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Celhdar ant i Molecular Neurobiology Vol . 14 . No. 3 1994

T e t r o d o t o x i n R e s is t a n t S o d i u m C h a n n e lsS h i g e r u Y o s h i d a ~

Received May 15 1994 revised June 10 1994KEY WORDS: tetrodotoxin; Na channels; neurons; muscles; development.

S U M M R Y

1 . T e t r o d o t o x i n ( T T X ) h a s b e e n w i d e l y u s e d a s a c h e m i c a l t o o l f o r b l o c k i n gN a + c h a n n e l s . H o w e v e r , r e p o r t s a r e a c c u m u l a t i n g t h a t s o m e N a c h a n n e l s a r er e s i s t a n t t o T T X i n v a r i o u s t i s s u e s a n d i n d i f f e r e n t a n i m a l s p e c i e s . S t u d y i n g t h es e n s it iv i ty o f N a c h a n n e l s t o T I X m a y p r o v i d e u s w i t h a n i n si g ht i n t o t h ee v o l u t i o n o f N a c h a n n e l s .

2 . N a c h a n n e l s p r e s e n t in T T X - c a r r y i n g a n i m a l s s u c h a s p u f fe r fi s h a n ds o m e t y p e s o f s h e ll fi sh , f r o g s , s a l a m a n d e r s , o c t o p u s e s , e t c ., a re r e s i s ta n t t o T T X .

3 . D e n e r v a t i o n c o n v e r t s T T X - s e n s i t i v e N a ch a n n e l s t o T T X - r e s is t a n t o n e si n s k e l e t a l m u s c l e c e l l s , i . e . , r e v e r t i n g - b a c k p h e n o m e n o n . A l s o , u n d i f f e r e n t i a t e ds k e l e t a l m u s c l e c e ll s c o n t a i n T T X - r e s i s t a n t N a c h a n n e ls . C a r d i a c m u s c l e c e l lsa n d s o m e t y p e s o f s m o o t h m u s c l e c e ll s a r e c o n s i d e r a b l y i n s e n si ti v e t o T I X .

4 . T I X - r e s i s t a n t N a c h a n n e l s h a v e b e e n f o u n d in c e ll b o d i e s o f m a n yp e r i p h e r a l n e r v o u s s y s te m ( P N S ) n e u r o n s i n b o t h i m m a t u r e a n d m a t u r e a n i m a ls .H o w e v e r , T I X - r e s i s t a n t N a c h a n n e ls h a v e b e e n r e p o r t e d in o n l y a f e w t y p e s o fc e n t r a l n e r v o u s s y s te m ( C N S ) . A x o n s o f P N S a n d C N S n e u r o n s a r e se n s it iv e toT T X . H o w e v e r , s o m e g lia l c e ll s h a v e T r X - r e s i s t a n t N a c h a n n e ls .

5 . P r o p e r t i e s o f T T X - s e n s i t i v e a n d T T X - r e s i s t a n t N a c h a n n e l s a r e d i f fe r e n t .L i k e C a 2 c h a n n e l s , T T X - r e s i s t a n t N a c h a n n e l s c a n b e b l o c k e d b y i n o r g a n i c( C o z , M n 2 , N i 2 ~-, C d 2 +, Z n ~ , L a 3 + a n d o r g a n i c ( D - 6 0 0 ) C a Z c h a n n e lb l o c k e r s . U s u a l l y , T T X - r e s i s t a n t N a c h a n n e l s s h o w s m a l l e r s i n g l e - c h a n n e lc o n d u c t a n c e , s l o w e r k in e t ic s , a n d a m o r e p o s i t i v e c u r r e n t - v o l t a g e r e l a t i o n t h a nT T X - s e n s i ti v e o n e s .6 . M o l e c u l a r a s p e ct s o f t h e T T X - r e s i s ta n t N a c h a n n e l h a v e b e e n d e s c r i b e d .T h e s t r u c t u r e o f t h e c h a n n e l h a s b e e n r e v e a l e d , a n d c h a n g i n g i ts a m i n o a c i d ( s)a l te r s t h e s e n s it iv i ty o f t h e N a c h a n n e l t o T T X .i Department of Physiology, Fukui Medical School, Fukui 910-11. Japan.

2270272.434 1/94/06 1~1.11227507.1X)/0 ~) 1994 Plenum PublishingCorporation

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8 Yoshida

7 . T T X - s e n s i t i v e N a c h a n n e l s s e e m t o b e u s e d p r e f e r e n t i a ll y i nd i f f e r en t i a t ed ce l ls an d i n h i g h e r an i m a l s i n s t ead o f TF X - r e s i s t a n t N a ch an n e l sf o r r a p i d a n d e f f e c ti v e p r o c e s s i n g o f i n f o r m a t i o n .8 . P o s s i b l e ev o l u t i o n c o u r s e s f o r N a an d C a 2 ch an n e l s a r e d i s cu s s ed w i t hr e g a r d t o o n t o g e n e s i s a n d p h y l o g e n e s i s .

