Biomembrane Electrochemistry - ACS Publications - American

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Electroconformational Coupling Enforced Conformational Oscillations of a Membrane Enzyme for Energy Transduction Tian Y. Tsong Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul, MN 55108

We have studied, at the molecular level, how a cell can interact with an applied electric field and how an endogenous electric field may be used by a cell to regulate its internal activities and to exchange information with other cells. Molecules of the cell membrane play an important role because an electric field, either of endogenous or exogenous origin, is greatly amplified at the site of a cell membrane. This field amplification by a membrane enables a cell to receive, process, and transmit weak electric signals. Several laboratories have shown that membrane adenosine triphosphatase can absorb energy from an applied electric field and use it to perform different types of chemical work. To interpret these results, an electroconformational coupling model has been proposed. The model postulates that there are at least two catalytically and electrically active forms of the enzyme: one with its cation binding site exposed to one side of the membrane and the other with its binding site exposed to the opposite side. The two forms exhibit different affinities for the ligand. An oscillating electric field of appropriate amplitude and frequency can turn the catalytic wheel of the enzyme by enforcing conformational oscillations among its different catalytic states. Analysis of the model reproduces many features of experimental results, including the observed optimal frequency for field stimulation. To obtain precise values of electric potential across a cell membrane, electric parameters of several cell membranes have also been measured.

0065-2393/94/0235-0561 $08.00/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

BIOMEMBRANE

562

ELECTROCHEMISTRY

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 10, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch026

SURFACES ARE ELECTRIFIED

( I ) , a n d a c e l l membrane is no exception. T h i s property o f a c e l l m e m b r a n e makes it w e l l suited for processing electric energy a n d signals. E a r l y studies o n electrical properties of c e l l membranes are d o c u m e n t e d i n the monographs of C o l e (2) a n d T i e n (3). M o r e recent studies have focused o n single-channel measurements of m e m b r a n e patches or p u r i f i e d channels reconstituted into bilayer l i p i d membranes ( B L M ) (see C h a p t e r 15 i n this volume). T h e reconstitution o f p u r i f i e d m e m b r a n e chan nels, enzymes, a n d receptors into l i p i d vesicles for biophysical characteriza tions is also a c o m m o n approach. O u r w o r k focuses o n the direct exposure o f cells, organelles, or proteoliposomes, i n suspension, to a direct current (dc) pulse, an alternating current (ac) field, or a dc-shifted radio-frequency field. F o r convenience, this last technique w i l l b e c a l l e d the p u l s e d electric field ( P E F ) m e t h o d (4, 5 ) .

Electric-Field-Induced Transmembrane Potential A P E F can induce a transmembrane electric potential, Δ ψ , or an effective electric field across the m e m b r a n e l i p i d bilayer o f E = A\\f /d, w h e r e d is the thickness o f the bilayer ( 5 - 8 n m ) . F o r a c e l l o f spherical shape, t h e relationship between Δ ψ a n d the a p p l i e d field, E , is (6-11) ι η

m

ιη

m

a

Δψ

ιη

= 1.5 H

c e l l

E cos θ

(1)

a

where R is the radius o f the c e l l a n d θ is the angle between the field fine a n d the n o r m a l to a point o f interest o n the c e l l m e m b r a n e . E q u a t i o n 1 is applicable to a dc type o f P E F o r an ac field ( 12). F o r an ac field o f the type E = E sin( = 1.5 R m

m

£ cos e / [ l + ( ω τ ) ] 2

ceI1

1/2

a

(2)

H e r e ω = 2ττ/, w h e r e / is the frequency o f the ac field. W h e n the maximal transmembrane potential is considered (Θ = 0°), eqs 1 a n d 2 become, respectively, Δ ψ ^ = 1.5 B

c e l l

Δ ψ ^ = 1.5 R

c e l l

E

(3)

a

£ °c/[l + ( ω τ ) ] a

2

1 / 2


(4)

26.

TSONG

Electroconformational

Coupling

563

This apphcability o f equations 1 - 4 has b e e n verified for many types o f cells and l i p i d vesicles b y

fluorescence

i m a g i n g techniques ( 7 - 9 ) a n d f o r t h e

measurement o f critical b r e a k d o w n field strength (10-12).


Interaction of a Membrane Protein with an Electric Field M o s t m e m b r a n e proteins are electrically active. I f a membrane-spanning protein has a conformational change that involves translocation o r displace ment o f charges, o r change i n t h e molar electric m o m e n t ( Δ Μ ) , t h e n a transmembrane electric field w i l l shift its c h e m i c a l e q u i h b r i u m to favor states that have higher electric moments ( M ) . T h e quantitative relationships f o r the effect o f an electric field o n the c h e m i c a l e q u i h b r i u m represented b y e q 5 are given i n eqs 6 - 8 (13, 14): β

e

?

l

P

(5)

2

Ki ,eiec = i2,o e x p [ Δ M E / R T ] K

2

^12,elec

=

e

(6)

^12,0

exp[rAM E /RT]

(7)

= * 2 i . o e x p [ ( r - 1) AMeEm/RT]

