Frequency Response of Membrane Components in Nerve Axon - ACS

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SHIRO TAKASHIMA Department of Bioengineering/D2, University of Pennsylvania, Philadelphia, PA 19104

The mechanism of nerve e x c i t a t i o n i s w e l l documented phenomenologically by the Hodgkin-Huxley equation (1,2. >_3) . According t o t h i s e m p i r i c a l f o r m u l a t i o n , the nerve impulse i s generated by two types of n o n - l i n e a r c u r r e n t s , i . e . , inward sodium and outward potassium c u r r e n t s . The magnitudes of these c u r r e n t s are c o n t r o l l e d by the membrane p o t e n t i a l , i n d i c a t i n g that there are v o l t a g e sensing mechanisms somewhere i n the membrane. F i r s t of a l l , the bulk o f nerve membranes i s hydrophobic. Therefore, f o r the charged sodium and potassium ions t o f l o w across the membrane, there must be s p e c i a l regions i n the membrane which have p o l a r groups p r o j e c t i n g i n t o them. These p o l a r regions have been c a l l e d i o n i c channels. They are h i g h l y s p e c i f i c and there are two types o f channels, one f o r sodium and another f o r potassium i o n s . These channels are normally c l o s e d a t the r e s t i n g s t a t e and, t h e r e f o r e , no ions can f l o w through them. However, when e l e c t r i c a l s t i m u l i are a p p l i e d , the f i e l d i s detected by the v o l t a g e sensor which, i n t u r n , sends a command to the gate t o open. I t i s s t i l l debatable whether there i s a v o l t a g e sensor which i s separate from the channel gate i t s e l f . N e v e r t h e l e s s , the f i e l d sensing d e v i c e , i n c l u d i n g the gate o r g a t i n g p a r t i c l e s , must be a group of charged p a r t i c l e s o r a group o f molecules having d i p o l e moments. I f the gating p a r t i c l e i s a charged molecule, the opening of gates must be an e l e c t r o p h o r e t i c movement o f these charges. I f the gate i s a group of d i p o l e moments, the opening of channels must be an o r i e n t a t i o n of these d i p o l a r s p e c i e s . Of these, d i p o l e o r i e n t a t i o n has been p r e f e r r e d by n e u r o p h y s i o l o g i s t s as the p o s s i b l e mechanism of channel opening. I n any case, the opening of channels i s i n i t i a t e d by the change of the f i e l d i n the membrane and the r a t e of the opening i s c o n t r o l l e d by the frequency response o r r e l a x a t i o n time of g a t i n g p a r t i c l e s . Of the two d i f f e r e n t types o f i o n i c channels, the opening of sodium channels i s considered as the f i r s t step of nerve e x c i t a t i o n . The opening of potassium channels i s a delayed r e sponse and lags behind that of sodium channels. There are 0097-6156/81/0157-0133$05.00/0 © 1981 American Chemical Society

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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roughly two ways to i n v e s t i g a t e the mechanism of channel opening. One method i s to study the g a t i n g current i n the time domain and the other i s to measure v o l t a g e dependent membrane capacitance i n the frequency domain. Measurements of g a t i n g c u r r e n t s i n the time domain have been done by s e v e r a l groups (4-9). The magnitude of the g a t i n g current i s s m a l l and i s of the order of 20-40 yA/cm . Because of t h e i r s m a l l magnitude, both sodium and potassium c u r r e n t s must be e i t h e r reduced o r blocked by v a r i o u s means, i n c l u d i n g use of t e t r o d o t o x i n (TTX). Based on the analyses done by these people, the g a t i n g current i s b e l i e v e d to be due to sodium channels r a t h e r than potassium channels. The g a t i n g current due to potassium channels was r e p o r t e d by Armstrong (10) recently. The time constant of the g a t i n g current i s a p p r o x i mately 80 to 110 ysec and i s almost independent of membrane potentials. T h e r e f o r e , the opening of sodium channels i s a slow process having a frequency response of a few k i l o - h e r t z . 2

