Photodynamic Modification of Excitable Cell Function - ACS Publications

Chapter 7

Photodynamic Modification of Excitable Cell Function John P. Pooler

Downloaded by GEORGETOWN UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch007

Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322

Photodynamic treatment of electrically excitable cells from nerve and muscle causes a dose-dependent perturbation in their signaling function. Nerve cell axons, particularly at neuromuscular junctions, may be induced to fire action potentials. Voltage clamp analyses of photomodified lobster axons reveal a block of sodium channels and an inhibition of inactivation gating in unblocked channels. Potassium channels (delayed rectifiers) are less susceptible, but also become blocked and have a slowed activation. An unidentified ion leakage is created that is probably responsible for triggering the light-induced firing. The susceptibility to firing observed in vertebrate neuromuscular junctions suggests that insect neuromuscular junctions may be a likely target for light-activated pesticides.

The nervous system has been i m p l i c a t e d as a key t a r g e t o f l i g h t a c t i v a t e d p e s t i c i d e s suggested in p a r t by b e h a v i o r a l a b n o r m a l i t i e s i n d i c a t i v e o f l o s s o f neuromuscular c o n t r o l Although t h e r e i s no d i r e c t e v i d e n c e to s u p p o r t t h i s c o n t e n t i o n , i t i s known t h a t t h e nervous systems o f a l l organisms a r e h i g h l y s u s c e p t i b l e to p e r t u r b a t i o n by t o x i n s and p h a r m a c o l o g i c a l agents (2), and nerve c e l l s from n o n - i n s e c t s p e c i e s have been shown t o be e a s i l y photomodified (3-9). The purpose o f t h i s c h a p t e r i s to d e s c r i b e e x i s t i n g s t u d i e s on photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s and p o i n t out how these f i n d i n g s might a p p l y to the a c t i o n s o f l i g h t activated pesticides. Nervous systems c a r r y out the f u n c t i o n o f s i g n a l i n g , from one r e g i o n o f a c e l l to another and from one end o f an organism to t h e other. A l l nervous systems a r e composed o f s i m i l a r elements and behave a c c o r d i n g to uniform p r i n c i p l e s at t h e c e l l u l a r l e v e l , i n much the same way t h a t complex e l e c t r o n i c d e v i c e s o f d i v e r s e f u n c t i o n are composed o f i d e n t i c a l elementary components ( 1 0 J . S t u d e n t s o f n e u r o b i o l o g y have u s u a l l y chosen s p e c i e s f o r study more

0097-6156/87/0339-0109$06.00/0 © 1987 American Chemical Society

Heitz and Downum; Light-Activated Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


110

LIGHT-ACTIVATED PESTICIDES

f o r c o n v e n i e n c e , such as ready a v a i l a b i l i t y and ease o f d i s s e c t i o n , than f o r any u n i q u e n e s s i n p r i n c i p l e o f o p e r a t i o n . Although s t u d i e s on the photodynamic m o d i f i c a t i o n s o f nervous systems have been c a r r i e d out on organisms o t h e r than t h o s e t h a t are t a r g e t s f o r l i g h t - a c t i v a t e d p e s t i c i d e s , one may c a u t i o u s l y e x t r a p o l a t e a c r o s s s p e c i e s and g e n e r a l i z e r e s u l t s from one s p e c i e s to a l l o t h e r s . Thus i t i s v e r y l i k e l y t h a t mechanisms s t u d i e d on t r a d i t i o n a l p r e p a r a t i o n s such as s q u i d n e r v e s and f r o g muscles a p p l y e q u a l l y to insect excitable c e l l s . The common elemental p r o c e s s e s found in a l l nervous systems i n c l u d e c o n d u c t i o n o f nerve i m p u l s e s ; the s t i m u l a t i o n or i n h i b i t i o n o f one c e l l by another at s y n a p t i c c o n t i g u i t i e s ; and spontaneous e x c i t a t i o n in a p p r o p r i a t e pacemaker and s e n s o r y t r a n s d u c e r c e l l s . Many o f t h e s e same events o c c u r in muscle t i s s u e as w e l l . D i s t u r b a n c e in any o f t h e s e p r o c e s s e s by photodynamic means i s potentially lethal. Historical

Background

Nerve-muscle p r e p a r a t i o n s . The f i r s t s u b s t a n t i a l i n v e s t i g a t i o n o f photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s was a s e r i e s o f s t u d i e s by L i p p a y i n i t i a t e d in the l a t e 1920s on n e r v e - m u s c l e p r e p a r a t i o n s from f r o g s , i . e . , i s o l a t e d f r o g s k e l e t a l muscles w i t h the d i s t a l p o r t i o n s o f t h e motor nerves t h a t i n n e r v a t e them s t i l l a t t a c h e d (1113). He found t h a t i l l u m i n a t i o n o f muscle s e n s i t i z e d w i t h any o f s e v e r a l xanthene s e n s i t i z e r s o r hematoporphyrin l e d to c o n t r a c t i o n s . Some c o n t r a c t i o n s were slow and s u s t a i n e d ; o t h e r s were r a p i d and spasm-like. L i p p a y proposed t h a t the spasms were t r i g g e r e d by l i g h t - i n d u c e d f i r i n g in the motoneurons. Research in subsequent y e a r s has amply c o n f i r m e d L i p p a y ' s h y p o t h e s i s ( 1 4 - 1 7 ) . Recordings i n motoneurons show t h a t a c t i o n p o t e n t i a l s o r i g i n a t e in the d i s t a l p o r t i o n s o f nerve near the muscle and t r a v e l a n t i d r o m i c a l l y away from the m u s c l e . They a l s o a c t i v a t e the neuromuscular s y n a p s e , triggering contraction. I f r e v e r s i b l e neuromuscular b l o c k i n g agents are a d d e d , the l i g h t - i n d u c e d c o n t r a c t i l e spasms can be b l o c k e d reversibly. In v e r t e b r a t e s neuromuscular t r a n s m i s s i o n i s a c c o m p l i s h e d by r e l e a s e o f a c e t y l c h o l i n e , s t o r e d in small v e s i c l e s w i t h i n the p r e s y n a p t i c nerve t e r m i n a l . A p r e s y n a p t i c a c t i o n p o t e n t i a l causes the emptying o f the c o n t e n t s o f many such v e s i c l e s i n t o the narrow space between the nerve membrane and muscle membrane, the a c e t y l c h o l i n e then a c t i n g to d e p o l a r i z e the muscle membrane. Normally the a c e t y l c h o l i n e i s r a p i d l y degraded immediately f o l l o w i n g the nerve impulse by a c e t y l c h o l i n e s t e r a s e p r e s e n t in h i g h c o n c e n t r a t i o n in the c l e f t a r e a . An a d d i t i o n a l f i n d i n g on s e n s i t i z e d n e r v e - m u s c l e p r e p a r a t i o n s from f r o g s i s a l i g h t - i n d u c e d d e c l i n e in the a c e t y l c h o l i n e s t e r a s e a c t i v i t y ( 1 5 ) . With a c e t y l c h o l i n e h y d r o l y z e d more s l o w l y , a l i n g e r i n g d e p o l a r i z i n g a c t i o n on the muscle c e l l o c c u r s . S i n g l e nerve a c t i o n p o t e n t i a l s , t h a t n o r m a l l y t r i g g e r s i n g l e muscle a c t i o n p o t e n t i a l s and subsequent c o n t r a c t i l e t w i t c h e s , now t r i g g e r m u l t i p l e a c t i o n p o t e n t i a l s and twitches. Thus the s t i m u l a t i n g a c t i o n o f nerve on muscle f o r both l i g h t - i n d u c e d and e l e c t r i c a l l y s t i m u l a t e d f i r i n g i s a m p l i f i e d . In i n s e c t s the neuromuscular t r a n s m i t t e r i s L - g l u t a m a t e , and i t s