(8)

e

fca.eh*

m

m

I n these equations, P a n d P are the two conformational states o f the transport p r o t e i n , a n d e q u i l i b r i u m constants (K) a n d rate constants (k) i n an electric field are shown to b e these constants i n zero field m u l t i p l i e d b y a nonlinear t e r m that is the product o f Δ Μ a n d the electric field across the m e m b r a n e , E . T h e r i n these equations is the apportionation constant a n d has a value b e t w e e n 0 a n d 1 (14). T h i s property o f a m e m b r a n e p r o t e i n has b e e n explored, a n d a m o d e l called electroconformational c o u p l i n g has b e e n proposed to interpret data o n the electric activation o f m e m b r a n e enzymes (13-17). A four-state membrane-facilitated transport m o d e l has b e e n ana lyzed a n d shown to absorb energy f r o m oscillating electric fields to actively p u m p a substrate u p its concentration gradient (see the section entitled Theory of Electroconformational Coupling). A n o t h e r m o d e of p r o t e i n - e l e c t r i c field interaction has b e e n proposed b y B l a n k (18-21). B l a n k considers that t h e effects o f a n electric field o n m e m b r a n e p r o t e i n mainly arise f r o m its effect o n the electric double layer at the w a t e r - m e m b r a n e interface. I n other words, an electric field can change the concentration o f ions near a m e m b r a n e p r o t e i n , w h i c h results i n a stimulation o r a r e d u c t i o n i n its activity. T h e surface compartmental m o d e l has b e e n used to interpret the ac stimulated adenosine triphosphate ( A T P ) splitting o f N a , K - A T P a s e (adenosine triphosphatase) a n d ribonucleic a c i d ( R N A ) synthesis i n various types o f cells (19-21). T w o types o f experiments have b e e n reported: O n e uses a l o w amplitude (less than 1 m V / c m ) ac field, a n d the other uses an ac amplitude larger than x

2

β

m


564

BIOMEMBRANE ELECTROCHEMISTRY

1 V / c m . L o w - f i e l d experiments

have b e e n

p r o m p t e d b y environmental

concern a n d h i g h - f i e l d experiments have b e e n done to understand molecular mechanisms o f field interactions w i t h c e l l membranes (14,

21,

The

22).

m e d i u m - f i e l d experiments s h o u l d also be relevant to the development o f biosensors and molecular electronics (23).

O u r experiments used moderately

h i g h field strength (a f e w volts to m a n y M o v o l t s p e r centimeter). T h e choice


o f a field strength was dictated b y m i m i c k i n g the endogenous field o f the enzyme u n d e r consideration. F o r example, i n h i b i t i o n o f the N a , K - A T P a s e o f h u m a n erythrocytes leads to a depolarization o f several millivolts (24),

which

implies that cation p u m p i n g b y the enzyme is responsible for an equivalent amount o f m e m b r a n e potential. T h e a p p l i e d field strength o f 20 V / c m is r e q u i r e d to generate 10 m V o f Δ ψ

Γη

i n a h u m a n erythrocyte. W h e n ¥ ¥ 0

λ

A T P a s e o f submitochondrial particles is considered, a 1 0 - k V / c m a p p l i e d field w o u l d be r e q u i r e d to generate about 200 m V o f Δψ synthesis

r e q u i r e d for A T P

Π1

(14).

U s e o f a high field to activate a m e m b r a n e enzyme was first reported b y W i t t et al. (25)

i n 1976. T h e y used dc pulses o f approximately 1 k V / c m a n d

o f 1-ms duration to induce A T P synthesis b y the chloroplast A T P a s e . F o l l o w i n g this initial work, there have b e e n m a n y reports o n 1 - k V / c m dc d u c e d A T P synthesis i n different A T P synthetic systems (see

field-in

the literature

cited i n references 13 a n d 14). T h e m a i n conclusion f r o m these studies is that an a p p l i e d field-induced transmembrane potential can facilitate A T P release f r o m the enzyme; w h e t h e r a P E F can affect enzyme turnover is not clear. Because 1 - k V / c m d c fields also cause severe Joule heating o f a sample suspension, thermal effects cannot be easily avoided except w h e n very short electric pulses (microseconds) are used. T h u s , the m e t h o d has l i m i t e d utility for electroactivation experiments. T h e P E F m e t h o d is, however, quite p o p u lar for the study o f electroporation a n d electrofusion o f c e l l membranes the chapter b y J. W e a v e r i n this volume), electroinsertion o f proteins (26),

a n d electrotransfection o f cells

(see

membrane

(27).

Electric Activation of Na, K-ATPase A summary o f the salient features o f o u r experimental results (28-30)

on Na,

K - A T P a s e follows. N a , K - A T P a s e o f h u m a n erythrocytes can respond to the stimulation o f an ac field of approximately 20 V / c m to p u m p N a , K , a n d R b +

+

+

against

their respective concentration gradients. T h e r e was no irreversible damage o f the membrane permeation barrier after the erythrocytes w e r e exposed for several hours to this magnitude o f ac field. A f t e r the field was t u r n e d off, the erythrocytes behaved very m u c h like fresh erythrocytes i n terms o f their c e l l shape, v o l u m e , a n d n o r m a l i o n p e r m e a t i o n properties.


26.