The other experiments, i . e . , frequency domain measurements of membrane capacitance and i t s v o l t a g e dependence, have been done by v a r i o u s i n v e s t i g a t o r s (11-18). The r a t i o n a l e behind these experiments i s the f o l l o w i n g . I f there are d i p o l a r p a r t i c l e s i n the membrane and i f they are capable of o r i e n t i n g i n response to a p p l i e d f i e l d s , t h i s movement must manifest i t s e l f as a frequency dependent membrane c a p a c i t a n c e , the i n d i c a t i o n of the presence of r e l a x a t i o n processes. T h e r e f o r e , the i n i t i a l step i n t h i s s e r i e s of experiments i s to e s t a b l i s h the membrane capacitance i n a wide frequency range and confirm the presence of frequency dependent c a p a c i t a n c e . Secondly, i f a p p l i c a t i o n of an e x t e r n a l v o l t a g e causes d i s l o c a t i o n of g a t i n g p a r t i c l e s from r e s t i n g p o s i t i o n to open p o s i t i o n , t h i s must manifest i t s e l f i n the change i n the magnitude of capacitance and i t s frequency dependence. Since time domain measurements i n d i c a t e the e x i s t e n c e of g a t i n g c u r r e n t s , i . e . , e s s e n t i a l l y a c a p a c i t i v e c u r r e n t , membrane capacitance must change upon a p p l i c a t i o n of e l e c t r i c a l pulses. As i s w e l l known, b i o l o g i c a l membranes c o n s i s t of two major components, i . e . , l i p i d s and p r o t e i n s . Although the progress i n the research on membrane p r o t e i n s i s very slow, i n v e s t i g a t o r s are beginning to f i n d that there are a v a r i e t y of p r o t e i n s i n b i o l o g i c a l membranes having molecular weights ranging from 20,000 to 300,000. Moreover, even the l i p i d part i s not uniform and monodisperse. There are s e v e r a l d i f f e r e n t types of l i p i d s i n b i o l o g i c a l membranes which form a b i l a y e r s t r u c t u r e . Under these circumstances, i t i s wise to s t a r t the i n v e s t i g a t i o n of membrane impedance by using simple systems r a t h e r than complex n a t u r a l membranes. A r t i f i c i a l l i p i d membranes, which resemble b i o l o g i c a l membranes except f o r the absence of p r o t e i n s , are r e a d i l y a v a i l able these days (19,20). T h e i r p h y s i c a l and e l e c t r i c a l charact e r i s t i c s are w e l l documented and these membranes are q u i t e s u i t a b l e f o r the study of membrane capacitance and i t s frequency dispersion.


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Membrane Components in Nerve Axon

Membrane Capacitance of A r t i f i c i a l Membranes The techniques used to form l i p i d b i l a y e r membranes are discussed by M u e l l e r and Rudin (19) i n d e t a i l . In t h e i r methods, l i p i d s are d i s s o l v e d i n alkane s o l v e n t s and the s o l u t i o n i s p a i n t e d to an aperture on the w a l l of a t e f l o n cup. The e n t i r e cup i s immersed i n an e l e c t r o l y t e s o l u t i o n such as NaCl or KC1. The t h i c k n e s s of the membrane, which i s m u l t i - l a y e r e d at the b e g i n n i n g , w i l l reduce w i t h time and f i n a l l y a b i l a y e r membrane i s formed w i t h a t h i c k n e s s of 30 to 60A depending upon the length of non-polar t a i l s of l i p i d molecules. Some s o l v e n t s such as n-decane are m i s c i b l e w i t h l i p i d s and form mixed membranes. However, some other s o l v e n t s , such as hexadecane, separate from l i p i d molecules when membranes are formed. E i t h e r s o l v e n t molecules form micro-lenses on the s u r f a c e of the membrane or they accumulate i n the torus region at the edge. In e i t h e r case, the presence of s o l v e n t molecules produces c e r t a i n e r r o r s . However, as w i l l be discussed l a t e r , use of hexadecane i s much b e t t e r than n-decane f o r our purpose. Thus, our experiments were a l l c a r r i e d out u s i n g hexadecane as the s o l v e n t . Although we used s e v e r a l l i p i d s , such as o x i d i z e d c h o l e s t e r o l , sphingomyelin and eggl e c i t h i n , we w i l l i l l u s t r a t e only the r e s u l t s obtained w i t h eggl e c i t h i n and hexadecane. Membrane capacitance and conductance were measured u s i n g the Wayne-Kerr admittance b r i d g e and measurements were made between 100 Hz and 20 KHz using a PAR l o c k - i n a m p l i f i e r model 124. One of the r e s u l t s obtained i s shown i n F i g u r e 1. The dotted curve shown i n t h i s f i g u r e i n d i c a t e s measured values a t h i g h frequencies and the downward slope a r i s e s from the presence of e l e c t r o l y t e s o l u t i o n s between the membrane and e l e c t r o d e s . The e r r o r due to s e r i e s r e s i s t a n c e was a l r e a d y discussed i n det a i l by Takashima and Schwan (15). The s o l i d l i n e i s obtained a f t e r the c o r r e c t i o n f o r t h i s e r r o r u s i n g the method d e s c r i b e d previously. F i r s t of a l l , the capacitance of lecithin-hexadecane membrane i s about 0.62 yF/cm . This value i s s m a l l e r than the capacitance of b i o l o g i c a l membranes, i . e . , 1 yF/cm . The d i f ference i s perhaps p a r t i a l l y due to the absence of p r o t e i n s i n a r t i f i c i a l membranes. In a d d i t i o n , i t i s known that the presence of s o l v e n t s decreases the values of membrane capacitance. F o r example, membranes formed by the Montal-Mueller Method (21), which are b e l i e v e d to be f r e e of s o l v e n t s , have a capacitance of 0.7 yF/cm (22). Thus, the capacitance of b i l a y e r membranes shown i n t h i s f i g u r e may be i n e r r o r by about 0.1 yF/cm because of the presence of s o l v e n t molecules. However, i t i s more important to note that membrane capacitance i s independent o f frequency, which provides unequivocal evidence that there i s no r e l a x a t i o n process i n l i p i d membranes i n t h i s frequency range. Coster and Smith (23) r e p o r t e d t h a t they observed a frequency d i s p e r s i o n of membrane capacitance of a r t i f i c i a l l a y e r s a t very 2