I N T R O D U C T I O N

T e t r o d o t o x i n ( T T X ) o r p u f f e rf i sh t o x i n is t h e m o s t c o m m o n l y k n o w n a n i m a ltox in . The puf fer f i sh , ca l l ed F u g u i n J a p a n , b e l o n g s t o t h e g e n u s T e t r a d o n o rF u g u an d i t co n t a i n s t h e t o x i n i n t h e o v a r i e s , t h e l i v e r , an d o ccas i o n a l l y t h e s k i n( K a o , 1 96 6; H a l s t e a d , 1 9 6 7 ). T e t r o d o t o x i n w a s n a m e d i n 18 9 4 b y t h e J a p a n e s es c i en t is t T a h a r a ; t h e s t r u c t u re o f T T X w a s d e c i d e d i n 1 96 4 a n d s y n t h e s i z e d i n1 9 7 0 ( s ee r ev i ew s b y K ao , 1 96 6; M o s h e r , 1 9 8 6 ) . T T X i s a v e r y p o t e n t t o x i n , e . g . ,o n l y 1 m g o f T F X c a n k i ll a s m a n y a s 7 0 0 0 m i c e , a n d I T X i s 1 6 0 ,0 0 0 t im e s m o r ep o t e n t t h a n c o c a i n e ( K a o , 1 9 6 6 ) .

S u r p r i s i n g l y , p u f f e r f i s h a r e ea t en i n A s i an co u n t r i e s , e s p ec i a l l y - i n J ap an .C a p t a i n J a m e s C o o k ( 1 7 7 7 ) d e s c r i b e d t h e T I ' X p o i s o n i n g i n d e t a i l w h i c h s t r u c kh i m a n d h i s c r e w i n 17 7 4 w h e n t h e y w e r e o n t h e i r s e c o n d v o y a g e a r o u n d t h ew o r l d . T h e y h a d l i v e r a n d r o e f o r s u p p e r a n d b e c a m e p o i s o n e d . F o r a h i s t o r y o fT T X p o i s o n i n g , s e e t h e e x c e l l e n t r e v i e w b y K a o ( 1 96 6 ).

I t is a ls o s u r p r is i n g t h a t T T X i s n o t s y n t h e s i zed b y p u f f e r fi s h b u t b y t h eb a c t e r i u m P s e u d o m o n a s s p ., w h i c h is p r e s e n t i n th e r e d a l g a a n d f o r m s p a r t o f t h ep u f f e r f i s h ' s d i e t ( Y a s u m o t o e t a L 1986) , o r V i b r i o i n t h e g u t ( N o g u c h i e t a l .1 9 8 6 ) . T h e r e f o r e c u l t u r e d p u f f e r f i s h , w h i c h a r e n o t f e d s u c h c o n t a m i n a t e d a l g a ,a r e n o n t o x i c .

S i n c e t h e d i s c o v e r y o f t h e s e l e c ti v e b lo c k i n g a c t io n o f T F X o n N a c h a n n e l s( K a o a n d F u h r m a n , 1 96 3; N a r a h a s h i e t a l . 1 9 6 4 ) , t e t r o d o t o x i n h a s b e e n w i d e l yu s e d a s a c h e m i c a l t o o l f o r s t u d y i n g i o n ic p r o p e r t i e s o f N a c h a n n e l s a n d o t h e rt y p e s o f ch an n e l s ( f o r r ev i ew s s ee K a o , 1 9 66 ; N a r ah as h i , 1 97 4; R i t ch i e an dR o g a r t , 1 9 7 7 ; C a t t e r a l l , 1 9 8 0 ; Y o s h i d a e t a l . 1 9 8 0 a ; U l b r i ch t , 1 9 8 1 ; K ao an dLev i n s o n , 1 98 6; P r i n ce , 1 9 8 8; N u m a , 1 9 8 9 ). I n d e ed , t h e p r o p e r t i e s o f N a an do t h e r i o n ic ch a n n e ls w o u l d n o t h a v e b e e n e l u c i d a te d w i t h o u t T T X .

T h i s re v i e w a r ti c le f o c u s e s o n T T X a n d d o e s n o t m e n t i o n s a x i to x i n ( S T X ) ,w h i ch is c o m m o n l y k n o w n a s a p a r a l y ti c s he ll fi sh p o i s o n . B o t h T r x a n d S T Xa r e g u a n i d i u m t o x in s a n d a c t o n N a c h a n n e l s . T h e m a j o r p o i n t o f th is a r ti c le i st o d e s c r i b e v o l t a g e - g a t e d a n d T T X - r e s i s t a n t N a c h a n n e l s i n m a m m a l i a n n e r v ea n d m u s c l e c e l l s , e s p e c i a l l y d o r s a l r o o t g a n g l i o n ( D R G ) c e l l s , o n w h i c h e x t e n s i v es t u d y h as b e en ca r r i ed o u t . I t i s g en e r a l l y co n s i d e r e d t h a t e l ec t r i c a l si g n a ls o fm a m m a l i a n e x c i ta b l e c e il s a r e f u n d a m e n t a l ly d e p e n d e n t o n v o l ta g e - g a t e d N a c h a n n e ls w h i ch a r e s e ns it iv e to T r x . H o w e v e r , e v i d e n c e h as b e e n a c c u m u l a ti n g

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t h a t s o m e s u c h c el ls a r e i n se n s it iv e t o T T X . D a t a o n n o n m a m m a l i a n c e ll s a r e a l s om e n t i o n e d t o c o n s i d e r t h e d e v e l o p m e n t o f T T X - r e s is t a n t N a c h a n ne ls .