TSONG

Coupling

565

A t , 4 °C, n o A T P - d e p e n d e n t N a , K , o r R b +

+

+

p u m p i n g activities

sensitive to ouabain w e r e detected. A n ac electric field stimulated active efflux o f N a

+

a n d influx o f K

and efflux o f K

+

+

and R b

+

b u t not the passive influx o f N a

+

a n d R b . T h i s ac-stimulated active transport o f cations was +

completely i n h i b i t e d b y 0.2 m M o f ouabain. A t 26 °C, the enzyme h y d r o l y z e d A T P to p u m p N a

+

a n d K . A n ac stimulation further increased the rate o f +


p u m p i n g . A t 37 °C, the A T P hydrolysis o f the enzyme was at its m a x i m u m a n d an ac stimulation d i d not escalate the rate o f active p u m p i n g o f these ions. T h e o p t i m a l field strength f o r activating b o t h the N a

+

a n d the K

p u m p s was 20 V / c m (peak-to-peak). T h i s a p p l i e d field w o u l d generate a Δ ψ

+

ιη

o f 12 m V , o r an effective transmembrane electric field, £ , o f 24 k V / c m . m

O t h e r field strengths l e d to r e d u c e d activities. W h e n the o p t i m a l field strength, 20 V / c m , was used, the o p t i m a l frequency for the N a K

+

or R b

+

+

p u m p was 1.0 M H z , whereas it was 1.0 k H z f o r the

p u m p . T h e w i d e separation o f the o p t i m a l frequencies f o r the

two p u m p s clearly indicates that they c a n f u n c t i o n independently o f each other. W i t h a 2 0 - V / c m field a n d a frequency o f 1 k H z , the K

p u m p , b u t not

+

the N a p u m p , w o u l d be activated. L i k e w i s e , at 1 M H z the N a p u m p , b u t +

not the K

+

p u m p , w o u l d be activated. W h e t h e r phosphorylation o f the

+

enzyme is responsible f o r c o u p l i n g the two p u m p s i n vivo remains to be tested. U n d e r o p t i m a l conditions, the net ac-stimulated N a 1 5 - 3 0 ions p e r enzyme p e r second a n d R b

p u m p i n g was

+

p u m p i n g was 1 0 - 2 0 ions p e r

+

enzyme p e r second. T h e ratio for the t w o was roughly 3:2. T h e agreement o f this value w i t h the generally observed transport ratio m a y b e coincidental. It is, however, possible to select a frequency o f the ac field f o r w h i c h the ratio of N a

+

to K

+

transport w o u l d be 3:2.

T h e ac-stimulated cation p u m p i n g apparently d i d not require hydrolysis o f A T P . A t 4 °C there was n o significant A T P hydrolysis, b u t there was stimulated activity. T h i s stimulated activity was almost identical i n A T P - c o n taining ( 6 0 0 - 8 0 0 - μ Μ A T P ) a n d A T P - d e p l e t e d ( < 1 0 - μ Μ A T P ) erythrocytes. T h e M i c h a e l i s - M e n t e n constant,

o f internal N a

K, M

+

for the R b

uptake a n d the N a efflux was 8 m M , a n d that f o r the external K +

+

+

was 1.5

m M , w i t h a H i l l coefficient o f approximately 1.5. These values agree w i t h A T P - d e p e n d e n t p u m p i n g activity o f the enzyme. O t h e r inhibitors o f the enzyme also h a d similar effects o n the ac-stimu lated activity. O u a b a i n ( 5 0 % i n h i b i t i o n at 0.35 μ Μ ) , o l i g o m y c i n (8.0 μ Μ ) , ouabagenin (3 μ Μ ) , a n d vanadate (20 μ Μ ) a l l i n h i b i t e d the ac-stimulated p u m p i n g activity. 4,4 -Bis(isothiocyano)-2,2 -distilbenesulfonate ,

,

inhibitor o f b a n d 3 p r o t e i n - m e d i a t e d L i / N a +

+

( D I D S ) , an

exchange, h a d no effect, n o r

d i d p h l o r e t i n at m i c r o m o l a r concentrations. These results show that N a , K - A T P a s e is responsible f o r the observed effects.



566


5

10

15

Stimul.

20

25

Voltage.

30

35

5

V/cm

10

15

Stlmul.

20

25

Voltage,

30

35

V/cm

Figure 1. Amplitude dependence of electric-field stimulated Na pumping (A) and Rb pumping (B). Na efflux was measured with the ac field at 1 MHz and Rb influx was measured at 1 kHz. 1 MHz and 1 kHz are the optimal frequencies of the Να pump and the Rb pump, respectively. Key: •, sample not stimulated with ac field; •, sample stimulated with ac field; • , sample not stimulated with ac field, in the presence of 0.2-mM ouabain; O , sample stimulated with ac field, in the presence of 0.2-mM ouabain. 1 amole / RBC-hr = 0.0108 mmol/ L of cells per hour, where RBC is red blood cell count. These measurements were done at 3 °C. (Adapted from reference 30.) +

+

+

+

+

+

Because the stimulated transport o f cations d i d not require A T P hydroly sis, the enzyme must b e able to absorb free energy f r o m the a p p l i e d electric field. O n l y a n oscillatory field o f d e f i n e d frequency a n d a m p l i t u d e was effective. Figures 1 a n d 2 show the dependence o f these i o n p u m p i n g activities o n the field strength a n d o n the frequency o f the a p p l i e d ac field, respectively. A n ac field c o u l d also stimulate A T P hydrolysis activity o f sheep k i d n e y N a , K - A T P a s e a n d E c t o nucleotide diphosphohydrolase, at 3 7 °C (31). A T P hydrolysis does not require energy input. T h e increase i n the A T P splitting activity reflects the sensitivity o f c h e m i c a l rate constants to a n electric field as i n eqs 7 a n d 8. F i g u r e 3 shows the A T P hydrolysis activity o f E c t o A T P a s e at 37 °C as a f u n c t i o n o f frequency w h e n a 5 . 0 - V / c m (peak-to-peak) ac field was used. A s m e n t i o n e d , B l a n k a n d Soo used 1 - m V / c m ac fields to stimulate the A T P splitting activity o f N a , K - A T P a s e ( 1 9 , 20). T h e i r results are also presented here.