7. POOLER

Photodynamic Modification of Excitable Cell Function

111

i n a c t i v a t i o n i s m a i n l y by uptake i n t o g l i a l c e l l s r a t h e r than by enzymatic d e g r a d a t i o n ( 1 8 J . Whether i n s e c t neuromuscular j u n c t i o n s a r e as s e n s i t i v e to photodynamic p e r t u r b a t i o n as t h o s e in f r o g s i s unknown. A d i r e c t photodynamic a c t i o n on muscle membrane, not i n v o l v i n g neuromuscular t r a n s m i s s i o n , has a l s o been d e s c r i b e d ( 1 9 J . The muscle s l o w l y d e p o l a r i z e s and may break i n t o spontaneous f i r i n g , t h e r e b y t r i g g e r i n g c o n t r a c t i o n w i t h o u t any nerve a c t i v i t y . The muscle a c t i o n p o t e n t i a l s a l s o become i n c r e a s e d i n d u r a t i o n . A second d i r e c t a c t i o n on muscle i s the slow c o n t r a c t u r e o r i g i n a l l y d e s c r i b e d by L i p p a y , and c o n f i r m e d by o t h e r s ( H ) . It o c c u r s even w i t h neuromuscular t r a n s m i s s i o n blocked and the muscle made i n e x c i t a b l e by d e p o l a r i z a t i o n w i t h e l e v a t e d e x t r a c e l l u l a r p o t a s s i u m . The b a s i s f o r the slow 1 i g h t - i n d u c e d c o n t r a c t u r e i s u n r e s o l v e d at present. These photodynamic s t u d i e s on n e r v e - m u s c l e p r e p a r a t i o n s were c a r r i e d out over a span o f many d e c a d e s , w i t h o u t b e n e f i t o f r e c e n t t e c h n i c a l advances o r o f i n s i g h t s i n t o p h y s i o l o g i c a l mechanism r e v e a l e d by these t e c h n i q u e s . While the f i n d i n g s o f a l l i n v e s t i g a t o r s a r e not i n t o t a l harmony, most agree t h a t the most s e n s i t i v e t a r g e t i s some r e g i o n o f the nerve t e r m i n a l . The l i g h t doses r e q u i r e d to cause major d i r e c t muscle e f f e c t s a r e l a r g e r than t h o s e needed to evoke l i g h t - i n d u c e d f i r i n g i n the motoneurons. All o f the r e p o r t e d l i g h t - i n d u c e d e f f e c t s are p h o t o d y n a m i c - - i . e . , r e q u i r e o x y g e n - - a n d seem to be independent o f the p a r t i c u l a r s e n s i t i z e r used. Minor c o n f l i c t s between r e s u l t s i n d i f f e r e n t s t u d i e s may r e f l e c t d i f f e r e n c e s in the d i s t r i b u t i o n o f s e n s i t i z e r w i t h i n the complex anatomy o f the muscle at the time o f illumination. G i a n t axons. Almost a l l i n v e s t i g a t i o n s on photodynamic m o d i f i c a t i o n o f the nervous system have been c a r r i e d out on s i n g l e g i a n t axons i s o l a t e d from marine animals such as s q u i d , c u t t l e f i s h , and l o b s t e r . Most o f the work p r i o r to 1970 was performed by C h a l a z o n i t i s and coworkers i n France and by Lyudkovskaya and coworkers in the USSR (3-8). The axon r e g i o n o f a nerve c e l l i s r e l a t i v e l y " s i m p l e " in t h a t i t s s i g n a l i n g f u n c t i o n i s l i m i t e d to c o n d u c t i n g i m p u l s e s , i n i t i a t e d at one end or i n o t h e r c e l l r e g i o n s , along i t s l e n g t h to s y n a p t i c t e r m i n a l s at the o t h e r end. However, d e s p i t e a s t e r e o t y p e d normal f u n c t i o n , axons are c a p a b l e o f r a t h e r complex b e h a v i o r . The e l e c t r o p h y s i o l o g y o f axons can p r e s e n t l y be s t u d i e d at s e v e r a l l e v e l s o f s o p h i s t i c a t i o n , from s i m p l e e x t r a c e l l u l a r r e c o r d i n g f o r d e t e c t i o n o f a c t i o n c u r r e n t s a s s o c i a t e d w i t h the passage o f nerve i m p u l s e s , to " p a t c h clamp" methods f o r o b s e r v i n g the b e h a v i o r o f i n d i v i d u a l macromolecular ion c h a n n e l s . The s t u d i e s o f C h a l a z o n i t i s , L y u d k o v s k a y a , and a few o t h e r s who preceded them many decades ago employed e i t h e r e x t r a c e l l u l a r r e c o r d i n g or s i m p l e i n t r a c e l l u l a r r e c o r d i n g w i t h a p i p e t e l e c t r o d e i n s e r t e d i n t o the axon. These methods permit d e t e c t i o n o f f i r i n g p a t t e r n s and s u b t h r e s h o l d membrane d e p o l a r i z a t i o n , but do not a l l o w one t o d e s c r i b e 1 i g h t - i n d u c e d changes i n membrane p e r m e a b i l i t y . Use o f the v o l t a g e clamp method, to be d i s c u s s e d f u r t h e r o n , has r e v e a l e d how i o n i c p e r m e a b i l i t i e s are p h o t o d y n a m i c a l l y changed.