N a C H A N N E L S I N T T X C A R R Y I N G A N I M A L S

T I X i s f o u n d n o t o n l y i n p u f fe r f i sh an d i ts r e l a t ed f is h es b u t a l s o i n o t h e rs p e c i e s o f a n i m a l s s u c h a s C o s t a R i c a n f ro g s , C a l i f o rn i a n s a l a m a n d e r s T a r i c h a ) ,A u s t r a l i an b l u e - r i n g ed o c t o p u s es , s h e l l f i s h , g o b y , an d s o me c r ab s , s t a r f i s h , an dan g e l f i s h ( R u s s e l , 1 9 6 5 ; K ao , 1 9 6 6 ; H a l s t ead , 1 9 6 7 ; M o s h e r an d F u h r man , 1 9 8 4 ;Y a s u m o t o e t a L , 1 9 8 6 ; P r i n ce , 1 9 8 8 ) . Th es e an i ma l s u s e TTX f o r s e l f - d e f en s e o ra t t a ck i n g o t h e r an i m a l s , e .g . , t h e A u s t r a l i an b l u e - r i n g e d o c t o p u s co n t a i n s T - I X i ni t s s a l i v a r y v en o m g l an d s an d u s e s t h e t o x i n f o r p a r a l y z i n g i t s p r ey , i n c l u d i n gh u m a n s ( M o s h e r a n d F u h r m a n , 1 9 84 ). I t s e e m s r e a s o n a b l e t h a t th e v o l t a g e - g a t e dN a c h a n n e l s o f t h e s e a n i m a l s, c a r r y in g T T X i n t h e ir t is s u e s, n e e d t o b ei n s en s i ti v e t o T T X t o s u rv i v e ( M o s h e r e t a L , 1 9 6 4 ; H ag i w ar a an d Tak ah as h i , 1 9 6 7 ;K i d o k o r o e t a l . , 1974) .

T I X - R E S I S T A N T N a + C H A N N E L S I N M U S C L E C E L L S

S k e l e t a l M u s d e Cel l sD e n e r v a t i o n c o n v e r t s q - T X - s e n si ti v e N a + c h a n n e l s i n s k e l e ta l m u s c l e c e ll s t o

T T X - r e s i s t a n t o n e s , i . e . , r e v e r t i n g - b a c k p h e n o m e n o n ( H a r r i s a n d T h e s l e f f , 1 9 7 1 ;R ed f e r n an d Th es l e f f , 1 9 71 ; P ~ tp p o n e , 1 9 8 0) . Th e k i n e t i c s o f co n v e r t e d N a c h a n n e l s b e c a m e s l o w e r ( P a p p o n e , 1 9 80 ). T I X - r e s i s t a n t N a + c h a n n e l s h a v e a l s ob e e n f o u n d i n u n d i f f e r e n ti a t e d o r d e v e l o p i n g m u s c l e c e ll s ( K i d o k o r o , 1 9 7 3 , 1 97 5;K an o , 1 9 7 5 ). I t is s u g g es t ed t h a t N a ch an n e l s i n u n d i f f e r en t i a t ed ce l ls d o n o tp o s s e s s s en s i t i v i ty t o q TX .

C a r d i a c M u s c l e Cel l sU s u a l l y , c a r d i a c N a + c h a n n e ls a r e m u c h l e ss s e n s it iv e t o T T X t h a n t h o s e i n

mo s t n eu r o n s an d s k e l e t a l mu s c l e ce l l s ( R i t ch i e an d R o g a r t , 1 9 7 7 ; C a t t e r aU , 1 9 8 0 ;B r o w n e t a L , 1981; Sat in e t a l . , 1 9 92 ). W h e n s e e n f r o m a p o i n t o f c e ll u la rd e v e l o p m e n t , a f r a c t io n o f N a c h a n n e l s w h i c h w e r e r e s i s ta n t t o T T X i ne m b r y o n i c c h ic k e n h e a r t c el ls b e c a m e se n s i ti v e t o T r x i n t h e l a te r s ta g e s( S h i g e n o b u a n d S p e r e l a k i s , 1 9 7 1 ; M c D o n a l d e t a l . , 1 9 7 3 ) . H o w e v e r , a c q u i r e ds en s i t i v i t y t o TTX i s n o t r i g i d . I n s o me ca s e s , f o r ex amp l e , i n ch i ck h ea r t c e l la g g r e g a t e s , N a c h a n n e l s e a s i l y c h a n g e d t h e ir T T X s e n s it iv i ty ( M c D o n a l d e t a l . ,1 9 73 ). E v e n i n o l d e r c h i ck h e a r t s , t h e T r X - s e n s i t i v e f a s t N a c h a n n e l s r e v e r t e db a c k t o t h e T T X - r e s i s t a n t s l o w N a + c h a n n e l s w h e n p l a c e d i n c u l t u r e ( S p e r e l a k i san d S h i g en o b u , 1 9 7 2 ) .

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2 3 Y o s h i d a

S m o o t h M u s c l e C e l l sS o m e t y p e s o f s m o o t h m u s c l e c e ll s a r e r e p o r t e d t o h a v e T T X - r e s is t a n t N a +

c h a n n e l s . A n e x a m p l e is t h e s m o o t h m u s c l e i s o l a te d f r o m s t o m a c h f u n d u s o f t h er a t ( M u r a k i e t a l . , 1 9 91 ). T h i s T T X - r e s i s t a n t N a c h a n n e l s h o w e d a m o r en e g a t i v e c u r r e n t - t o - v o l t a g e l - V ) r e l a t io n t h a n t h e q T X - s e n s i t i v e N a c h a n n e lp r e s e n t i n s m o o t h m u s c l e s u c h a s t h a t i n g u i n e a p i g u r e t e r .