Theory of Electroconformational Coupling T o interpret experimental data o n ac activation o f N a , K - A T P a s e , FQFJ^ A T P a s e , and other related m e m b r a n e p h e n o m e n a based o n the concept o f electrocon-



26.


TSONG

2

3

4

5

6

7

567

Coupling

8

0

Log (Freq. in Hz)

1

2

3

4

5

6

7

Log (Freq. in Hz)

Figure 2. Frequency dependence of electric-field-stimulated Να pumping (A) and Rb pumping (B). Afield strength of 20 V/cm (peak-to-peak) was used in both cases. Symbols are the same as in Figure 1. These measurements were done at 3 °C. (Adapted from reference 30.) +

+

3000

0

1

2 3 4 5 Log Frequ./Hz

6

Figure 3. Frequency dependence of electric-field-stimulated ATP hydrolysis activity of a highly purified Ecto nucleotide diphosphohydrolase. The ΑΤΈ splitting activity of the detergent-solubilized Ecto enzyme was measured at 37 °C in the absence of ac field (Δ), in the presence of 5.0-V/cm (peak-to-peak) ac field (O), and in the presence of 10-mM vanadate (A). Buffer: 0.1% of detergent NP40, 30-mM histidine, 4-mM ATP, 4-mM Mg , at pH 7.4. The optimal amplitude of stimulation is 5.0 V/cm with a 10-kHz ac field. (Adapted from reference 31.) 2+


8

568

BO IMEMBRANE ELECTROCHEMS ITRY

formational c o u p l i n g discussed earlier, w e have used a four-state m e m b r a n e transport, reaction 9 , to analyze thermodynamics of i o n p u m p i n g i n an oscillating electric field (13-17):


r 'out

1 ?

-

T

S

(9)

in

TS 2

Results o f o u r analysis indicate that any M i c h a e l i s - M e n t e n type o f enzyme has the ability to b e c o m e a free energy transducer u n d e r certain specified conditions (14).

I n the absence o f an electric field, reaction 9 is a facilitated

m e m b r a n e transport system; that is, the transporter, T , can carry the sub strate S across the m e m b r a n e f r o m the compartment w i t h higher concentra tion o f S (out) to the compartment w i t h l o w e r concentration o f S (in). H o w e v e r , w h e n an ac field is t u r n e d on, Τ can p u m p S f r o m i n to out, u p the concentration gradient. A n enzyme or transporter must be able to interact w i t h an electric

field.

This simply means that there is charge displacement i n its structure that is associated w i t h the T

x

to T

2

transition. A n electric field favors a state w i t h

greater electric m o m e n t (assuming T ) than a state w i t h smaller electric 2

m o m e n t (assuming T j ) . I f the Tj^ to T

2

transition changes the molar electric

m o m e n t of the transporter b y Δ Μ , an electric field o f E β

m

w i l l effect a shift

i n the e q u i l i b r i u m according to e q 6. W h e n an oscillating electric

field,

E

a

= E

a c

sin(a>0, is applied, the

enzyme w i l l be f o r c e d to oscillate a m o n g its various conformational states. These oscillations w i l l drive the catalytic cycle of reaction 9 to t u r n either clockwise or counterclockwise. T h e direction o f flux depends o n the affinity o f Τ for S. I f T

2

has higher affinity for S

i n

than T

x

has for S

o u t

, t h e n the ac

field w i l l t u r n the w h e e l clockwise to p u m p S f r o m right to left. Otherwise, the ac w i l l t u r n the w h e e l counterclockwise to p u m p S f r o m left to

right.

F i g u r e 4 shows an example of ac field driven conformational oscillations of reaction 9 . State occupancies o f different species of the transporter are plotted as a function o f the ac cycle. A s seen, each species oscillates w i t h periodicity coincident w i t h the ac frequency. I n other words, the enzyme oscillation is enforced by the ac field. F i g u r e 5 reproduces the frequency dependence o f N a

+

and R b

+

p u m p i n g b y N a , K - A T P a s e using suitable

kinetic parameters. F o r this process to occur efficiently, the frequency of the ac field must match the kinetic characteristics of reaction 9 . N u m e r i c a l analysis indicates


26.

TSONG

Electroconformational Coupling

569


0.625 Γ

1.5 2.0 TIME (t / f) Figure 4. ac-induced conformational oscillations of enzyme states in reaction 9. The state occupancy of T S is plotted as a function of the ac cycle (A). The net clockwise flux of S is integrated (B). The set of parameters used for the calculation are given in Liu et al (30). 2

that the system can b e t u n e d to an o p t i m a l set o f conditions f o r a maximal energy conversion. F o r a neutral ligand, the efficiency o f energy conversion (i.e., the ratio o f p o w e r absorption a n d p o w e r output) approaches the theoretical m a x i m u m 1 0 0 % ( i n practice, it is less than 100%) i f (10)


570


"C

40.

ο OQ

œ ω

£ο

30.

20.