112


C h a l a z o n i t i s and Lyudkovskaya both demonstrated t h a t s e n s i t i z e r - t r e a t e d g i a n t axons would commence r e p e t i t i v e f i r i n g d u r i n g i l l u m i n a t i o n , preceded by a l a t e n t p e r i o d d u r i n g which a slow depolarization occurred. The f i r i n g c o n t i n u e d a f t e r c e s s a t i o n o f i l l u m i n a t i o n f o r v a r y i n g p e r i o d s o f t i m e , but then s t o p p e d . Sometimes the membrane p o t e n t i a l would jump to a s t e a d y d e p o l a r i z e d l e v e l d u r i n g i l l u m i n a t i o n f o r s e v e r a l seconds and then r e t u r n to a v a l u e near the normal r e s t i n g p o t e n t i a l . In a s e p a r a t e study o f photodynamic m o d i f i c a t i o n o f g i a n t a x o n s , Lyudkovskaya d e s c r i b e d rather d i f f e r e n t r e s u l t s (8). In t h i s c a s e no l i g h t - i n d u c e d f i r i n g was r e p o r t e d . I n s t e a d Lyudkovskaya found major p e r t u r b a t i o n s i n the shape o f e l e c t r i c a l l y s t i m u l a t e d a c t i o n p o t e n t i a l s . They d e v e l o p e d a l o n g p l a t e a u between the r i s i n g and f a l l i n g p h a s e s , and the d u r a t i o n i n c r e a s e d from a t y p i c a l 1 o r 2 ms up to hundreds o f milliseconds. Both C h a l a z o n i t i s and Lyudkovskaya d e s c r i b e d the l i g h t - i n d u c e d f i r i n g as r e v e r s i b l e , w h i l e the p r o l o n g a t i o n o f e l e c t r i c a l l y s t i m u l a t e d a c t i o n p o t e n t i a l s was s a i d to be irreversible. In 1968 P o o l e r i n i t i a t e d an i n v e s t i g a t i o n o f photodynamic m o d i f i c a t i o n o f g i a n t axons from l o b s t e r s ( 2 0 ) . He found an i r r e v e r s i b l e prolongation of e l e c t r i c a l l y stimulated action p o t e n t i a l s , as d i d L y u d k o v s k a y a , but no l i g h t - i n d u c e d f i r i n g . In a subsequent c l a r i f y i n g study d e s i g n e d to r e c o n c i l e the c o n f l i c t s i n r e p o r t e d f i n d i n g s , he found t h a t l i g h t - i n d u c e d f i r i n g c o u l d be i n i t i a t e d i n l o b s t e r g i a n t axons i f the b a t h i n g s o l u t i o n was f r e e o f calcium (21). C h a l a z o n i t i s and Lyudkovskaya had used such a c o n d i t i o n in t h e i r s t u d i e s d e s c r i b i n g 1 i g h t - i n d u c e d f i r i n g . Nerve c e l l s become h y p e r e x c i t a b l e i n s o l u t i o n s d e v o i d o f c a l c i u m , but a l s o d e t e r i o r a t e s l o w l y i f a c a l c i u m - f r e e c o n d i t i o n i s m a i n t a i n e d (22!). Thus g i a n t axons may be p h o t o d y n a m i c a l l y induced i n t o f i r i n g in conditions of low-calcium h y p e r e x c i t a b i l i t y . The f i r i n g i s a p p a r e n t l y r e v e r s i b l e , in t h a t f i r i n g c e a s e s at some p o i n t f o l l o w i n g t e r m i n a t i o n o f i l l u m i n a t i o n and may u s u a l l y be r e s t a r t e d by another dose o f l i g h t . The r e v e r s i b i l i t y i s a p p a r e n t , but not r e a l , because the c e s s a t i o n o f f i r i n g i s due in p a r t to d e t e r i o r a t i o n (and o t h e r f a c t o r s t o be d e s c r i b e d l a t e r ) r a t h e r than to removal o f t h e p h o t o m o d i f i c a t i o n t h a t i n i t i a t e s the f i r i n g . V o l t a g e Clamp A n a l y s i s o f Membrane Channel F u n c t i o n T e c h n i c a l background. Much o f our p r e s e n t u n d e r s t a n d i n g o f e x c i t a b l e c e l l f u n c t i o n stems from the s t u d i e s o f H o d g k i n , Huxley and K a t z , who a p p l i e d the v o l t a g e clamp t e c h n i q u e to g i a n t axons from s q u i d ( 2 3 J . When under v o l t a g e clamp the membrane p o t e n t i a l in a r e s t r i c t e d small area o f membrane i s c o n t r o l l e d by e l e c t r o n i c feedback. U s u a l l y the membrane p o t e n t i a l i s held a t a n e g a t i v e l e v e l from which a sequence o f s t e p d e p o l a r i z a t i o n s a r e a p p l i e d . At each v a l u e o f p o t e n t i a l to which the membrane i s c l a m p e d , the e l e c t r o c h e m i c a l d r i v i n g f o r c e f o r ion f l u x i s f i x e d . T h e r e f o r e the time c o u r s e o f c u r r e n t f l o w by any ion s p e c i e s r e f l e c t s the time c o u r s e o f membrane p e r m e a b i l i t y to t h a t i o n , u s u a l l y e x p r e s s e d in terms o f c o n d u c t a n c e (see E q u a t i o n 1) ( 2 4 ) . The t o t a l c u r r e n t t h r o u g h an axon membrane may be r e s o l v e c F i n t o t h r e e main components: sodium c u r r e n t , p o t a s s i u m c u r r e n t , and a n o n - s p e c i f i c leakage


7. POOLER

113


current. C u r r e n t s are r e l a t e d to membrane conductance and d r i v i n g f o r c e s as expressed in E q u a t i o n 1, where V i s t h e membrane p o t e n t i a l , Ε i s the r e v e r s a l p o t e n t i a l o f the a p p r o p r i a t e i o n , and g i s the c o n d u c t a n c e .