T T X - R E S I ST A N T N a + C H A N N E L S IN N E U R O N S

P e r ip h e ra l N e r v o u s S y s t e m P N S ) N e u r o n s : D o r s a l R o o t G a n g l io n C e l lsS i n c e t h e d i s c o v e r y o f T T X - r e s i s t a n t N a + c h a n n e l s u s in g t h e i n tr a c e ll u la r -

r e c o r d i n g m e t h o d i n m a m m a l i a n n e u r o n s , e . g ., m o u s e d o r s a l r o o t g a n g l io n( D R G ) c e l l s ( M a t s u d a e t a L , 1 9 7 6 ) , e x t e n s i v e s t u d i e s h a v e b e e n c a r r i e d o u t o nD R G c e ll s b e c a u s e o f e a s e o f a c c e s s a n d t h e s p h e r ic a l s h a p e o f t h e c e ll b o d y . Inr e c e n t y e a rs , m o r e o v e r , t h e r e h a s b e e n r e n e w e d i n te r e s t in D R G c e ll s u s in gw h o l e - ce l l an d s i n g l e - ch an n e l r eco r d i n g t ech n i q u es f o r an a l y s i s i n d e t a i l . Th i ss e c t io n , t h e r e f o r e , o u t l i n e s t h e s t u d i e s o n D R G c e ll s.

T T X - r e s i s t a n t N a + c h a n n e l s w e r e f o u n d f ir s t i n c u l t u r e d D R G c e l ls t a k e nf r o m f e t a l m i c e ( M a t s u d a e t a L , 1 97 6, 1 9 7 8) . M o s t D R G c e ll s g e n e r a t e d aT T X - r e s i s t a n t s p i k e w i t h N a + a n d C a 2+ c o m p o n e n t s , i .e ., N a + c h a n n e l s w e r ei ns e n si ti ve t o q T X . T r X - r e s i s t a n t N a c h a n n e ls w e r e a ls o d e t e c t e d i n D R G c e ll so f th e a d u l t m o u s e b u t i n a s m a l l e r n u m b e r ( Y o s h i d a e t a l . , 1978) .

A c c o r d i n g t o m o r p h o l o g i c a l , p h y s i o l o g i c a l , a n d p h a r m a c o l o g i c a l p r o p e r t i e s ,D R G c e l l s h a v e b e e n c l a s s i f i e d i n t o s u b g r o u p s b y m a n y r e s e a r c h e r s . O u rc l as s if ic a t io n o f m o u s e D R G c e l ls i s d e s c r i b e d h e r e a s a n e x a m p l e . W e c l a s s if ie dD R G c e ll s i n t o t h r e e m a j o r g ro u p s : ( 1 ) l a rg e o r m e d i u m - s i z e d n e u r o n s w h i c he l ic i t a q ~ X - s e n s i t i v e N a s o m a s p i k e w i th a m y e l i n a t e d a x o n a n d a f a s tc o n d u c t i o n v e l o c it y , ( 2) s m a l l n e u r o n s w h i c h g e n e r a t e a T T X - r e s i s t a n t N a + s o m as p i k e w i th a n u n m y e l i n a t e d a x o n a n d a s lo w c o n d u c t i o n v e l o c i ty , a n d ( 3 ) s m a l l( o c c a s i o n a l l y l a rg e ) n e u r o n s w i th a g e n e r a l l y u n m y e l i n a t e d a x o n w h i c h p r o d u c e aT T X - r e s is t a n t s o m a s p i k e w i th N a + a n d C a 2+ c o m p o n e n t s ( M a t s u d a e t a l . , 1978;Y o s h i d a e t a l . , 1 9 7 8 ; Y o s h i d a a n d M a t s u d a , 1 9 7 9 ) . E v e n i n c e l l s i n w h i c h t h ec o n t r i b u t i o n o f C a 2+ c h a n n e l s t o t h e a c t i o n p o t e n t i a l w a s n e g l i g ib l e u n d e rp h y s i o l o g i ca l co n d i t i o n s , r a i s i n g t h e co n cen t r a t i o n o f a d i v a l en t c a t i o n ( C a 2 +,S rz+, o r B a 2 ) l ed t o ac t i o n p o t en t i a l s , i n d i ca t in g t h a t C a 2+ ch an n e l s a r e p r e s en ti n m o s t D R G c e ll s in v a ry i n g p r o p o r t i o n s ( Y o s h i d a a n d M a t s u d a , 1 980 ; F e d u t o v ae t a l . , 1 9 91 ). A l t h o u g h t h e r e is s o m e d e b a t e o n h o w t o c l as s if y n e u r o n s i n tos u b g r o u p s , s im i la r re s u lt s h a v e b e e n o b t a i n e d f r o m D R G c e ll s o f o t h e r a n i m a ls p e c i e s s u c h a s f r o g ( M o r i t a a n d K a t a y a m a , 1 98 9; C a m p b e l l , 1 9 9 2 ), r a t ( L a w s o n e ta l . , 1 9 8 4 ; H a r p e r a n d L a w s o n , 1 9 8 5 a , b ; C a f f r e y e t a L , 1 9 9 2 ; O g a t a a n dT a t e b a y a s h i , 1 9 9 2 a , 1 9 9 3 ) , a n d g u i n e a p i g ( F u k u d a a n d K a m e y a m a , 1 9 8 0 ) . I t i st o b e n o t e d , h o w e v e r , t h a t n o a p p r e c i a b le T I X - r e s i s t a n t N a + c h a n n e ls h a v e

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lTX -R es i s t an t N a Channe l s 231

b e e n d e t e c t e d i n c h i c k D R G c e l l s ( D i c h t e r a n d F i s c h b a c h , 1 9 7 7 ; P e t e r s e n , e t a l .1987) .