X


3

10. >

Ο

< Log (Freq. in Hz) Figure 5. Simulation of the ac-stimulated cation pumping of Figure 2 by the electroconformational coupling model. The filled circles are experimental points and the solid lines are calculated curves according to reaction 9. (Reproduced with permission from reference 32. Copyright 1991.)

Here r

œ n f

, r , and r e

b

are, respectively, the rate o f the T to T x

T S ) transition, the rate o f the rise o f electric 2

binding of S

i n

to T

2

(or S

o u t

field,

2

(or T j S to

a n d the rate o f the

to Τ ) . W h e n this inequality is met, the system χ

w i l l respond to the oscillating field at nearly e q u i l i b r i u m conditions and, as a result, the efficiency o f energy conversion reaches its maximal value. A n y m e m b r a n e transport system m a y b e classified into one o f the six categories:

1. A transporter without gating charges a n d a neutral ligand. 2. A transporter w i t h gating charges a n d a neutral ligand. 3. A transporter without gating charges a n d a charged ligand. 4. A t r a n s p o r t e r - l i g a n d complex without gating charges; that is, the gating charges o f the transporter are neutralized b y the charges o f the ligand. 5. A transporter a n d its complex w i t h the ligand having gating charges o f opposite signs. 6. A transporter w i t h gating charges o f the same sign as the charged ligand.

System 1 is electrically inactive a n d cannot b e c o u p l e d to a n ac field f o r energy transduction. A l l other systems absorb energy f r o m a n ac field to actively transport the ligand. System 2 has b e e n examined i n greater detail


26.

TSONG


elsewhere (13-17).

571

Coupling

F o r systems 2 a n d 6, the efficiency o f energy transduc

tion can approach 1 0 0 % w h e n a large-amplitude ac field is used a n d w h e n the c o n d i t i o n specified b y e q 10 is satisfied. T h e maximal efficiency is only 5 0 % for systems 3 a n d 4. I n these two systems, reverse energy transduction (i.e., the conversion o f c h e m i c a l potential energy into an electric

energy)

cannot be achieved. I n system 5, the maximal efficiency is a mere 8.7%. T h e


active p u m p i n g of ligand i n systems 3, 4, a n d 5 is mainly due to

the

rectification. I n a l l cases except system 1, the active flux depends strongly o n the frequency o f the field a n d o n the concentration o f the ligand, a n d there are windows b o t h o n the frequency a n d the concentration axes. B y measuring the windows, rate constants a n d e q u i l i b r i u m constants o f reaction 9 can be d e t e r m i n e d (32).

F i g u r e 6 gives a three-dimensional relationship o f the active

flux, the ac frequency, a n d the ligand concentration. T h e presence

of a

concentration w i n d o w remains to be verified by experiment. E n e r g y transduction b y a m e m b r a n e p r o t e i n is not l i m i t e d to electric signals. A n y potential energy that can interact w i t h a p r o t e i n a n d that exhibits similar properties o f an oscillating field s h o u l d also be able to transmit energy to the p r o t e i n to drive an endergonic reaction (14, T

x

to T

2

22).

F o r example, i f the

transition involves a v o l u m e change or a change i n its area i n the

l i p i d bilayer, it s h o u l d be able to transduce acoustic signals. T h e f o r m e r is called P V c o u p l i n g , and the latter is called 7A c o u p l i n g (33).

W e have

investigated h o w such a mechanosensitive membrane transport system can absorb energy f r o m an acoustic signal to p u m p an i o n and, thus, convert acoustic energy into electric potential energy (33).

L i k e w i s e , temperature

oscillations a n d concentration oscillations can also be used for energy a n d signal transductions. T h e temperature oscillations require a A H (enthalpy o f reaction) a n d the concentration oscillations require a AG reaction) for the T

x

to T

2

transition

(free energy o f

(14).

Measurements of Membrane Electric Parameters F o r the analysis o f data using the electroconformational c o u p l i n g m o d e l , w e n e e d to k n o w electric parameters o f c e l l membranes. T h e potential generated b y an ac

field

transmembrane

can be calculated using the

Schwan

equation (eq 2). T o test whether the equation is applicable to c e l l m e m branes, w e have measured the critical b r e a k d o w n potential, Δ ψ

ο Λ

, of myeloma

cell membranes u s i n g an ac field o f frequencies f r o m 100 H z to 300 k H z . W h e n an a c - i n d u c e d Δ ψ pores a n d a dye

fluorescence

ιη

equaled Aif/ , the m e m b r a n e was p u n c t u r e d w i t h crit

p r o b e ( p r o p i d i u m iodide) permeated the cell. T h e

b o u n d specifically to D N A and gave rise to a r e d color (emission

m a x i m u m 639 n m ) . F i g u r e 7 gives an example o f the measurement o f E using p r o p i d i u m i o d i d e . T h e field intensity that i n d u c e d a Δ ψ

0Γίί

, E


c r i t

c r i t

, was

572



A

Β

-2.

V — 1

-1.

-0.5

.

ι

.

.

.

1

0.

0.5

1.

1.5

2.

2.5

log(f/k ) 14

Figure 6. The ac-stimulated flux, ] u, as a function of dimensionless frequency, f /k , and ligand concentration A (A) and contour maps of the same plot (B). Data from Markin and Τ song (32). lm

14

plotted as a f u n c t i o n o f the ac frequency i n F i g u r e 8. These curves fitted nicely to the Schwan equation, w h i c h indicates that the Schwan equation is applicable to cell membranes (12). T h e dielectrophoresis m e t h o d has also b e e n used to obtain the m e m b r a n e capacitance a n d the m e m b r a n e dielectric constant (34). T h e electroconformational c o u p l i n g m e t h o d measures the force acting o n a c e l l b y a n o n u n i f o r m oscillating electric field. D e p e n d i n g o n the frequency



26.