I

= g (V - E)

(1)

The n o n - s p e c i f i c leakage conductance i s c o n s i d e r e d to be c o n s t a n t i n a normal membrane, w h i l e sodium and potassium c o n d u c t a n c e s a r e c l e a r l y v o l t a g e - and t i m e - d e p e n d e n t . T h e i r r e s p o n s e to a s t i m u l u s u n d e r l i e s the c h a r a c t e r i s t i c nerve impulse when the membrane p o t e n t i a l i s not c o n t r o l l e d by e l e c t r o n i c f e e d b a c k . I n v e s t i g a t i o n subsequent to the c l a s s i c s t u d i e s by H o d g k i n , Huxley and K a t z demonstrates t h a t the i o n i c conductance in a g i v e n f i n i t e r e g i o n o f membrane i s the sum o f the conductance o f a l l the i n d i v i d u a l i o n c h a n n e l s i n t h a t membrane. Each channel i s a p o l y p e p t i d e c o n t a i n i n g a pore spanning the membrane t h a t has a r e g i o n o f r e s t r i c t i o n , g i v i n g r i s e to i o n i c s e l e c t i v i t y ( 2 5 J . Most c h a n n e l s a l s o c o n t a i n one o r more s e p a r a t e "gate" r e g i o n s t h a t o c c l u d e the pore under some c o n d i t i o n s and open to a n o n - o c c l u d i n g c o n f o r m a t i o n under o t h e r c o n d i t i o n s . In t y p i c a l v o l t a g e clamp experiments the membrane r e g i o n under study c o n t a i n s up to a m i l l i o n channels. Ion conductances change smoothly over time i n r e s p o n s e to a s t e p change in p o t e n t i a l . T h i s smooth change r e p r e s e n t s the ensemble average o f many i n d i v i d u a l c h a n n e l s , each b e i n g e i t h e r open or c l o s e d . Thus the time c o u r s e o f the ensemble average r e f l e c t s the time c o u r s e o f p r o b a b i l i t y t h a t a g i v e n channel type i s o p e n . Sodium c h a n n e l s . When an axon membrane i s d e p o l a r i z e d , many sodium c h a n n e l s open ( a c t i v a t e ) but then c l o s e ( i n a c t i v a t e ) w i t h a somewhat slower time c o u r s e . Most i n v e s t i g a t o r s f e e l t h a t a c t i v a t i o n i s the opening o f a gate t h a t i s c l o s e d when the membrane i s p o l a r i z e d , and i n a c t i v a t i o n i s the c l o s i n g o f a p h y s i c a l l y s e p a r a t e gate t h a t i s n o r m a l l y open when the membrane i s p o l a r i z e d . When the membrane i s r e p o l a r i z e d t h e r e i s a r e v e r s a l o f the events t h a t o c c u r d u r i n g depolarization. The a c t i v a t i o n g a t e c l o s e s and the i n a c t i v a t i o n gate opens. Photodynamic M o d i f i c a t i o n

o f L o b s t e r G i a n t Axons

Block o f sodium c h a n n e l s . Photodynamic p e r t u r b a t i o n o f sodium channel f u n c t i o n can be expressed in terms o f changes i n g a t i n g v a r i a b l e s and maximum c o n d u c t a n c e . V o l t a g e clamp a n a l y s i s o f p h o t o m o d i f i e d l o b s t e r g i a n t axons r e v e a l s a d e c r e a s e in the maximum sodium c o n d u c t a n c e . During i l l u m i n a t i o n a t a c o n s t a n t dose r a t e t h e d e c r e a s e f o l l o w s a s i m p l e s u r v i v a l c u r v e , i . e . , an e x p o n e n t i a l time c o u r s e toward a zero asymptote ( 2 6 ) . The s i m p l e s t i n t e r p r e t a t i o n of t h i s b e h a v i o r i s t h a t i n c r e a s i n g numbers o f i n d i v i d u a l c h a n n e l s become t o t a l l y b l o c k e d . At l a r g e d o s e s e s s e n t i a l l y a l l c h a n n e l s a r e b l o c k e d and no measurable sodium c u r r e n t flows d u r i n g a depolarization. The r a t e c o n s t a n t f o r the development o f b l o c k d u r i n g i l l u m i n a t i o n v a r i e s l i n e a r l y w i t h dose r a t e and depends h e a v i l y on the s p e c i e s o f s e n s i t i z e r . When d i f f e r e n t s e n s i t i z e r s are compared under c o n d i t i o n s o f equal absorbed dose r a t e s , the r a t e


114



o f channel block becomes a u s e f u l assay t o compare t h e i r p o t e n c y . For example, w i t h i n the f l u o r e s c e i n f a m i l y o f s e n s i t i z e r s r o s e bengal i s by f a r the most potent ( 2 7 ) . P e r t u r b a t i o n o f sodium channel g a t i n g . I l l u m i n a t i o n a l s o causes a complex d i s t u r b a n c e o f the i n a c t i v a t i o n gate i n n o n - b l o c k e d c h a n n e l s , l e a d i n g to a prolonged flow o f sodium c u r r e n t i n r e s p o n s e to d e p o l a r i z a t i o n ( 2 6 ) . F o l l o w i n g any f i n i t e l i g h t d o s e , t h e r e f o r e , t h e r e a r e t h r e e s u b p o p u l a t i o n s o f sodium c h a n n e l s : normal, b l o c k e d , and unblocked w i t h m o d i f i e d i n a c t i v a t i o n g a t i n g . The r e l a t i v e numbers o f t h e s e s u b p o p u l a t i o n s a r e shown s c h e m a t i c a l l y i n F i g u r e 1 as a f u n c t i o n o f l i g h t d o s e . Note t h a t the number with p e r t u r b e d i n a c t i v a t i o n g a t i n g r i s e s w i t h l i g h t dose up t o a maximum but then f a l l s at high doses because t h e m a j o r i t y o f t h e c h a n n e l s become blocked. D i s t u r b a n c e o f sodium channel i n a c t i v a t i o n r e v e a l s i t s e l f i n the k i n e t i c s o f i n a c t i v a t i o n and i t s v o l t a g e dependence. At a l i g h t dose s u f f i c i e n t to b l o c k about 50% o f the c h a n n e l s , the r a t e at which unblocked open c h a n n e l s i n a c t i v a t e d u r i n g a d e p o l a r i z a t i o n d e c r e a s e s by r o u g h l y 50% ( 2 6 ) . At the same t i m e , the s t e a d y - s t a t e i n a c t i v a t i o n versus voltage r e l a t i o n i s d i s t o r t e d . The s t e a d y - s t a t e v o l t a g e dependence f o r a c t i v a t i o n and i n a c t i v a t i o n g a t i n g may be expressed in terms o f the g a t i n g parameters m and h o f the HodgkinHuxley model a c c o r d i n g to E q u a t i o n s 2 and 3 , where V i s membrane p o t e n t i a l , V and V. a r e the membrane p o t e n t i a l s a t which the g a t i n g parameters are at h a l f maximum, and t h e k ' s i n d i c a t e t h e s t e e p n e s s o f the v o l t a g e dependence: m

œ

= 1/(1

+

exp((V - V J / k J )

