug or enzyme carriers for the
targeting of specific cell types have been described (26,39), but intact
erythrocytes should be better carriers (though not for enzymes). The
ELECTRIC
FIELD
ON
CELLS
0
#95
Time after injection (d)
|0
20
30
lOO
e~
10
o
e~
1
m
o
o.1
100
2 6 0 / / 1,000
I
Time after injection (min)
Fig. 4.
Dissipation of sucrose tracer from mouse
circulation.
Sucrose was loaded in electric-pulsetreated mouse erythrocytes which were subsequently
resealed by the method described in the text. These
erythrocytes were then injected back into the mouse
circulation~ and the dissipation of radioactivity
from the circulation was monitored: O ~ completely
resealed sample; 9 ~ incompletely reseated sample; A
free sucrose administered directly.
Upper abscissa
is for the data in O and 9
and lower abscissa is
for the data in A.
(After Kinosita and Tsong (38)).
advantage is t h a t e r y t h r o c y t e s from one's own body will not be
rejected.
In practice, it would be necessary to modify the erythrocytes for a controlled release or for the targeting of specific cell
types.
The problem that we encountered was that most drugs are
p e r m e a n t to c e l l membranes~ a l b e i t slowly (Except for surfacer e c e p t o r drugs, can one imagine drugs that are not permeant to
membranes, yet are effective?).
This, however, would be a common
problem, whether liposomes, intact erythrocytes, or ghosts are used.
The problem could be alleviated if drugs can be modified to include a
sugar moiety, since we know sucrose did not leak out once it was
entrapped within the erythrocytes.
As m e n t i o n e d , not all cell types r e q u i r e the resealing step.
Dictyostelium, yeast, mouse L cells, or sea-urchin eggs are able to
496
TSONG
sustain a voltage perforation of membranes without lysis (40-43).
Rossignol et al. (40) were able to poke sea-urchin eggs with pores
that allowed permeation of Ca2+ into the eggs.
W h e n the medium
contained CaZ+, the cells developed partial fertilization envelopes.
Without the voltage treatment, the presence of Ca2+ in the medium
did not trigger development of these partial fertilization envelopes.
This means t h a t the development of the fertilization envelope in
f e r t i l i z a t i o n required Ca2+, although this Ca2+ could be supplied
through sperm or from the mobilization of a Ca 2+ store within the
egg.
Knox et al. (41) have used the method to load Dictyostelium
with radioactive ATP.
The ceils developed normally after loading.
Neumann and coworkers (44,45) have employed the voltage-perforation
method to load a restriction fragment of DNA, which contained a gene
for t h y m i d i n e kinase, into mouse lyoma cells.
The efficiency of
loading was thousands of times higher than that of using conventional
methods ( 4 5 ) , and the loaded gene was expressed.
The example
i l l u s t r a t e s the p o t e n t i a l application of the high-voltage-pulsation
method for genetic engineering. In fact, for genetic engineering, the
voltage-pulse method is simpler than the cell-fusion method which we
will mention briefly at the end of this article.
Specific sites of pore formation in voltage-treated erythrocytes
The question of whether the voltage perforation of the membrane
occurred at random or at specific sites is especially relevant when one
is dealing with a biological membrane which is full of ion-transport
apparatus in its structure.
One may suspect that certain membrane
channels or transport pumps are more susceptible to voltage perforation than other parts of a cell membrane. Lipid bilayers break down
at a transmembrane potential between 100 and 300 mV, and, if the
composition of the bitayers is right, the membrane can reseal within
microseconds (17,36,46).
The fact that a high-voltage breakdown of
biological membranes is irreversible in this time scale means that
protein components are likely candidates for the voltage action.
In
order to test this idea, a device was set up to measure the time
evolution of transmembrane current during and after the voltage
pulsation (47).
The device took advantage of the fact that the
resistance of a dense cell suspension was high when cell membranes
were intact, but decreased upon pore formation.
By measuring the
excess conductance of the cell suspension when a voltage pulse was
applied, it was possible to follow the kinetics of the pore formation.
The transmembrane current was observed only if the magnitude of the
voltage pulse exceeded the threshold value, and the kinetics of pore
formation were biphasic. A reaction in less than I ps was interpreted
as being due to the formation of pores, while the slower reaction, in
50 ps, was due to the expansion of pores. Various transport inhibitors
were then added to the red-cell suspension to see whether they could
block or reduce the signal of the voltage-induced transmembrane
current.