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Membrane capacitance (Curve 1 ) and conductance (Curve 2) at different membrane potentials


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low frequencies below 10 Hz. However, the meaning of t h i s d i s p e r s i o n i s not c l e a r l y known and i t may have been due to the presence of s o l v e n t molecules which tend to form some type of boundaries w i t h i n the membrane. The c h a r a c t e r i s t i c frequencies of sodium and potassium currents are of the order of 200 Hz t o 3 KHz. As has been d i s c u s s e d , l i p i d b i l a y e r s do not e x h i b i t any r e l a x a t i o n processes i n t h i s frequency range, i n d i c a t i n g t h a t l i p i d molecules may not p l a y an important r o l e i n the permeation of ions through i o n i c channels. The v o l t a g e dependence of l i p i d b i l a y e r capacitance has been s t u d i e d by White and Thompson (24). They noted t h a t the presence of n-decane i n these membranes gives r i s e to a f a l s e v o l t a g e dependence of membrane capacitance. On the other hand, L a t o r r e et a l . (25), u s i n g s o l v e n t - f r e e membranes, demonstrated t h a t the capacitance of these membranes i s only n e g l i g i b l y dependent on e x t e r n a l v o l t a g e s . U n l i k e the experiments by White and Thompson, we used hexadecane i n s t e a d of n-decane as the s o l v e n t . The capacitances of l e c i t h i n b i l a y e r s at v a r i o u s membrane p o t e n t i a l s are shown i n F i g u r e 2. C l e a r l y , membrane capacitance i s i n d e pendent of a p p l i e d p o t e n t i a l s , i n d i c a t i n g t h a t 1) the presence of hexadecane does not produce a v o l t a g e dependent capacitance and 2) the membrane capacitance of l e c i t h i n b i l a y e r s i s i t s e l f independent of e x t e r n a l p o t e n t i a l . As s t a t e d b e f o r e , hexadecane does not form mixed membranes w i t h l e c i t h i n and they are spat i a l l y separated when the membrane i s formed. This may be the reason why hexadecane does not produce the a r t i f a c t as n-decane does. In any event, these observations c l e a r l y i n d i c a t e that l e c i t h i n b i l a y e r does not e x h i b i t a frequency dependent c a p a c i tance between 100 Hz and 20 KHz and e x t e r n a l l y a p p l i e d p o t e n t i a l s have no e f f e c t on the membrane capacitance. Membrane Capacitance of Nerve Membrane Giant axons from s q u i d have a l a r g e diameter ranging from 300 to 700 ym. Because of t h i s l a r g e s i z e , we are able to i n s e r t metal e l e c t r o d e s d i r e c t l y i n t o the axon and measure capac i t a n c e and conductance across the membrane. This i s the most unequivocal method to measure transmembrane capacitance and i s f a r b e t t e r than the use of e x t e r n a l e l e c t r o d e s as done p r e v i o u s l y by Cole and C u r t i s (11). In s p i t e of the s i m p l i c i t y and ease of t h i s technique, there are s t i l l a few unsolved problems which w i l l be discussed l a t e r . F i g u r e 3 shows one of the exemplary r e s u l t s of nerve membrane capacitance and conductance measurements. Comparison of t h i s r e s u l t w i t h the one shown i n F i g u r e 2 r e a d i l y demonstrates that there are c o n s i d e r a b l e d i f f e r e n c e s between these two s e t s of curves. F i r s t of a l l , membrane capacitance of nerves has a negative frequency dependence below 300 Hz, namely capacitance i n c r e a s e s w i t h frequency i n t h i s r e g i o n . This i s a behavior which cannot be e x p l a i n e d by a simple RC c i r c u i t and i n d i c a t e s the presence