(2)

h

e

= 1/(1

+

exp((V - V ) / k ) )

(3)

E q u a t i o n 3 may be r e v i s e d as i n E q u a t i o n 4: h

w

h

h

to i n c l u d e a n o n - i n a c t i v a t e d

= (1 - f ) / ( l

•

exp((V - V ) / k ) ) h

h

fraction,

+ f

f,

(4)

New experiments on l o b s t e r axons show t h e development o f a f o o t i n the i n a c t i v a t i o n c u r v e such t h a t some c h a n n e l s f a i l to i n a c t i v a t e at a l l at p o t e n t i a l s near z e r o (see F i g u r e 2 ) . A s i m i l a r b e h a v i o r has been found on squid g i a n t axons ( 2 8 ) . P o s s i b l e photodynamic p e r t u r b a t i o n o f a c t i v a t i o n g a t i n g i s small a t b e s t and below t h e r e s o l u t i o n o f t h e measurements. The a c t i v a t i o n parameter v a l u e s f o r p h o t o m o d i f i e d axons a r e not s i g n i f i c a n t l y d i f f e r e n t from t h o s e o f normal axons ( F i g u r e 2 ) . An e a r l i e r i n v e s t i g a t i o n r e v e a l e d no change in the k i n e t i c s o f a c t i v a t i o n e i t h e r (26). Thus i t appears t h a t p h o t o m o d i f i c a t i o n o f sodium channel g a t i n g i s l i m i t e d to the i n a c t i v a t i o n component. I n a c t i v a t i o n g a t i n g i s a complex p r o c e s s . Inactivation o c c u r r i n g as the c l o s u r e o f open c h a n n e l s d u r i n g a l a r g e d e p o l a r i z a t i o n may be d i f f e r e n t from i n a c t i v a t i o n o c c u r r i n g d u r i n g a small d e p o l a r i z a t i o n t h a t does not a c t i v a t e many c h a n n e l s ( 2 9 ) . T h i s second form o f i n a c t i v a t i o n i s c a l l e d c o n d i t i o n e d inactivation because i t i s measured by " c o n d i t i o n i n g " the membrane w i t h a small



7. POOLER


Relative

Light

115

Dose

F i g u r e 1. Schematic i l l u s t r a t i o n o f photodynamic m o d i f i c a t i o n o f sodium c h a n n e l s showing d i v i s i o n o f t o t a l p o p u l a t i o n i n t o three f r a c t i o n s . B e f o r e i l l u m i n a t i o n a l l c h a n n e l s are n o r m a l , w h i l e a f t e r l a r g e l i g h t doses a l l are b l o c k e d . The unblocked c h a n n e l s w i t h m o d i f i e d i n a c t i v a t i o n r e a c h a maximum at an intermediate l i g h t dose.

Membrane

Potential

(mv)

F i g u r e 2. Steady s t a t e a c t i v a t i o n and i n a c t i v a t i o n v e r s u s membrane p o t e n t i a l f o r normal axons ( c o n t i n u o u s c u r v e s ) and p h o t o m o d i f i e d axons (dashed c u r v e s ) . The c u r v e s a r e p l o t s o f E q u a t i o n s 2 and 4 u s i n g mean parameter v a l u e s o b t a i n e d from 10 measurements each on normal axons and p h o t o m o d i f i e d axons s e n s i t i z e d w i t h 5 μΜ a c r i d i n e orange and i l l u m i n a t e d f o r a time s u f f i c i e n t to b l o c k 50% o f the sodium c o n d u c t a n c e .


116



d e p o l a r i z a t i o n and then t e s t i n g f o r the amount o f i n a c t i v a t i o n d u r i n g a subsequent l a r g e d e p o l a r i z a t i o n . New experiments show the k i n e t i c s o f c o n d i t i o n e d i n a c t i v a t i o n on p h o t o m o d i f i e d l o b s t e r g i a n t axons to be u n a l t e r e d , and f u r t h e r m o r e , the k i n e t i c s o f r e c o v e r y from i n a c t i v a t i o n (the opening o f the i n a c t i v a t i o n g a t e upon repolarization) are not p e r t u r b e d e i t h e r ( F i g u r e 3 ) . Therefore, photodynamic m o d i f i c a t i o n o f sodium channel g a t i n g i s not o n l y l i m i t e d to i n a c t i v a t i o n , but to t h a t a s p e c t observed as the c l o s u r e o f open c h a n n e l s . P e r t u r b a t i o n o f potassium channel s and l e a k a g e . There are many k i n d s o f potassium c h a n n e l s i n e x c i t a b l e c e l l s . The predominant t y p e o f p o t a s s i u m channel i n axons i s c a l l e d a d e l a y e d rectifier. I t s g a t i n g has been modeled by the η parameter o f the Hodgkin-Huxley model ( 2 4 ) . More r e c e n t study o f d e l a y e d r e c t i f i e r s r e v e a l s the e x i s t e n c e o f a slow component i n the k i n e t i c s not d e a l t w i t h i n the Hodgkin and Huxley a n a l y s i s , s u g g e s t i n g two p o p u l a t i o n s o f d e l a y e d r e c t i f i e r (30). A d e s c r i p t i o n o f photodynamic p e r t u r b a t i o n o f d e l a y e d r e c t i f i e r s i s p r e s e n t l y i n c o m p l e t e because o f u n c e r t a i n t i e s i n t h e b e h a v i o r o f normal c h a n n e l s . N e v e r t h e l e s s , c e r t a i n f a c t s are apparent. F i r s t , p o t a s s i u m c h a n n e l s i n axons are b l o c k e d by photodynamic t r e a t m e n t . The s u s c e p t i b i l i t y , however, i s l e s s than t h a t o f sodium c h a n n e l s . I f the s u r v i v a l o f unblocked potassium and sodium c h a n n e l s i s compared under i d e n t i c a l r e a c t i o n c o n d i t i o n s , the r a t e o f potassium channel decay i s o n l y about 20% as g r e a t (21). S e c o n d , the a c t i v a t i o n g a t i n g o f potassium c h a n n e l s i s d i s t u r b e d ( f o r sodium c h a n n e l s i t i s n o t ) . The r a t e o f a c t i v a t i o n i s d e c r e a s e d and the slow component becomes more p r o m i n e n t . It i s not c l e a r whether t h i s r e p r e s e n t s a s e l e c t i v e b l o c k o f the f a s t component, making the o v e r a l l k i n e t i c s appear s l o w e r , o r whether the a c t i v a t i o n i s t r u l y slowed. In any event the o v e r a l l e f f e c t i s a d e c r e a s e in the l e v e l o f potassium conductance reached a t a g i v e n time f o l l o w i n g the s t a r t o f a s t e p d e p o l a r i z a t i o n . The m o d i f i c a t i o n t h a t p o t e n t i a l l y has the g r e a t e s t s i g n i f i c a n c e f o r axon f u n c t i o n — a n i n c r e a s e in l e a k a g e — h a s not been studied s y s t e m a t i c a l l y . T h i s i s because t h e v a s t m a j o r i t y o f v o l t a g e clamp s t u d i e s o f photodynamic m o d i f i c a t i o n have been c a r r i e d out on l o b s t e r g i a n t a x o n s , on which l e a k a g e cannot be measured f o r t e c h n i c a l reasons ( 3 1 ) . However, v o l t a g e clamp s t u d i e s performed on s q u i d g i a n t axons sïïôw a c o n s i s t e n t i n c r e a s e in leakage (unpublished). I n t e r p r e t a t i o n o f Nerve C e l l