A s f o r a x o n s , s p i k e c o n d u c t i o n a l o n g t h e a x o n w a s b l o c k e d b y T T X i n allt y p e s o f m o u s e D R G c e ll s i n p h y s i o lo g i c a l s o l u t io n ( Y o s h i d a a n d M a t s u d a , 1 9 79 ).H o w e v e r , p r o p a g a t i n g B a 2 o r S r 2 a c ti o n p o t e n t i a ls w e r e o b s e r v e d in a p a r t o ft h e u n m y e l i n a t e d a n d m y e l i n a t e d a x o n s , i n d i c a ti n g t h a t v o l t a g e - g a t e d C a 2c h a n n e l s a r e a l s o p r e s e n t i n t h e a x o n ( Y o s h i d a a n d M a t s u d a , 1 98 0; Y o s h i d a e t a L1 9 8 0 b ) . T h e s e f i n d i n g s w e r e s u p p o r t e d b y a s t u d y u s i n g a p a t c h - p i p e t t e o n t h el a r g e g r o w t h c o n e m e m b r a n e , i .e ., 13 f i b e r t e r m i n a l, o f th e c u l t u r e d r a t D R G c e ll s( Y a m a g u c h i , 1 9 92 ). T h e g r o w t h c o n e h a d v o l t a g e - g a t e d N a , C a 2 , a n d K c h a n n e l s , a n d t h e N a c h a n n e l s w e r e s e n s it iv e t o T T X .

O t h e r P e r i p h e r a l N e r v o u s S y s t e m N e u r o n sT T X - r e s i s t a n t N a c h a n n e l s h a v e b e e n f o u n d i n o t h e r P N S n e u r o n s s u c h a s

b u l l f ro g s y m p a t h e t i c ( J o n e s , 1 9 8 7) a n d p a r a s y m p a t h e t i c n e u r o n s ( C l a r k e t a l .1 9 9 0 ), t ri g emi n a l an d c i li a r y g an g l i o n ce l ls o f q u a i l em b r y o s ( S ch l i ch te r e t a l .1 9 9 1 ) , r a t an d r ab b i t n o d o s e g an g l i o n ce l l s ( B o s s u an d F e l t z , 1 9 8 4 ; B accag l i n i an dC o o p e r , 1 9 8 2 ; I k e d a e t a l . 1 9 8 6 ; I k ed a an d S ch o f i e l d , 1 9 8 7 ; S t an s f e l d an d Wal l i s ,1 9 8 5 ) , r a t an d ca t p e t r o u s g an g l i o n ce l l s ( B o s s u an d F e l t z , 1 9 8 4 ; G a l l eg o , 1 9 8 3 ) ,an d r a t s u p e r i o r ce r v i ca l n eu r o n s ( S ch o f i e l d an d I k ed a , 1 9 8 8 ) . R es u l t s s i m i l a r t ot h o s e w it h D R G c e ll s h a v e b e e n o b t a i n e d f r o m t h e se p r e p a ra t io n s . F o r e x a m p l e ,T T X - r e s i s t a n t N a c h a n n e ls i n a d u l t r a t n o d o s e g a n g li o n c e l ls w e r e b l o c k e d b yC a 2 c h a n n e l b l o c k e r s ( C d 2 , C o 2 , a n d D 6 0 0 ) a n d e x h i b i t e d a s l o w e r t im ec o u r s e t h a n T T X - s e n s i t i v e o n e s ( B o s s u a n d F e l t z , 19 84 ; I k e d a e t a L 1986 ; Ikedaand Schof i e ld , 1987 ; C lark e t a l . 1990) .

e n t r a l N e r v o u s S y s t e m N e u r o n sI n t e n s i v e s t u d y h a s b e e n c a r r i e d o u t o n c e n t r a l n e r v o u s s y s t e m ( C N S )

n e u r o n s . H o w e v e r , n o t m a n y T I X - r e s i s ta n t N a c h a nn e l s h a v e b e e n r e p o r t e dc o m p a r e d w i th P N S n e u r o n s. R a t h e r , T T X is u s e d t o b lo c k N a + c u r r e n ts t o w o r ko n o t h e r t y p e s o f c h a n n e l s i n C N S n e u r o n s .A c h a i n o f s e g m e n t a l g a n g l ia f o r m s t h e C N S o f t h e l e e c h , a n d a c t i o np o t e n ti a ls o f t h e R e t z i u s g a n g li o n n e u r o n d e p e n d o n T T X - r e s i s ta n t N a c h a n n e ls( K l e i n h a u s a n d P r i c h a r d , 1 9 76 ). A s f o r m a m m a l s , T T X - r e s i s t a n t v o l t a g e - g a t e dN a ch a n n e l s w e r e a c t i v a te d b y n e u r o t e n s i n i n d o p a m i n e r g i c n e u r o n s o f t h e r a tm e s e n c e p h a l o n ( M e r c u r i e t a l . 1 9 93 ) a n d b y o x y t o c i n i n v a ga l n e u r o n s o f t h e r a tb r a i n s t e m ( R a g g e n b a s s a n d D r e i f u s s , 19 9 2) . M o r e o v e r , a p p r o x i m a t e l y h a l f o f t h en e u r o n s i n t h e r a t v e n t r a l t e g m e n t a l a r e a s h o w e d o n l y a n o r d i n a r y T T X - s e n s i t i v eN a + c u r r e n t , b u t t h e r e m a i n i n g n e u r o n s e x h i b i t e d a T l X - r e s i s t a n t N a c u r r e n t a sw e l l ( O g a t a a n d T a t e b a y a s h i , 1 99 2 c) . F u r t h e r m o r e , a c u t e l y d i ss o c i a te d n e u r o n sf r o m t h e m e d i a l e n t o r h i n a l c o r t e x o f t h e r a t e x h i b i t e d a T I X - r e s i s t a n t N a c u r r e n t in a d d i t i o n t o th e T T X - s e n s i t iv e N a c u r r e n t ( W h i t e e t a l . 1 9 9 3 ) . Th eT T X - r e s i s t a n t N a + c u r r e n t w a s m o r e s e n s it iv e t o i n o r g a n i c C a 2 c h a n n e l b l o c k e r s( C d 2 +, La 3 +, an d Z n 2 + ) t h a n t h e T T X - s e n s i t i v e o n e .