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FREQUENCY Hz Figure 3. Membrane capacity (Curve 1 ) and conductivity (Curve 2) of squid giant axon at various frequencies. Note anomalous behavior at low frequencies.

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Figure 4. Schematic of the field distribution at various distances from the point of current injection: λ is the space constant, x , x and r are membrane resistance and resistances of internal and external media, respectively. The dimension of r is ohm cm; those of r, and r are ohms. m

h

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of an i n d u c t i v e element i n the membrane (26). Membrane c a p a c i tance reaches a peak value of 1 yF/cm between 300 Hz and 1 KHz. This i s the value of capacitance which has been accepted u n i v e r s a l l y f o r most b i o l o g i c a l membranes (26,27). Above t h i s f r e quency, membrane capacitance dereases from 1 yF/cm to a p p r o x i mately 0.6 yF/cm between 1 and 20 KHz. This gradual decrease may be, at l e a s t p a r t i a l l y , due t o a r e a l d i s p e r s i o n of membrane capacitance. However, there i s d i s t u r b i n g evidence that a por t i o n of t h i s frequency dependent capacitance i s due to a f r i n g e e f f e c t a t the t i p o f the i n t e r n a l a x i a l e l e c t r o d e . Suppose one i n j e c t s a current a t one p o i n t o f the membrane, and monitors the voltage along the x-coordinate of the axon. The peak v o l t a g e values w i l l decay e x p o n e n t i a l l y along the f i b e r as shown i n Figure 4. Thus, 2

2

2

V(x) = V(0) exp - |χ/λ| where V(0) and V(x) a r e the peak v o l t a g e s at χ and 0. space constant and i s given by:

λ

2

λ i s the

r

=

; (υ + r 1 2 where rm> r * , and r - are membrane r e s i s t a n c e and r e s i s t a n c e s of i n t e r n a l and e x t e r n a l media o f the axon. I f the v o l t a g e at the t i p of the i n t e r n a l e l e c t r o d e i s V(0), the f i e l d w i l l taper o f f according to eq. ( 1 ) , g i v i n g r i s e to an a x i a l component i n a d d i t i o n to the r a d i a l d i s t r i b u t i o n o f the f i e l d . Therefore, the magnitude of the f r i n g e capacitance depends on the space constant, which i n t u r n i s a f u n c t i o n of membrane r e s i s t a n c e r . Furthermore, the space constant i s known to depend on frequency (28) and decreases with i n c r e a s i n g f r e q u e n c i e s . Thus, the s t r a y capacitance due to f r i n g e e f f e c t decreases as the frequency i n c r e a s e s . A c c o r d i n g l y , the presence of f r i n g e e f f e c t a t the t i p of the i n t e r n a l e l e c t r o d e produces a frequency dependent capacitance. The frequency dependence of membrane capacitance as shown i n F i g u r e 4 i s , t h e r e f o r e , a t l e a s t p a r t i a l l y due to the f r i n g e e f f e c t at the t i p of the i n t e r n a l electrode. In order to reduce the r e l a t i v e c o n t r i b u t i o n o f s t r a y capacitance, we use a long i n t e r n a l e l e c t r o d e with a length of 27 qm. As shown i n F i g u r e 5, the membrane capacitance per u n i t area i s shown a t v a r i o u s lengths of the i n t e r n a l e l e c t r o d e . F o r example, i f the length of the i n t e r n a l e l e c t r o d e i s 5 mm (Point 1), the membrane c a p a c i t y per u n i t area becomes as l a r g e as 2 yF/cm due to the presence of a r e l a t i v e l y l a r g e s t r a y c a p a c i tance. However, i f the length of the i n t e r n a l e l e c t r o d e i s 27mm, the capacitance p e r u n i t area decreases to 0.9 yF/cm because the s t r a y capacitance c o n t r i b u t e s a r e l a t i v e l y s m a l l value. Since the r e s u l t s shown i n Figure 4 were obtained with a r