Of roughly one dozen of transport inhibitors tested, only
ouabain, a potent
inhibitor of ( N a , K ) A T P a s e , p a r t i a l l y blocks the
m e m b r a n e c u r r e n t in a m a n n e r c o n s i s t e n t with the d r u g ' s physiological
e f f e c t (48).
The h a l f - s a t u r a t i o n of the ouabain e f f e c t was 0.15 pM.
At a s a t u r a t i o n
c o n c e n t r a t i o n , ouabain blocked 30% of the t o t a l
v o l t a g e - i n d u c e d m e m b r a n e c u r r e n t . This would m e a n t h a t roughly 30%
ELECTRIC FIELD ON CELLS
#97
of the membrane current resulted from perforation of the (Na,K)
channels.
Another 70% of the transmembrane current came through
pores which have not been identified.
Investigators using a Coulter
Counter often observe a change in the cell size distribution when the
applied electric-field strength exceeds a certain value. It is important, however, to distinguish between the electric size and the physical
size of the cells. The former is used in Coulter counting, and depends
not only on the true dimension of the cells, but also on the conductance properties of the cell membranes. When membranes become
conductive, their electric size diminishes.
The physical size, on the
other hand, reflects the true volume and shape of the cells.
Only
when the physical size is unchanged can one measure voltage-induced
transmembrane conductance.
The number of pores per c e l l could be estimated if certain
assumptions are made.
Pore size was determined by the permeability
of membranes to oligosaccharides, and membrane conductance was
measured in KCI solution. If one assumed that membrane conductance
after voltage-perforation of the red cells was due to the movement of
K + ion9 the number of pores could be estimated.
For erythrocytes
treated with a 3.7-kV/cm 20-1Js pulse in isotonic saline, this number
was around 200 per celt (#7). Since the pore sizes were most likely
non-uniform, this estimate must be taken as a rough approximation.
The number of (Na, K)ATPases in erythrocytes is around 300 per cell
(#9).
In this regard~ it is interesting to note that hypotonic shock
induces only a single pore in the red cells (50,51).
2. Voltage Activation of Membrane-Bound ATPases
(Na,K)ATPase of erythrocytes
Living cells maintain a steady-state transmembrane potential by an
asymmetric distribution of ionic species on both sides of the membrane.
This maintenance of a membrane potential is an energyconsuming process, and it usually requires ATP-dependent pumping
a c t i v i t y of the membrane. One of these energy-dependent ionic pumps
is the (Na,K)ATPase.
In the erythrocyte, for each ATP consumed,
the enzyme pumps out 3 Na + ions from the cytoplasm in exchange of
2 K + ions (52).
Because of this unbalanced charge transport, the
enzyme is electrogenic (53).
For human erythrocytes under normal
c o n d i t i o n s , the a c t i v a t i o n of ( N a , K ) A T P a s e h y p e r p o l a r i z e s the
membrane by several m i l l i v o l t s (53).
In the previous section we
mentioned that high-voltage pulses of several kilovolts per centimeter
could induce irreversible membrane pores~ and 30% of these pores
might have occurred at the (Na,K)ATPase sites.
Because of the
e l e c t r o g e n i c character of the pump, it is not surprising that the
enzyme should be perforated by a voltage pulse.
The question has
been asked, why, after the opening of the pump, should i t be irreversibly denatured; and, if we can prevent its denaturation, would it be
possible for us to reversibly activate this pump by an a r t i f i c i a l l y
imposed transmembrane potential? Because a few kV/cm can generate
roughly 1 V of transmembrane potential, and this value is two orders
of magnitude larger than the physiological potential of the erythrocytes, it is likely that once the pores were opened, a strong transmembrane current would follow, which could g e n e r a t e intense local
#98
TSONG
heating which might then modify the protein structure irreversibly. It
follows that only by imposing a membrane potential comparable to the
physiological potential (i.e. 10 mY) might we be able to activate the
enzyme without denaturing the protein.
Teissie and I then employed a low-voltage AC stimulation method
to induce a membrane current that was f u l l y reversible, and 25% of
this membrane current was inhibited by ouabain (5#).
Because the
concentration for half inhibition by ouabain agreed with the inhibition
constant for the (Na,K)ATPase, and the effect of ouabain diminished
if 10 mM K + was present in the incubation medium, we concluded that
the ouabain-sensitive part of the membrane current was due to the
reversible opening of (Na,K)ATPase (5#).