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T T X - R E S I S T A N T N a + C H A N N E L S I N G L I A L C E L L SV e r t e b r a t e g li al c el ls h a v e v o l t a g e - g a t e d N a c h a n n e l s ( s e e r e v i e w b y B a r r e se t a l . 1 99 0). D i f f e r e n t r e s u lt s h a v e b e e n o b t a i n e d f o r t h e T F X s e n s it iv i ty o f N a c h a n n e l s i n t h e s e c e l ls . F o r e x a m p l e , p o o r s e n s i t iv i ty o f N a + c h a n n e l s t o T T X w a sr e p o r t e d f o r c u l t u r e d r a t a s t r o c y t e s ( B e v a n e t a L 1 98 5) a n d acu t e ly i s o l a t edS c h w a n n c e il s i n a d u l t r a b b i t s ( C h i u , 1 98 7). O n t h e o t h e r h a n d , N a c h a n n e l sw e r e h i g h ly s e n si ti v e t o T T X i n a c u t e l y d i s s o c ia t e d a s t r o c y t e s o f r e t i n a l g a n g l io n

( B a r r e s e t a L 1 9 8 9 ) an d Sch wan n ce l l s i n cu l t u re (Sh rag e r e t a l . 1985).F u r t h e r m o r e , e x p r e s s i o n o f T T X - s e n si ti v e a n d T T X - r e s is t a n t N a c h a n n e l s w a sc o n f i n e d t o m o r p h o l o g i c a l l y d i s t i n g u i s h a b l e s u b t y p e s o f a s t r o c y t e s a n d t h e i rk i n e t ic s w e r e d i f f e r e n t l y c o n t r o l l e d b y p r o t e i n k i n a s e C , i n d i c a t in g t h a t T I ' Xs e n s it iv i ty o f t h e N a c h a n n e l i s r e l a t e d t o t h e f u n c t i o n o f t h e s e c e ll s ( T h i o a n dSo n th e im er , 1 9 9 3 ) .

D I F F E R E N C E S I N P R O P E R T I E S O F T T X - R E S I S T A N T A N D T T X -S E N S I T I V E N a C H A N N E L S

S e n s i t iv i t y to C a 2 C h a n n e l B l o c k e r sU s u a l l y T T X - r e s i s t a n t N a c h a n n e l s a r e b l o c k e d b y b o t h i n o r g a n i c ( C o 2 ,M n 2 +, N i 2 , C d 2 +, Z n ~ +, L a 3 +) an d o rg an i c (D-6 0 0 ) C a ~+ ch a n n e l b lo ck e r s

( K o s t y u k e t a l . 1 9 8 1 ; I k e d a a n d S c h o f ie l d , 1 9 8 7; R o y a n d N a r a h a s h i , 1 9 9 2; W h i t ee t a l . 1 99 3). H o w e v e r , T r X - r e s i s t a n t N a c h a n n e l s in a d u l t r a t n o d o s e g a n g l i o nce l ls w e re l e ss s en s i t i v e t o i n o rg a n i c C a 2+ ch an n e l b lo ck e r s t h an C a 2+ ch an n e l sp r e s e n t i n t h e s a m e p r e p a r a t i o n ( I k e d a a n d S c h o f ie l d , 1 98 7).

C h a n n e l K i n e t i c sE l e c t r o p h y s i o l o g i c a l p r o p e r t i e s o f T I ' X - r e s i s t a n t N a + c h a n n e l s i n D R G c e ll s

h a v e b e e n i n v e s t i g a t e d i n m o r e d e t a i l u s i n g t h e w h o l e - c e l l a n d s i n g l e - c h a n n e lr e c o r d i n g te c h n i q u e s , a n d i t h a s b e e n s h o w n t h a t t h e k i n e t i c s o f T I ' X - r e s i s t a n tN a + c h a n n e l s a r e u s u a l ly d i f f e r e n t f r o m T T X - s e n s i ti v e N a c h a n n e l s .

I n c o m p a r i s o n w i t h T T X - s e n s i t i v e N a c h a n n e l s , l -T X - r es is ta n t N a c h a n -n e l s s h o w a l o w e r s i n g l e - c h a n n e l c o n d u c t a n c e , s l o w e r a c t i v a t i o n a n d i n a c t i v a t i o np r o c e s s e s , a n d a s h i f t i n a m o r e d e p o l a r i z e d d i r e c t i o n o f t h e c u r r e n t - v o l t a g er e l a ti o n , t h e c o n d u c t a n c e - v o l t a g e c u r v e , a n d t h e s t e a d y - s t a te i n a c t iv a t io n c u r v ei n b o t h i m m a t u r e a n d a d u l t D R G n e u r o n s ( P a p p o n e , 1 9 80 ; K o s t y u k e t a l . 1981;M c L e a n e t a l . 1 9 8 8 ; Om r i an d M e i r i , 1 9 9 0 ; Sch war t z e t a l . 1 9 9 0 ; Fed u lo v a e t a l .1 9 9 1 ; R o y an d Narah as h i , 1 9 9 2 ; Og a t a an d T a t eb ay as h i , 1 9 9 2 b , 1 9 9 3 ; C a f f r ey e ta l . 1992 ; Ca m pb el l , 1993; E l l io t t and E l l io t t , 1993), se nso ry c ran ia l gan g l ion ce l l s(B o s s u an d Fe l t z , 1 9 8 4 ) , an d r a t s k e l e t a l m u s c l e ce l l s (W e i s s an d Ho rn , 1 9 8 6 ) . Inc o n t r a s t , n o s i gn i fi c an t d i ff e r e n c e s o r e v e n r e v e r s a l i n t h e p o l a r i t y o f d i f f e r e n c e s int h e c u r r e n t - v o l t a g e r e l a t i o n s h i p w e r e r e p o r t e d i n t h e f r o g n o d e o f R a n v i e r( B e n o i t e t a L 1 98 5), g u i n e a p ig D R G c e il s ( P e t e r s e n e t a l . 1987), bul l frog