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New York Academy of Sciences Figure 5. Membrane capacity per unit area of squid giant axon as a function of the length of the electrodes (29) ((upper scale) electrode length in mm; (lower scale) inverse of length in mm' ) 1

-40 0 40 MEMBRANE POTENTIAL mV

120

Figure 6. Membrane capacitance of squid giant axon at various membrane poten tials. Membrane potential was shifted by injecting currents. The abscissa shows actual potential across the membrane in mV.


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long i n t e r n a l e l e c t r o d e (27 mm), the r e l a t i v e c o n t r i b u t i o n o f s t r a y capacitance i s s m a l l . I t may be s a f e , t h e r e f o r e , to conclude that the frequency dependence of measured capacitance i s due t o a r e a l d i s p e r s i o n o f membrane capacitance. Once the f r e quency dependence of membrane capacitance i s e s t a b l i s h e d , the time constant o r c h a r a c t e r i s t i c frequency o f the r e l a x a t i o n w i t h i n the membrane can be e a s i l y determined. As can be seen, the center frequency i s about 3 KHz, which gives r i s e t o a time constant o f 53 ysec. The o r i g i n o f the frequency dependent membrane capacitance i s not w e l l understood, although i t i s l i k e l y to be due to membrane p r o t e i n s . However, there are a v a r i e t y of proteinous components i n the membrane and most of them are not d i r e c t l y r e l a t e d to i o n i c channels. Therefore, i t i s v i r t u a l l y impossible to p h y s i c a l l y separate the capacitance due t o g a t i n g p r o t e i n s from capacitances due t o other p r o t e i n s . However, i f the frequency dependent capacitance i s r e l a t e d t o gate p r o t e i n s , the c a p a c i tance must change with an i n c r e a s e o r decrease i n the membrane potential. I t i s known that i o n i c channels are not t i g h t l y c l o s e d a t the r e s t i n g s t a t e (membrane p o t e n t i a l near -60 mV). Only when the membrane p o t e n t i a l i s reduced to -120 mV, are a l l * i o n i c channels n e a r l y completely c l o s e d , and the membrane becomes r e a l l y p a s s i v e . Under these circumstances, the membrane w i l l be t i g h t l y packed and p r o t e i n s as w e l l as l i p i d s w i l l be locked i n t o a t i g h t l y c o n s t r a i n e d s t a t e l e a d i n g t o a small capacitance. Figure 6 shows the membrane capacitances at v a r i o u s p o t e n t i a l s . As one can see, the membrane capacitance at -60 mV ( r e s t i n g s t a t e ) i s 0.95 yF/cm , while h y p e r - p o l a r i z e d membrane has an even s m a l l e r capacitance. On the other hand, i o n i c channels open w i t h d e p o l a r i z a t i o n , i . e . , channel p r o t e i n s become r e l a t i v e l y f r e e t o r o t a t e because of removal o f the negative b i a s v o l t a g e . Therefore, the membrane capacitance must i n c r e a s e w i t h d e p o l a r i z i n g p o t e n t i a l s i f the c o n t r i b u t i o n of g a t i n g p r o t e i n s to membrane capacitance i s s i g n i f i c a n t . As shown i n t h i s f i g ure, c l e a r l y the membrane capacitance i n c r e a s e s when the membrane p o t e n t i a l decreases from -60 mV t o s m a l l e r v a l u e s . I t i s important t o note that the i n c r e a s e i n capacitance reaches a peak value a t a p o t e n t i a l of +20 mV. This i s the value of the membrane p o t e n t i a l when sodium channels are almost wide open. Beyond t h i s v a l u e , the magnitude of sodium current begins t o decrease. The peak value o f membrane capacitance around 20-30 mV c l e a r l y r e f l e c t s the behavior o f sodium channels. 2