Serpersu and I monitored the movement of Rb + and Na + ions in
both d i r e c t i o n s of AC-stimulated red cells, and compared it with
u n s t i m u l a t e d samples and o u a b a i n - p r e t r e a t e d , stimulated samples.
Indeed, at 3~
a voltage-stimulated Rb + uptake was observed with an
AC field of 16 V/cm, and this uptake was completely inhibited by
ouabain (Fig. 5A).
The uptake was against a chemical concentration
g r a d i e n t , i n d i c a t i n g t h a t a 'pump' a c t i v i t y was involved.
The
surprising result was that no Rb + efflux or Na + movement in either
direction could be stimulated with an AC field (55).
The voltageinduced Rb + u p t a k e was sensitive to the field strength and AC
frequency, excluding 3oule heating as the cause of the observed effect.
The o p t i m u m s t i m u l a t i o n occurred at 20 V/cm (that generated a
transmembrane potential of 12 mV), and at 1 kHz (Fig. 5B,C).
At
3~
in a low-ionic-strength medium, the maximum uptake was 26
amole/RBC-s, or roughly 22 ions per pump per s, if one assumes that
the uptake was mediated by the (Na,K) pump (Fig. 5B and C).
That the voltage-stimulated Rb + uptake involved the a c t i v i t y of
(Na,K)ATPase has now been established by several c r i t e r i a . First, the
u p t a k e was c o m p l e t e l y inhibited by ouabain, a potent inhibitor of
(Na,K)ATPase.
Second, although the Rb + uptake required no Na +
efflux activity of the enzyme, it did require the presence of internal
Na + and external K + ions.
The K m of cellular Na + was roughly 5
raM, and the K m of external K + was 1.5 raM, consistent with the
values for (Na,KIATPase activity.
Third, the uptake was against a
c h e m i c a l c o n c e n t r a t i o n g r a d i e n t (12.5 mM external vs 100 mM
internal). Thus, a pump activity must be involved. Yet, the question
of whether the e f f e c t of ouabain was specific has been raised.
The
data in Fig. 5A,B,C indicate, however, t hat t here was no non-specific
e f f e c t of ouabain (see the background Rb + uptake in the presence and
absence of ouabain).
The above result is non-orthodox for the following reason.
The
Na + and K+ pumping activities of (Na,K)ATPase are believed to be
coupled, that is, they do not function separately (56). Yet, our result
shows t h a t at 3~
when t h e r e was no d e t e c t a b l e Na + pumping
sensitive to ouabain, t her e was a voltage-stimulated Rb + uptake which
was mediated by the (Na,K)ATPase. Vanadate inhibited Na + pumping
at 25~
but it did not block AC-stimulated Rb + uptake.
These
r e s u l t s led us to suggest that whereas the transport of Na + ion
requires the consumption of ATP, the m ovem ent of K+ ion is probably
driven by an e l e c t r i c a l potential.
This potential is generat ed by the
electrogenic component of the (Na,K)ATPase activity.
Work in our
ELECTRIC FIELD ON CELLS
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TSONG
l a b o r a t o r y has shown t h a t electrogenic transport ATPases can be
s t i m u l a t e d by electric fields.
Sarcoplasmic Ca-ATPase is another
example (57) of this process. The electrical pulsation method allows
for the biochemical study of these transport ATPases.
Voltage-driven ATP synthesis by mitochondrial ATPase
Not only can an a r t i f i c i a l l y imposed transmembrane potential
activate transport ATPases, it can also induce synthesis of ATP by
ATP synthetases. Witt and coworkers have reported that chloroplasts
exposed to an electric field on the order of 1 kV/cm synthesized ATP
in the dark.
Thus, electrical energy could be converted to the
c h e m i c a l - b o n d energy of ATP (58) by this light-energy-transducing
system.
Since then investigators from different laboratories have
reported similar observations using different energy-transducing systems
(59-61).
The following example is taken from our own work, and
serves to illustrate the requirement of the activity of FoFIATPase for
the voltage induction of ATP synthesis in submitochondrial particles
(61).