TSONG

573

Electroconformational Coupling

Figure 7. Measurement of the cntical breakdown potential of a myeloma cell membrane using an ac field. The chemical formula of propidium iodide is shown in the upper figure. The middle figure shows a typical myeloma cell under the microscope (bright field). The relative positions of the platinum electrodes are indicated. The lower figure gives some photographs taken at different times after a cell was electroporated by an ac field E . Within 1-3 s, two narrow, fluorescent bands appeared at the two loci facing the electrodes (the leftmost photo). The next three photos, from left to right, were taken at 20 s, 1 min, and 3 min, respectively, after the application of a 200-ms ac field of 1 kV/cm at 100 kHz. (Reproduced with permission from reference 12. Copyright 1990.) c r i t

of the ae, the c e l l may b e attracted either toward higher o r lower intensity. A t a critical frequency, f

field

, the net force acting o n the c e l l is zero;

CTit

that is, the c e l l stops m o v i n g i n either direction. A c c o r d i n g to Saver ( 3 5 ) , this frequency depends o n electric parameters o f the c e l l m e m b r a n e a n d the conductivity o f the external m e d i u m . Experimentally, f

crit

is d e t e r m i n e d i n

m e d i a o f different conductivities. B y regression analysis the dielectric c o n stant a n d the m e m b r a n e conductivity c a n b e d e t e r m i n e d ( 3 4 ) .



574


LOG (FREQUENCY IN Hz)

Figure 8. Critical ac field strength, E , for electroporation of myeloma cells as a functions of ac frequency. Data were obtained in media of different resistivities: 52,600 Ω cm (O); 7050 Ω cm (Φ); 2380 Ω cm(u). The curves drawn through these data points were obtained by calculation (O) or optimization ( · and • ) according to the Schwan equation. (Reproduced with permission from reference 12. Copyright 1990.) crit

Developmental Bibliography T h e conceptual development o f the theory o f electroconformational c o u p l i n g can b e traced b y referring to the following list o f publications. 1987 Tsong, T . Y . ; A s t u m i a n , R. D . E l e c t r o c o n f o r m a t i o n a l c o u p l i n g a n d m e m b r a n e protein function. Prog. Biophys.

Mol. Biol. 50, 1 - 4 5 .

Tsong, T . Y . ; C h a u v i n , F . ; A s t u m i a n , R . D . Interaction o f m e m b r a n e proteins w i t h static a n d dynamic electric fields v i a electroconformational c o u p l i n g . I n Mechanistic Fields with Living

Approaches

Systems;

to Interaction

of

Electromagnetic

Blank, M.; Findl, E . , E d s . ; P l e n u m : New

York; p p 1 8 7 - 2 0 2 .


26.

TSONG

Westerhoff,


575

Coupling

H. V.; K a m p , F . ; Tsong, T . Y . ; A s t u m i a n , R .

D. Interaction

between enzyme catalysis a n d nonstationary electric fields. I n nistic Approaches

to Interaction

of Electromagnetic

Fields with

MechaLiving

Systems; Blank, M.; Findl, E., E d s . ; P l e n u m : N e w Y o r k ; p p 2 0 3 - 2 1 5 . 1988


Tsong, T . Y . A c t i v e cation p u m p i n g o f N a , K - A T P a s e a n d S R C a - A T P a s e i n d u c e d b y an electric field. Methods Enzymol.

157, 2 4 0 - 2 5 1 .

Tsong, T . Y . ; A s t u m i a n , R . D. Electroconformational c o u p l i n g : An efficient mechanism for energy transfection b y m e m b r a n e - b o u n d A T P a s e s . Ann. Rev. Physiol.

50, 2 7 3 - 2 9 0 .

Tsong, T . Y . ; T o m i t a , M.; Lo, M. M. S. Pre-selection o f B-lymphocytes b y antigen for fusion to m y e l o m a cells b y p u l s e d electric field m e t h o d . I n Molecular

Mechanisms

of

Membrane

Fusion;

O k i , S. et a l . , E d s . ;

P l e n u m : N e w York; p p 2 2 3 - 2 3 6 . A s t u m i a n , R . D.; C h o c k , P . B . ; C h a u v i n , F.; Tsong, T . Y . E l e c t r o c o n f o r m a tional c o u p l i n g a n d the effects o f static a n d dynamic electric fields o n membrane

transport. I n Electromagnetic

Fields

and

Biomembranes;

Markov, M.; Blank, M., Eds.; Plenum: New York; pp 59-74. 1989 Tsong, T . Y . Electroporation o f c e l l membranes: Its mechanisms a n d applica tions. I n Electroporation and Electrofusion in Cell Biology; N e u m a n n , E. et al, E d s . ; P l e n u m ; N e w York; p p 1 4 9 - 1 6 3 . L o , M. M. S.; Tsong, T . Y . P r o d u c i n g monoclonal antibodies b y electrofusion. I n Electroporation and Electrofusion in Cell Biology; N e u m a n n , E. et al., E d s . ; P l e n u m : N e w York; p p 2 5 9 - 2 7 0 . Tsong, T . Y . D e c i p h e r i n g the language o f cells. Trends in Biochem. 89-92.