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s y m p a t h e t i c ( J o n e s , 1 9 8 7 ) , a n d p a r a s y m p a t h e t i c ( C l a r k e t a L 1 9 9 0 ) n eu r o n s , an da h u m a n n e u r o b l a s t o m a c e l l l i n e L A - N - 5 ( W e i s s a n d S i d e l l , 1 9 9 1 ) . I n c o n n e c t i o nw i t h th is , it s h o u l d b e r e m e m b e r e d t h a t a s in g l e -p o i n t m u t a t i o n i n t h e a m i n o a c i ds e q u e n c e o f t h e r a t b r a i n N a c h a n n e l d e c r e a s e d i ts s e n si ti v it y t o T F X a n di n d u c e d a h y p e r p o l a ri z i n g s h if t in th e c u r r e n t - v o l t a g e r e l a ti o n ( N o d a e t a l . 1989).

A s f o r C N S n eu r o n s , T TX - r e s i s t an t N a ch an n e l s a r e k i n e t i ca ll y i n d is t in g u -i s h a b le f r o m T T X - s e n s i t iv e o n e s i n n e u r o n s o f th e r a t m e d i a l e n to r h i n a l c o r t e x( W h i t e e t a L 1 9 9 3 ) a n d t h e r a t v e n t r a l t e g m e n t a l a r e a ( O g a t a a n d T a t e b a y a s h i ,1992c) .I t is in t e r e st in g t h a t o n l y T I X - r e s i s t a n t N a c h a n n e l s w e r e f o u n d w h e n a d u l tr a t D R G c e ll s w e r e c u l tu r e d i n t h e a b s e n c e o f s e r u m a n d n e r v e g r o w t h f a c to r( A g u a y o e t a l . 1 9 9 1 ) . T h e m a j o r e f f e c t o f n e r v e g r o w t h f a c t o r w a s c o n c l u d e d t ob e an acce l e r a t i o n i n k in e t i c s an d acq u i s i t io n o f s en s it iv i t y to T F X o f N a ch an n e l s ( O m r i an d M e i r i, 1 9 90 ). I n ad d i t i o n , t h e p r o p o r t i o n o f N a ch an n e l s o ff a s t a n d s l o w k in e t ic s w a s d e p e n d e n t o n t e m p e r a t u r e i n t h e f r o g n o d e o f R a n v i e r ,an d i t w a s s u g g es t ed t h a t f o r m s o f t h e N a ch an n e l w e r e r e l a t e d t o d i f f e r en t li p ide n v i r o n m e n t s o f t h e p r o t e i n c o n s t it u ti n g t h e c h a n n e l ( B e n o i t e t a L 1985) .