Submitochondrial particles (SMP) were prepared from rat liver and
beef heart. The size of these particles depended on the preparation,
and was roughly 80 nm in diameter for the rat liver SMP and 200 nm
for the beef heart SMP. SMP suspended in a medium containing I-3
mM ADP and I-3 mM 32Pi were exposed to voltage pulses in the
range of 5-35 kV/cm and of duration 5-I00 ps. ATP formed was then
assayed by two independent methods, the luciferin/luciferase bioluminescence measurement and the radioactivity counting of [32p]ATP
formed.
In all experiments, either i mM NaCN or 10 pg/ml of
rotenone was present in the sample to completely inhibit the respiration. It was found that ATP synthesis was induced when the electric
field exceeded I0 kV/cm.
A threshold membrane potential of roughly
60-I00 mV was required for the synthesis (61).
The yield of ATP
reached 10 ATP per F0F I complex per 30-kV/cm 100-ps pulse. Fig.
6A shows ATP synthesis vs applied-field strength and Fig. 6B shows
the ATP yield when 30-kV/cm 100-ps pulses were used in the experiment with beef heart SMP (62). The synthesis was shown to be due
to effects of the electric field, and was unrelated to 3oule heating by
variation of the ionic strength of the medium, as was discussed earlier
in our experiment with erythrocyte hemolysis (see Figs. I A and IB).
That the electric-field-induced ATP synthesis is biologically relevant
was demonstrated by studying the effects of FoFIATPase inhibitors,
the e l e c t r o n - t r a n s p o r t - c h a i n uncouplers, and other ionophores.
Oligomycin, N , N ' - d i c y c l o h e x y l carbodiimide (DCCD), potent and
specific inhibitors of FoFIATPase , completely blocked the voltage
induction of ATP synthesis.
Proton-conducting ionophores FCCP
(carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and 2,4-dinitrophenol also blocked the synthesis.
Other ionophores, such as
v a l i n o m y c i n , nigericin, and monensin, displayed varying degrees of
effectiveness in inhibiting the synthesis. These observations indicate
that while the synthesis required the activity of FoFIATPase, the
energy source for the synthesis was derived from the voltage-generated
transmembrane potential.
ELECTRIC FIELD ON CELLS
501
Fig. 6.
(A) Voltage400
induced ATP synthesis
by beef heart submitochondrial
particles
(SMP).
SMP
(mean ~
300
d i a m e t e r of 200 nm)
were suspended in a
medium containing 3 mM
sodium phosphate, 1 mM
200
MgCl2, 2.5 mM NaADP, 5 |
mM NaHEPES,
20 mM
glucose,
71 I U / m l
hexokinase, 2 mM NaCN,
1 mg/ml BSA, 0.25 M
100
sucrose, 10.8 cpm/pmol
32p i. 4.8 mg of SMP in
0.70 ml was exposed to
three
successive
0
v o l t a g e p u l s e s of a
given field strength
E|eo~.rio Field, kV/om
(with a pulse decay
time of i00 ~s).
The
amount of Pi esterified
5
was determined and is
B
expressed as pmoles of
ATP per mg proteins per
4
pulse.
The difference
between the pulsed and
unpulsed levels of ATP
is plotted against the ~
3
magnitude
of
the
applied electric field E
(in
kV/cm).
The
2
initial temperature of
the sample was 15~
and the final temperI
ature did not exceed
25ec.
Each
point
represents the average
of t h r e e
separate
9
I
I
I
I
experiments. The error
bars give the standard
Number of Pu]ee8
deviations.
(B) ATP synthesis depended on the number of applied electric
pulses. 30-kV/cm pulses with a field decay time of I00 ps were
used.
Experimental conditions were similar to A.
See text for
details. (After Knox & Tsong (62)).
I
I
I
I
The kinetics of the synthesis were investigated by changing the
electric-pulse duration and monitoring the ATP yield.
Electric pulses
of duration shorter than I0 ps were found to be ineffective, and there
was a minimum pulse width of roughly 15 IJs required for the ATP
.SO2
TSONG
synthesis (61,62).
The synthesis reached a steady state within #0 ps.
Other experiments have demonstrated that the ATP d e t e c t e d did not
originate from the nucleotide-translocating activity of the membrane.
Neither did it come from the release of tightly bound nucleotides. In
fact, as was mentioned, the yield under our experimental conditions
reached l0 ATP per enzyme complex per 30-kV I00-ps pulse. Thus, a
few turnovers of the enzyme occurred within 100 ps. An important
consideration in this type of experiment is the distinction between a
synthesis driven by a chemical potential and a synthesis driven by an
electrical potential.