From these o b s e r v a t i o n s , i t became apparent that the f r e quency dependent capacitance must be due, a t l e a s t p a r t i a l l y , to g a t i n g p a r t i c l e s , and, i n p a r t i c u l a r , t o those of sodium channels. I f the capacitance change shown i n F i g u r e 6 i s indeed due t o sodium channels, then the change must be a f f e c t e d by TTX, which i s known t o block sodium channels s e l e c t i v e l y . Figure 7 shows the membrane capacitance at v a r i o u s p o t e n t i a l s . As can be seen, TTX e f f e c t i v e l y e l i m i n a t e s the v o l t a g e dependence of


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-100

-50 0 50 MEMBRANE POTENTIAL mV

Figure 7. Membrane capacitance of squid giant axon at various membrane potentials. The external medium contained 3 X 10~ M TTX in order to block the sodium current. 7


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the membrane p o t e n t i a l , o f f e r i n g unequivocal evidence that the v o l t a g e dependent capacitance i s due to sodium channels. From these o b s e r v a t i o n s , we can draw the f o l l o w i n g c o n c l u sions. 1) The membrane capacitance of nerve axon i s dependent upon frequency, i n d i c a t i n g the presence of a r e l a x a t i o n process i n themembrane even a t r e s t . 2) The membrane capacitance decreases with h y p e r p o l a r i z a t i o n , when the membrane has a t i g h t l y packed s t r u c t u r e . 3) The membrane capacitance i n c r e a s e s w i t h d e p o l a r i z a t i o n , when i o n i c channels are wide open. 4) The v o l t a g e dependent capacitance i s due to sodium channels. I t i s w e l l known that both sodium and potassium currents through nerve membranes are slow processes having a frequency response o f a few k i l o - h e r t z or l e s s . However, there are a t l e a s t two p o s s i b i l i t i e s behind t h i s o b s e r v a t i o n . 1) The f r e quency response of channels i s f a s t , but i o n i c c u r r e n t s are diffusion limited. That i s , the r a t e of channel opening i s f a s t , but d i f f u s i o n o f ions i s slow and r a t e l i m i t i n g . 2) Opening and c l o s i n g of i o n i c channels are i n t r i n s i c a l l y slow and rate limiting. Case (1) suggests the presence of f a s t components i n nerve membrane. In t h i s case, h i g h frequency p e r t u r b a t i o n may i n t e r f e r e with the e x c i t a t i o n mechanism. However, as has been discussed, the c h a r a c t e r i s t i c frequency of channel p r o t e i n s i s i n the range of a few k i l o - h e r t z . Under these circumstances, opening or c l o s i n g of i o n i c channels may be the r a t e l i m i t i n g step i n the nerve e x c i t a t i o n . In t h i s case, e x t e r n a l high f r e quency f i e l d s w i l l have no e f f e c t on nerve e x c i t a t i o n unless they cause s u b s t a n t i a l r e t a r d a t i o n of i o n i c movements. These c o n s i d e r a t i o n s l e a d us to a c o n c l u s i o n that nerve exc i t a t i o n i s a very slow process and the membrane does not cont a i n elements which has a frequency response of MHz o r GHz. The only molecular s p e c i e s which may have such a frequency response are water molecules. However, the r o l e of water i n nerve e x c i t a t i o n i s t o t a l l y unknown and does not o f f e r , at present, any e v i dence f o r the p o s s i b l e mechanism of i n t e r a c t i o n between microwaves and nerve membranes. The author i s supported by ONR N00076-C-0642. The author i s indebted to Dr. H. P. Schwan f o r h i s v a l u a b l e suggestions.