Set. 14,

Tsong, T . Y . ; Liu, D. S.; C h a u v i n , F.; A s t u m i a n , R . D. Resonance electrocon formational c o u p l i n g : A proposed mechanism for energy a n d signal transduction b y membrane proteins. Biosci. Rep. 9, 1 3 - 2 7 . Tsong, T . Y . Electroconformational c o u p l i n g : A fundamental process o f biomolecular electronics for signal transduction. I n Molecular Electronics; H o n g , F . , E d . ; P l e n u m : N e w York; p p 8 3 - 9 5 . Tsong, T . Y . ; A s t u m i a n , R . D . C h a r g e - f i e l d interactions i n c e l l membranes and electroconformational coupling: Transduction o f electric energy b y membrane A T P a s e . I n Charge-Field Effects in Biosystems; A l l e n , M . J., E d . ; P l e n u m : N e w York; V o l . II, p p 1 6 7 - 1 7 7 . A s t u m i a n , R . D . ; Robertson, B . ; Tsong, T . Y . C h a r g e - f i e l d interactions i n c e l l membranes a n d electroconformational coupling: T h e o r y f o r the interac tions between dynamic electric fields a n d membrane enzymes. I n Charge-Field Effects in Biosystems; A l l e n , M . J., E d . ; P l e n u m : N e w York; V o l . II, p p 1 7 9 - 1 9 0 .


576


Robertson, Β.; A s t u m i a n , R . D . ; Tsong, T . Y . N o n l i n e a r effects o f p e r i o d i c electric fields o n m e m b r a n e proteins. I n Charge-Field

Effects in Biosys-

tems; A l l e n , M . J., E d . ; P l e n u m : N e w York; V o l . II, p p 1 9 1 - 2 0 9 . A s t u m i a n , R . D . ; C h o c k , P . B . Tsong, T . Y . ; Westerhoff, H . V . Effects o f ;

oscillations a n d energy-driven fluctuations o n the dynamics o f enzyme


catalysis a n d free energy transduction. Phys. Rev. A 39, 6 4 1 6 - 6 4 3 5 . 1990 L i u , D . S.; A s t u m i a n , R . D . ; Tsong, T . Y . A c t i v a t i o n o f N a a n d the R b p u m p i n g modes o f N a , K - A T P a s e b y an oscillating electric field. /. Biol. Chem. 265, 7 2 6 0 - 7 2 6 7 . Tsong, T . Y . E l e c t r i c m o d u l a t i o n o f m e m b r a n e proteins: E n f o r c e d conforma tional oscillation a n d cellular energy a n d signal transductions. Rev. Biophys.

Biophys.

Chem.

Annu.

19, 8 3 - 1 0 6 .

D e B u s t r o s , Α.; L e e , R . Y . C o m p t o n , D . ; Tsong, T . Y . ; B a y l i n , S. B . ; N e l k i n , ;

B . D . D i f f e r e n t cis-acting D N A elements regulate expression o f the h u m a n calcitonin gene i n c u l t u r e d lines o f l u n g carcinoma a n d medullary thyroid carcinomas. Mol. Cell Biol. 10, 1 7 7 3 - 1 7 7 8 . X i e , T . D . ; S u n , L . ; Tsong, T . Y . Study o f mechanisms o f electric

field

i n d u c e d D N A transfection I: D N A entry b y surface b i n d i n g a n d d i f f u sion through m e m b r a n e pores. Biophys.

J. 58, 1 3 - 1 9 .

X i e , T . D . ; Tsong, T . Y . Study o f mechanisms o f electric field-induced D N A transfection II: Transfection b y low-amplitude, low-frequency alternat i n g electric fields. Biophys.

J. 58, 8 9 7 - 9 0 3 .

M a r l d n , V . S.; T s o n g , T . Y . ; A s t u m i a n , R . D . ; Robertson, B . E n e r g y transduc tion between a concentration gradient a n d an alternating electric /. Chem.

field.

Phys. 93, 5 0 6 2 - 5 0 6 6 .

Marszalek, P . ; L i u , T . S.; Tsong, T . Y . Schwan equation a n d transmembrane potential i n d u c e d b y alternating electric

field.

Biophys.

J. 58, 1 0 5 3 -

1058. Tsong, T . Y . O n electroporation a n d some related c e l l m e m b r a n e p h e n o m ena. Bioelectrochem.

Bioenerg.

24, 2 7 1 - 2 9 5 .

T o m i t a , M . ; Tsong, T . Y . Selective p r o d u c t i o n o f h y b r i d o m e cells: Preselec tion o f B-lymphocytes b y antigen f o r electrofusion w i t h m y e l o m a cells. Biochim.

Biophys.

Acta 1055, 1 9 9 - 2 0 6 .

1991 Prudovsky, I.; Tsong, T . Y . F u s i o n o f

fibroblasts

w i t h differentiated a n d

non-differentiated l e u k e m i a cells resulting i n blockage o f D N A synthe sis. Dev. Biol. 144, 2 3 2 - 2 3 9 . Marszalek, P . ; Zielinsky, J . J.; F i k u s , M . ; Tsong, T . Y . D e t e r m i n a t i o n o f electric parameters o f c e l l membranes b y a dielectrophoresis m e t h o d . Biophys.

J. 59, 9 8 2 - 9 8 7 .


26.