The former is simply the product of a certain
concentration of ATP according to the chemical equilibrium constant
in e f f e c t when ADP and Pi are mixed) and would have no relevance to
our discuss[0n here.
The p h o s p h o r y l a t i o n p o t e n t i a l , defined as
( A T P ) / ( A D ~ ) ( P i ) , is 5 x 10=6 M=t for a chemically equilibrated
system, and ;is between 200 and 800 M=t in actively respiring ceils
(63).
In our experiment, a value of roughly 2.5 M-I was obtained
(62).
Obviously this was not the highest value we could obtain, as
the quantity of ATP synthesized had not yet reached a plateau with
the increasing number of applied pulses (Fig. 6B).
Voltage induction of ATP synthesis has now been demonstrated in
c h l o r o p l a s t s , m i t o c h o n d r i a , and E s c h e r i c h i a
coli
(6#).
These
experiments, are important for clarification of some basic concepts in
bioenergetics.
According to the chemiosmotic hypothesis of Mitchell
(65), a crucial, and possibly the final product of electron transport is
the f o r m a t i o n of a proton gradient across the inner mitochondrial
m e m b r a n e . The proton electrochemical potential thus generated is
then utilized by FoFtATPase for the synthesis of ATP.
The proton
electrochemical potential is composed of two terms, the ApH and the
membrane potential, A0. The experiments described here, in principle,
allow estimation of the relative contribution of the two terms, if ApH
can be measured in microsecond time range.
In a photosynthetic
system, Witt and coworkers have used electrochromic properties of
membrane protein to monitor the generation of a membrane potential.
Surprisingly, they have observed that although ATP synthesis paralleled
the rise of membrane potential and reached a steady-state level in 500
ps, the appearance of a measurable pH change occurred much slower,
in a 100-ms time range (66,67).
We are continuing our investigation
Of the voltage induction of ATP synthesis in microsecond time scale
by using very short electric pulses.
3.
Other R e c e n t D e v e l o p m e n t s
A recent finding that has a t t r a c t e d much attention among cell
biologists is that high electric pulses can induce fusions of clustered
ceils. Neumann and coworkers have demonstrated that D i c t ] ] o s t e l i u m
d i s c o i d e u m in certain developmental stages can be induced to fuse
when exposed to electric pulses (#3). Other workers have shown that
electric pulses can induce cell fusions in various cell types when these
cells are in a close contact (6,61,65,69). 3T3 cells, for example, in a
confluent monolayer culture, have been shown to fuse by exposing
them to electric pulses (61). To bring contact of ceils, various tricks
have been used.
The most fascinating is the use of the dielectrophoresis technique ( l l , 6 g ) .
Cells exposed to a nonuniform electric
ELECTRIC FIELD ON CELLS
503
field in the r a n g e of 10-100 V/cm and in the megacycle range
aggregate and orient themselves to form 'pearl-chain' (68).
When an
electric pulse is applied through the pearl-chain, these cells fuse to
form an elongated cell rod (68).
Giant ceils can also form with
randomly clustered celIs.
Although fused cells are viable in many cases (68,69), application
of this technique for genetic engineering remains to be proven.
It
would seem that the technique which uses electric pulses to induce
cell uptake of DNA, as we discussed earlier, is a simpler and a more
viable way of introducing genetic materials into a cell (t~4,~5). On
the other hand, the cell fusion method should be especially suitable for
producing hybridoma cells in immunochemistry research (69).
A low electric field in the range of millivolts per centimeter has
been found to stimulate tissue or bone regeneration (6,70,71).
Many
patients have benefited directly from this discovery. Electric fields in
t h e s a m e m i l l i v o l t s - p e r - c e n t i m e t e r range are also found to align
tubulin polymerization (72).
Since the field strength is too weak to
induce dipole orientation , the e f f e c t must be a highly cooperative
phenomenon.
One can reasonably expect that other e f f e c t s of an
electric field will continuously be found, and these e f f e c t s will have
direct relevance to the life science research.
Acknowledgements
I thank my associates, Dr. E. H. Serpersu and Mr. B. E. Knox, and
my f o r m e r a s s o c i a t e s , Drs. K. Kinosita and 3. Teissie, for their
excellent scientific insight and fine work. Other coileagues, either in
the past or at p r e s e n t , associated with my laboratory have also
c o n t r i b u t e d to the progress of the project.
This work has been
supported by the United States NIH Grant GM28795.
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