Experiments

Two o f the key o b s e r v a t i o n s on nerve c e l l s seem to be in c o n f l i c t . Many s t u d i e s d e s c r i b e photodynamic m o d i f i c a t i o n as e x c i t a t o r y : nerve c e l l s a r e induced to f i r e d u r i n g l i g h t . Yet v o l t a g e clamp a n a l y s i s shows t h a t sodium c h a n n e l s become b l o c k e d by 1 i g h t — c l e a r l y an i n h i b i t o r y a c t i o n . ( L o c a l a n e s t h e t i c s work by b l o c k i n g sodium channels in axons.) The r e s o l u t i o n l i e s i n the f a c t t h a t axons have f a r more than the minimum d e n s i t y o f sodium c h a n n e l s r e q u i r e d to s u s t a i n a c t i o n p o t e n t i a l s ; thus events t h a t s t i m u l a t e an axon to f i r e may proceed even though sodium c h a n n e l s are b e i n g b l o c k e d simultaneously. ( I f they aVl_ become b l o c k e d , then f i r i n g w i l l



POOLER


Membrane

Potential

< mv )

F i g u r e 3. Time c o n s t a n t f o r c o n d i t i o n e d i n a c t i v a t i o n and removal o f i n a c t i v a t i o n in normal axons ( t r i a n g l e s y m b o l s , c o n t i n u o u s l i n e s ) and p h o t o m o d i f i e d axons ( p l u s s y m b o l s , dashed lines). Each p o i n t i s the mean o f 11 or more o b s e r v a t i o n s . R e a c t i o n c o n d i t i o n s are the same as i n F i g u r e 2. The l a c k o f photodynamic e f f e c t stands in c o n t r a s t to the s l o w i n g o f i n a c t i v a t i o n when a s s e s s e d as the c l o s u r e o f open c h a n n e l s ( 2 6 ) .


118



cease.) The l i g h t - i n d u c e d f i r i n g appears to be t r i g g e r e d by a 1 i g h t - i n d u c e d d e p o l a r i z a t i o n t h a t a c t s as a s t i m u l u s analogous to a sensory g e n e r a t o r p o t e n t i a l . The d e p o l a r i z a t i o n , i n t u r n , i s p r o b a b l y a r e s u l t o f the i n c r e a s e i n l e a k a g e . However, t h i s i n t e r p r e t a t i o n must be c o n s i d e r e d s p e c u l a t i v e u n t i l more d e t a i l i s l e a r n e d about t h e l i g h t - i n d u c e d l e a k a g e - - s p e c i f i c a l l y the ion species involved. In t h e o r y , d e p o l a r i z a t i o n can be brought about by a d e c r e a s e in p e r m e a b i l i t y to an ion s p e c i e s w i t h an e q u i l i b r i u m p o t e n t i a l more n e g a t i v e than the r e s t i n g p o t e n t i a l , such as potassium. K o h l i and B r y a n t ' s b r i e f r e p o r t on photodynamic d e p o l a r i z a t i o n o f s k e l e t a l muscle c e l l s (19) s t a t e s t h a t the e f f e c t was not seen when c h o l i n e was s u b s t i t u t e a f o r sodium, s u g g e s t i n g t h a t the d e p o l a r i z a t i o n i s caused by an anomalous r i s e in sodium permeability. L i g h t - i n d u c e d f i r i n g i s easy to produce on n e r v e - m u s c l e p r e p a r a t i o n s , but w i t h much more d i f f i c u l t y on g i a n t axons from marine a n i m a l s . Most, and p o s s i b l y a l l , o f the l i g h t - i n d u c e d f i r i n g d e s c r i b e d by Lyudkovskaya and C h a l a z o n i t i s was on g i a n t axon p r e p a r a t i o n s made h y p e r e x c i t a b l e by l o w e r i n g the c a l c i u m c o n c e n t r a t i o n in the r e a c t i o n medium. Pooler described l i g h t induced f i r i n g on l o b s t e r axons i n c a l c i u m - f r e e m e d i a , but not w i t h high calcium c o n c e n t r a t i o n s p r e s e n t . On n e r v e - m u s c l e p r e p a r a t i o n s the t e r m i n a l p o r t i o n s o f the motoneurons e v i d e n t l y p o s s e s s a h i g h s u s c e p t i b i l i t y t o l i g h t - i n d u c e d f i r i n g even i n c o n d i t i o n s o f normal calcium. The r e a s o n s f o r t h i s a r e p r e s e n t l y unknown. The r e v e r s i b i l i t y o f l i g h t - i n d u c e d f i r i n g d e s c r i b e d by C h a l a z o n i t i s and Lyudkovskaya was shown to be an apparent r e v e r s i b i l i t y , due in p a r t to a slow d e t e r i o r a t i o n i n c a l c i u m - f r e e solutions. I f a nerve c e l l i s h y p e r e x c i t a b l e and t e e t e r i n g on the edge o f f i r i n g because o f low c a l c i u m a n d / o r a l i g h t - i n d u c e d d e p o l a r i z a t i o n , any small i n f l u e n c e can t r i g g e r f i r i n g or s t o p existing f i r i n g . Prolonged d e p o l a r i z a t i o n induces another form o f i n a c t i v a t i o n known as slow i n a c t i v a t i o n , not o r d i n a r i l y seen on the time s c a l e o f an a c t i o n p o t e n t i a l ( 2 5 J . A 1 ight-induced d e p o l a r i z a t i o n o r a l o n g t r a i n o f a c t i o n p o t e n t i a l s may cause slow i n a c t i v a t i o n , thus e f f e c t i v e l y r a i s i n g the f i r i n g t h r e s h o l d and h a l t i n g f i r i n g , but w i t h o u t r e v e r s i n g the p h o t o m o d i f i c a t i o n . M o d i f i c a t i o n o f g a t i n g in unblocked c h a n n e l s a l s o c o n t r i b u t e s to p e r t u r b a t i o n s in f i r i n g b e h a v i o r . The slowing o f i n a c t i v a t i o n k i n e t i c s and the f o o t in the i n a c t i v a t i o n c u r v e ( F i g u r e 2) both l e a d to a d i s t o r t i o n in shape and c o n s i d e r a b l e p r o l o n g a t i o n o f a c t i o n p o t e n t i a l d u r a t i o n , up to hundreds o f m i l l i s e c o n d s in some c a s e s . A v e r y l o n g a c t i o n p o t e n t i a l can i t s e l f s e r v e as a prolonged s t i m u l u s to nearby axon segments t h a t have r e c e i v e d a lower l i g h t d o s e . In most experiments o n l y a small segment i s i l l u m i n a t e d , t h u s p e r m i t t i n g e l e c t r o t o n i c i n t e r a c t i o n s between the m o d i f i e d r e g i o n and s u r r o u n d i n g unmodified segments. The f i n d i n g t h a t the c l o s u r e o f open c h a n n e l s i s s l o w e d , w h i l e the k i n e t i c s o f c o n d i t i o n e d i n a c t i v a t i o n remain n o r m a l , s u p p o r t s a p r e v i o u s l y s t a t e d c o n t e n t i o n (32) t h a t normal i n a c t i v a t i o n may o c c u r as two independent p r o c e s s e s — o n e t h a t o c c u r s o n l y f o l l o w i n g a c t i v a t i o n o f c h a n n e l s , and one t h a t i s independent o f a c t i v a t i o n .