TSONG


577

Coupling

M a r k i n , V . S.; Tsong, T . Y . Reversible mechanosensitive i o n p u m p i n g as a part o f mechanoelectrical transduction. Biophys.

J. 59, 1 3 1 7 - 1 3 2 4 .

M a r k i n , V . S.; Tsong, T . Y . F r e q u e n c y a n d concentration windows for the electric activation o f a m e m b r a n e active transport system. Biophys.

J.

59, 1 3 0 8 - 1 3 1 6 . M a r k i n , V . S.; Tsong, T . Y . Electroconformational c o u p l i n g o f i o n transport i n


oscillating electric field: Rectification versus p u m p i n g . Bioenerg.

Bioelectrochem.

26, 2 5 1 - 2 7 6 .

References 1. Bockris, J. O ’ M . ; Reddy, Α. Κ. N. Modern Electrochemistry; Plenum: New York, 1970; Vols. I and II. 2. Cole, K. S. Membranes, Ions and Impulses; University of California Press: Berkeley, CA, 1972. 3. Tien, H. T. Bilayer Lipid Membranes; Dekker: New York, 1974. 4. Tsong, T. Y. Biosci. Rep. 1983, 3, 487. 5. Kinosita, K.; Tsong, T. Y. Proc. Natl. Acad. Sci. 1977, 74, 1923. 6. Schwan, H. P. In Biological Effects and Dosimetry of Nonionizing Radiation; Grandolfo, M.; Michaelson, S. M.; Rindi, Α., Eds.; Plenum: New York, 1983; p 213. 7. Gross, D.; Loew, L. M.; Webb, W. W. Biophys. J. 1986, 50, 339. 8. Kinosita, K.; Ashikava, I.; Staita, N.; Yoshimura, H.; Itoh, H.; Nagayama, K.; Ikegami, A. Biophys. J. 1988, 53, 1015. 9. Ehrenberg, B.; Farkas, D. L.; Fluhler, Ε. N.; Lojewska, Z.; Loew, L. M. Biophys. J. 1987, 51, 833. 10. Teissie, J.; Tsong, T. Y. Biochemistry 1981, 20, 1548. 11. El-Mashak, M. E.; Tsong, T. Y. Biochemistry 1985, 24, 410. 12. Marszalek, P.; Liu, D. S.; Tsong, T. Y. Biophys. J. 1990, 58, 1053. 13. Tsong, T. Y.; Astumian, R. D. Bioelectrochem. Bioenerg. 1986, 15, 457. 14. Tsong, T. Y. Annu. Rev. Biophys. Biophys. Chem. 1990, 19, 83. 15. Robertson, B.; Astumian, R. D. Biophys. J. 1990, 57, 689. 16. Astumian, R. D.; Chock, P. B.; Tsong, T. Y.; Westerhoff, Η. V. Phys. Rev. A 1989, 39, 6416. 17. Markin, V. S.; Tsong, T. Y.; Astumian, R. D.; Robertson, B. J. Chem. Phys. 1990, 93, 5062. 18. Blank, M. J. Electrochem. Soc. 1987, 134, 1112. 19. Blank, M.; Soo, L. Bioelectrochem. Bioenerg. 1989, 22, 313-322. 20. Blank, M.; Soo, L. Bioelectrochem. Bioenerg. 1990, 24, 51-61. 21. Mechanistic Approaches to Interactions of Electric and Electromagnetic Fields with Living Systems; Blank, M.; Findl, E., Eds.; Plenum: New York, 1987. 22. Tsong, T. Y. Trends Biochem. Sci. 1989, 14, 89. 23. Tsong, T. Y. In Molecular Electronics; Hong, H. T., E d . ; Plenum: New York, 1989; p 83. 24. Hoffman, J. F.; Kaplan, J. H.; Callahan, T. J. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1979, 38, 2440. 25. Witt, H. T.; Schlodder, E.; Graber, P. FEBS Lett. 1976, 69, 272. 26. Mouneimne, Y.; Tosi, P. F.; Gazitt, Y.; Niolau, C. Biochem. Biophys. Res. Commun. 1989, 159, 34.



578

BIOMEMBRANE

ELECTROCHEMISTRY

27. Neumann, Ε.; Sowers, Α. Ε.; Jordan, C. A. Electroporation and Electrofusion in Cell Biology; Plenum: New York, 1989. 28. Serpersu, E. H.; Tsong, T. Y. J. Membrane Biol. 1983, 74, 191. 29. Serpersu, E. H.; Tsong, T. Y. J. Biol. Chem. 1984, 259, 7155. 30. Liu, D. S.; Astumian, R. D.; Tsong, T. Y. J. Biol. Chem. 1990, 265, 7260. 31. Liu, D. S. et al. Abstract, F A S E B Annual Meeting, 1991. 32. Markin, V. S.; Tsong, T. Y. Biophys. J. 1991, 59, 1308. 33. Markin, V. S.; Tsong, T. Y. Biophys. J. 1991, 59, 1317. 34. Marszalek, P.; Zielinsky, J. J.; Fikus, M.; Tsong, T. Y. Biophys J. 1991, 59, 982. 35. Sauer, F. In Interactions between Electromagnetic Fields and Cells; Chiabrera, Α.; Grattarola, M.; Vivani, R., Eds.; Plenum: New York, 1985; p 181. RECEIVED

17,

f o r review January

29,

1991.

ACCEPTED

revised manuscript August

1992.


Biomembrane Electrochemistry - ACS Publications - American

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