Abstract The frequency response of various chemical constituents of nerve membrane was studied. Biological membranes in general consist of lipids and proteins. Firstly, impedance characteristics of artificial lipid bilayer membranes are examined using lecithin-hexadecane preparations. It was observed that the capacitance of plain lipid membranes was independent of frequency between 100 Hz and 20 KHz, Moreover, application of external voltages has no effect up to 200 mV. Secondly, membrane capacitance and conductance of nerve axon were investigated. There are three components in nerve membranes, i . e . , conductance, capaci-


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tance and inductance. Of these, inductive elements are known to arise from K + current. The remaining components, capacitance and conductance, have a characteristic frequency of 2-3 KHz, indicating the presence of structures which undergo a relaxation in this frequency range. Furthermore, the capacitance of nerve membrane depends on membrane potential, increasing with depolarization and decreasing with hyperpolarization. Therefore, the frequency dependent capacitance may be due to dipolar components in or near ionic channels. These observations clearly show that the components which are related to nerve excitation have a frequency response of a few kilohertz. Therefore, it us unlikely that high frequency fields of MHz or GHz interfere with these slow processes. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Hodgkin, A.L. and Huxley, A.F. J. Physiol., 1952, 117, 500. Adelman, W.L. "Biophysics and Physiology of Excitable Membranes"; Van Nostrand-Reinhold: New York, 1971. Plonsey, R. "Bioelectric Phenomena"; McGraw-Hill Publ. Co.: New York, 1969. Armstrong, A.M. and Bezanilla, F. Nature, 1973, 242, 459. Bezanilla, F. and Armstrong, A.M. Science, 1974, 183, 753. Keynes, R.D. and Rojas, E. J. Physiol., 1974, 239, 393. Keynes, R.D. and Rojas, E. J. Physiol., 1976, 255, 157. Meves, H. J. Physiol., 1974, 243, 847. Nonner, W., Rojas, E. and Stampfli, R. Pflugers Arch. Ges. Physiol., 1975, 354, 1. Gilly, W.R. and Armstrong, A.M. Biophys. J., 1980, 29, 485. Cole, K.S. and Curtis, H. J. Gen. Physiol., 1941, 22, 649. Cole, K.K. and Curtis, H. J. Gen. Physiol., 1941, 25, 29. Taylor, R.E. J. Cell. Comp. Physiol., 1965, 66, 21. Matsumoto, N., Inoue, I. and Kishimoto, U. Japanese J. Physiol., 1970, 20, 516. Takashima, S. and Schwan, H.P. J. Membrane Biol., 1974, 17, 51. Takashima, S. J. Membrane Biol., 1976, 27., 21. Takashima, S., Yantorno, R. and Novack, R. Biochim. Biophys. Acta, 1977, 469, 74. Fishman, H.M., Moore, L.E. and Poussart, D.J.M. Biophys. J., 1977, 19, 177. Mueller, P., Rudin, D.O., Tien, H.T. and Westcott, W.C. Nature (London), 1962, 194, 979. Mueller, P. and Rudin, D.O. "Lab-Tech, in Membrane Biophysics"; Passaw, H. and Stampfli, R., Eds., Springer-Verlag: Berlin, 1969. Montai, M. and Mueller, P. Proc. Natl. Acad. Sci. USA, 1972, 69, 3561. White, S. Biochim. Biophys. Acta, 1968, 196, 354.


9.

23. 24. 25. 26. 27. 28. 29.

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Coster, H.G.L. and Smith, J.R. Biochim. Biophys. Acta, 1964, 373, 151. White, S. and Thompson, T.E. Biochim. Biophys. Acta, 1973, 323, 7. Alvarez, O. and Latorre, R. Biophys. J., 1978, 21, 1. Cole, K.S. "Membrane, Ions and Impulses"; University of California Press: Berkeley, Ca., 1968. Schwan, H.P. "Electrical Properties of Tissues and Cell Suspensions"; Advances in Biol, and Med. Phys.; Lawrence, J.H. and Tobias, C.A., Eds., Academic Press: New York, 1957. Tasaki, I. and Hagiwara, S. Am. J. Physiol., 1957, 188, 423. Takashima, S. and Yantorno, R. In "Electrical Properties of Biological Polymers, Water, and Membranes"; Takashima, S., Ed.; Ν.Y. Acad. Sci.: New York, 1977, 309.

RECEIVED October 31,

1980.


Frequency Response of Membrane Components in Nerve Axon - ACS

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