7. POOLER


119

Chemical mechanisms. Channel f u n c t i o n i s v e r y e a s i l y p e r t u r b e d by a v a r i e t y o f p h a r m a c o l o g i c a l agents t h a t b l o c k a n d / o r modify g a t i n g . The open pore r e g i o n o f a channel t h a t d i s c r i m i n a t e s between d i f f e r e n t ion s p e c i e s (the s e l e c t i v i t y f i l t e r ) i s p r o b a b l y no more than a few Angstroms i n d i a m e t e r and i t w o u l d n ' t take a g r o s s s t r u c t u r a l a b n o r m a l i t y in t h i s p a r t o f a channel to b l o c k i o n movement ( 2 5 ) . The p h o t o c h e m i c a l mechanisms u n d e r l y i n g m o d i f i c a t i o n o f axon f u n c t i o n are not w e l l u n d e r s t o o d , however. V o l t a g e clamp experiments i n d i c a t e t h a t d i f f e r e n t s e n s i t i z e r s v a r y g r e a t l y in t h e i r p o t e n c y , but b r i n g about the same k i n d s o f a c t i o n s q u a l i t a t i v e l y , i m p l y i n g a common mode o f a c t i o n . (Lyudkovskaya d e s c r i b e d some s e n s i t i z e r - s p e c i f i c a c t i o n s i n n o n - v o l t a g e clamped a x o n s , but t h e s e have not been seen i n v o l t a g e clamp e x p e r i m e n t s . ) The major m o d i f i c a t i o n o f sodium c h a n n e l s — b l o c k and p e r t u r b a t i o n o f i n a c t i v a t i o n - - a r e p r o b a b l y independent p r o c e s s e s o c c u r r i n g in parallel. For a g i v e n degree o f channel b l o c k , however, the p e r t u r b a t i o n o f i n a c t i v a t i o n can v a r y somewhat depending on r e a c t i o n conditions. For example, r o s e b e n g a l , i n c o n t r a s t to o t h e r s e n s i t i z e r s , i s so e f f e c t i v e at s e n s i t i z i n g channel b l o c k t h a t the r e l a t i v e l y low l i g h t doses r e q u i r e d to b l o c k 50% o f the c h a n n e l s may not p e r t u r b i n a c t i v a t i o n measurably (28, 3 3 ) . Thus s e n s i t i z e r a c c e s s i b i l i t y to a b l o c k i n g s i t e and an i n a c t i v a t i o n s i t e may v a r y somewhat from s e n s i t i z e r to s e n s i t i z e r . An i n c r e a s e in l e a k a g e c o u l d r e s u l t from m o d i f i c a t i o n o f e x i s t i n g c h a n n e l s , c r e a t i o n o f new pathways through o t h e r i n t e g r a l membrane p r o t e i n s or by a p e r t u r b a t i o n in t h e l i p i d b i l a y e r s t r u c t u r e . There are l i m i t e d d a t a p o i n t i n g to s i n g l e t oxygen as an intermediate. On l o b s t e r axons d e u t e r i u m o x i d e and a z i d e were a b l e to enhance and i n h i b i t channel b l o c k by about 50% e a c h , u s i n g r o s e bengal or e o s i n as s e n s i t i z e r s ( 3 4 ) . On s q u i d a x o n s , w i t h r e a g e n t s p e r f u s e d i n s i d e the c e l l , B - c a r o î ë n e e f f e c t i v e l y b l o c k e d s e n s i t i z a t i o n by methylene b l u e but not r o s e bengal (28). U n c e r t a i n t i e s in t h e d i s t r i b u t i o n o f r e a g e n t s w i t h i n tfie complex anatomy o f a c e l l u l a r system c l o u d s the i n t e r p r e t a t i o n o f t h e s e experiments. A p p l i c a t i o n to L i g h t - A c t i v a t e d P e s t i c i d e s S i n c e none o f the i n v e s t i g a t i o n on photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s has been performed on i n s e c t s p e c i e s , any e x t r a p o l a t i o n to l i g h t - a c t i v a t e d p e s t i c i d e s must o f n e c e s s i t y be very s p e c u l a t i v e . However, i f s e n s i t i z e r s i n c o n t a c t w i t h i n s e c t s can permeate t o e x c i t a b l e membranes ( i . e , they a r e not stopped by major d i f f u s i o n b a r r i e r s ) , i t seems v e r y l i k e l y t h a t the k i n d s o f m o d i f i c a t i o n s found in o t h e r l i f e forms would a l s o o c c u r in i n s e c t s . W i t h i n a whole organism t h e r e a r e many p h o t o m o d i f i a b l e s i t e s . So l o n g as the s e n s i t i z e r i s not photobleached then a l l s i t e s w i l l become m o d i f i e d at s u f f i c i e n t l y h i g h l i g h t d o s e s . To be o f p h o t o t o x i c i m p o r t a n c e , however, some must e x h i b i t p h o t o t o x i c p o t e n t i a l at r e l a t i v e l y low l i g h t d o s e s . W i t h i n a network o f nerve c e l l s an a r r a y o f elementary p r o c e s s e s can be p e r t u r b e d . At a g i v e n l i g h t dose some w i l l be f a r more m o d i f i e d than o t h e r s because o f d i f f e r e n t p r e - i l l u m i n a t i o n a s s o c i a t i o n s w i t h s e n s i t i z e r and d i f f e r e n t e f f e c t i v e quantum y i e l d s . The consequence o f a g i v e n



120


modification w i l l also vary. (To use an analogy: A car can sustain a crushed bumper better than a broken spark plug.) At some point the accumulating modifications w i l l become manifestly toxic and survival of the organism threatened. Of the known photodynamic perturbations in excitable c e l l s , which ones have both a high s u s c e p t i b i l i t y to modification and a pivotal role in signaling? The obvious choice, block of sodium channels, i s probably not crucial because of the large excess number of channels r e l a t i v e to the minimum required to propagate action potentials. Only after many other perturbations have occurred i s i t l i k e l y that enough sodium channels would be blocked to halt propagation. Block of potassium channels i s also not l i k e l y to be crucial because of their r e l a t i v e l y low s e n s i t i v i t y to block. Interference with sodium channel inactivation may be more important because the prolongation of action poential duration that results from the interference decreases the maximum frequency of f i r i n g during a burst of action potentials. Even t h i s , however, seems less important than l i g h t induced f i r i n g at neuromuscular junctions. In vertebrate species each muscle c e l l is a "slave" to the motoneuron that innervates i t and light-induced f i r i n g translates d i r e c t l y into muscle contraction. Extraneous muscle contraction interferes with locomotion. In turn, the disturbed locomotion translates into interference with feeding, escape from predators and reproduction. Whether this occurs in insects remains unknown, however. While neuromuscular transmission in insects has many mechanistic s i m i l a r i t i e s to that in vertebrates (35) the different transmitter substance employed, method of transmitter removal, and the difference in innervation pattern (18) makes this an open question. As noted in the introduction, one of the important elemental processes occurring in a l l nervous systems is spontaneous generation of excitation. The control of insect walking, in which muscles are alternately stimulated and i n h i b i t e d , i s thought to originate in a group of pacemaker c e l l s that undergo rhythmic o s c i l l a t i o n s in membrane potential (36). The frequency of o s c i l l a t i o n is continuously modulate? by synaptic input. Such l a b i l e c e l l s should be easily perturbed by photodynamic means. While no photodynamic studies have been carried out on insect pacemaker c e l l s , i t seems very l i k e l y that these c e l l s might be among the most susceptible to the action of light-activated pesticides.

Literature Cited 1. 2. 3. 4. 5. 6. 7.

Callaham, M. F . ; Lewis, L. Α.; Holloman, M. E.; Broome, J. E.; Heitz, J . R. Comp. Biochem. Physiol. 1975, 51C, 123-128. Narahashi, T. Physiol. Rev. 1974, 54, 813-889. Chalazonitis, N. These Sciences, Paris 1954, Ser. A, No. 2994, Order 3866, 1-116. Chalazonitis, N.; Chagneux, R. Bull. Inst. Oceanogr. (Monaco) 1961, 58(1223), 1-20. Chalazonitis, N. Photochem. Photobiol. 1964, 3, 534-559. Lyudkovskaya, R. G. Biofizika 1961, 6, 300-306. Lyudkovskaya, R. G.; Kayushin, L. P. Biofizika 1959, 4, 404412.


7. POOLER 8. 9. 10. 11. 12. 13. 14. 15.


16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.


121

Lyudkovskaya, R. G.; Kayushin, L. P. Biofizika 1960, 5, 663670. Bostock, H. J . Physiol. 1982, 332, 57P-58P. Kuffler, S. W.; Nicholls, J . G . ; Martin, R. A. From Neuron to Brain, 2nd ed. Sinauer: Sunderland, MA, 1984. Lippay, F. Pflug. Arch. 1929, 222, 616-639. Lippay, F. Pflug. Arch. 1930, 224, 587-599. Lippay, F . ; Wechsler, L. Pflug. Arch. 1930, 224, 600-607. Rosenblum, W. I. J . Cell. Comp. Physiol. 1960, 55, 73-79. Burmistrov, Y. M.; Lyudkovskaya, R. G. Biofizika 1968, 13, 5565. Lyudkovskaya, R. G.; Pevzner, L. P. Biofizika 1964, 9, 580588. Sazonenko, Μ. Κ. Biofizika 1963, 8, 681-689. Usherwood, P. N. R.; Cull-Candy, S. G. In Insect Muscle; Usherwood, P. N. R., Ed.; Academic: London, 1975; pp. 207-280. Kohli, R. P.; Bryant, S. H. Experientia 1964, 20, 368-369. Pooler, J . P. Biophys. J . 1968, 8, 1009-1026. Pooler, J . P.; Oxford, G. S. J . Membr. Biol. 1973, 12, 339348. Adelman, W. J.; Adams, J. J . Gen. Physiol. 1959, 42, 655-664. Hodgkin, A. L.; Huxley, A. F . ; Katz, B. J . Physiol. 1952, 116, 424-448. Hodgkin, A. L.; Huxley, A. F. J . Physiol. 1952, 117, 500-544. Hille, B. Ionic Channels of Excitable Membranes. Sinauer: Sunderland, MA, 1984. Pooler, J . P. J. Gen. Physiol. 1972, 60, 367-387. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1979, 30, 491-498. Oxford, G. S.; Pooler, J . P.; Narahashi, T. J . Membr. Biol. 1977, 36, 159-173. Goldman, L. Q. Rev. Biophys. 1976, 9, 491-526. Dubois, J . M. Prog. Biophys. Molec. Biol. 1983, 42, 1-20. Pooler, J . P.; Valenzeno, D. P. Biophys. J . 1983, 44, 261-269. Oxford, G. S.; Pooler, J . P. J . Gen. Physiol. 1975, 66, 765779. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1978, 28, 219-226. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1979, 30, 581-584. Pichon, Y. In Insect Neurochemistry and Neurophysiology; Borkovec, A. B.; Kelly, T. J., Eds.; Plenum: New York, 1984; pp. 23-50. Shepherd, G. S. Neurobiology. Oxford: New York, 1983.

RECEIVED November 20, 1986


Photodynamic Modification of Excitable Cell Function - ACS Publications

Recommend Documents