M O L E C U L A R A S P E C T S O F T H E T T X - R E S I S T A N T N a C H A N N E L

Th e N a ch an n e l p u r i f ied f r o m t h e e l ec t r i c ee l i s a g l y co p r o t e i n w i th am o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 2 1 5 ,0 0 0 a n d is c o m p r i s e d o f a p p r o x i m a t e l y2 0 0 0 a m i n o a c id s , 5 0 0 s u g a r r e s i d u e s , a n d 5 0 f a t ty a c i d c h ai ns . H o w e v e r , N a +c h a n n e l s i s o l a t e d f r o m m a m m a l i a n s k e l e t a l m u s c l e a n d b r a i n h a v e m o r e c o m p l e xs u b u n i t s t r u c tu r e s c o n s is t in g o f a - a n d / 3 - s u b u n i t s ( N o d a e t a l . 1984, 1986; Numa,1 9 8 9) . M o s t o f t h e f u n c t i o n a l p r o p e r t i e s , i n c l u d in g t h e s en s i ti v i ty t o TTX , o f t h eN a c h a n n e l r e s id e in t h e l a rg e a - s u b u n i t ( N o d a e t a l . 1986; Cat teral l , 1988;B a r ch i , 1 98 8; T r i m m er an d A g n e w , 1 98 9; M an d e l , 1 99 2).T T X is a m e m b r a n e i m p e r m e a n t a n d p o s i ti v el y c h a r g e d g u a n id i u m t ox in a n di t b l o ck s N a ch an n e l s f r o m t h e ex t r ace l l u l a r s i d e . I t i s t h o u g h t t h a t t h e p o s i t iv e l yc h a r g e d g u a n i d i u m m o i e t y o f T T X i n te r a c ts w i t h th e n e g a t i v e ly c h a r g e d a m i n oa c i d s p r e s e n t n e a r o r w i th i n t h e m o u t h o f t h e c h a nn e l . I n d e e d , w h e n t h e a m i n oac i d s w h i ch f o r m t h e n eg a t i v e l y ch a r g ed l in in g a t t h e m o u t h o f t h e N a ch an n e lw e r e r e p l a c e d b y p o s i ti v e ly c h a r g e d s a m e a m i n o a c i d s o r b y s o m e o t h e r a m i n oa c id s , t h e s e n s it iv i ty o f t h e N a c h a n n e l t o T r X w a s c o m p l e t e l y a b o l i s h e d ( T e r l a ue t a l . 1 99 1). T h u s , a T F X m o l e c u l e p h y s i c a ll y o c c l u d e s th e c h a n n e l p o r e b yp l u g g i n g t h e m o u t h o f t h e N a + ch an n e l w i t h t h e a i d o f e l ec tr i c a l fo r ce . Th i sp l u g g in g s c h e m e i n d ic a t e s t h a t T T X b l o c k s a f l ow o f io n s t h r o u g h t h e N a c h a n n e l w i t h o u t i n t e r fe r i n g w i t h t h e m o v e m e n t o f t h e g a t e o f t h e c h a n n e l( A r m s t r o n g a n d B e z a n i ll a , 1 97 4). I t is c o m m o n l y a s s u m e d t h a t o n e T I X m o l e c u l es u ff ic e s t o b l o c k o n e N a + c h a n n e l , a l t h o u g h i t h a s b e e n p r o p o s e d t h a t t w o T T Xm o l e c u l e s a r e n e c e s s a r y t o b l o c k t h e c h a n n e l ( B e n o i t e t a l . 1985).I n t e re s t in g l y , T r X - s e n s i t i v e N a + c h a n n e ls c a n b e e a s i ly r e n d e r e d i n se n s it iv et o th e t o x in b y t r e a t m e n t w i t h t r i m e t h y l o x o n i u m ( T M O ) , w h i c h m e t h y l a t e sc a r b o x y l i c a c i d s o f th e T T X r e c e p t o r . T h e s in g l e -c h a n n e l c o n d u c t a n c e o f s u c h

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c o n v e r t e d N a c h a n n e l s h a s b e e n s h o w n t o b e r e d u c e d ( S i g w o r t h a n d S p a l d i n g ,1980 ; Spa ld ing , 1980 ; W ei ss and H or n , 1986) .

T h r e e d i s ti n c t m R N A s ( t y p e s I, II , a n d I I I ) e n c o d i n g N a c h a n n e l s h a v eb e en i s o l a t ed f r o m t h e r a t b r a i n , an d o n l y t h e t y p e I N a ch an n e l l a ck ed T -F Xs en s i t i v i t y ( N o d a e t a l . 1 9 86 ). S e e n f r o m a s t r u c t u ra l p o i n t o f v ie w , g l u t a m a t er e s i d u e 3 8 7 s e e m s t o b e a n e s s e n t ia l a m i n o a c id i n th e T T X - b i n d i n g s it e o f t h eN a c h a n n e l b e c a u s e r e p l a c in g r e s i d u e 3 8 7 w it h t h e u n c h a r g e d a m i n o a c i dg l u t a m i n e g r e a t ly r e d u c e d t h e s e n s it iv i ty o f t h e N a c h a n n e l ( N o d a e t a l . 1989) .A s f o r t h e c a r d i a c N a c h a n n e l, t h e a m i n o a c i d a t p o s i t io n 3 7 4 c o n t r o l l e d t h es en s i t iv i t y o f t h e ch an n e l t o TT X an d C d 2 ( S a t i n e t a l . 1992).

I n n o r m a l s k e le t al m u s c l e, o n l y m R N A e n c o d i n g T T X - s e n s i ti v e N a c h a n n e ls is e x p r e ss e d . H o w e v e r , e x p r es s io n o f m R N A e n c o d i n g t he T T X -r e s is t a n t N a c h a n n e l s i n c r e a s e d a f t e r d e n e r v a t i o n a n d d e c l i n e d a s r e i n n e r v a t i o na p p e a r e d ( Y a n g e t a l . 1 9 91 ). M o r e o v e r , a N a c h a n n e l s u b t y p e , m i c r o I, i s o l a te df r o m a d e n e r v a t e d r a t s k e l e t a l m u s c l e c D N A l i b r a r y , w a s n o t e x p r e s s e d i n b r a i no r h e a r t b u t w a s fo u n d a t lo w le v e l s i n n e o n a t a l s k e l e t a l m u s c l e a n d i n c r e a s e d t om a x i m u m l e v el s i n a d u l t t i ss u e , p a r a ll e li n g t h e e x p r e s s i o n o f T F X - s e n s i t i v e N a c h a n n e l s ( T r i m m e r e t a L 1 99 0) . S u r p r is i n g ly , d e n e r v a t i o n o f ad u l t m u s c l e w asa l s o fo l l o w e d b y a r i s e i n m i c r o I m R N A , a t a t i m e w h e n T l ~ X - r e si s ta n t N a c h a n n e ls r e a p p e a r . F u r t h e r m o r e , S k M 2 m R N A , a n m R N A e n c o d i n g t h e c~-s u b u n i t o f t h e T T X - r e s i s ta n t N a c h a n n e l i n d e n e r v a t e d r a t s k e l e t a l m u s c l e , w a sn o t d e t e c t a b l e i n n o r m a l l y i n n e r v a t e d a d u l t s k e l e t a l m u s c l e b u t w a s a b u n d a n t i nr a t c a r d i a c m u s c l e ( K a l l e n e t a L 1 99 0). T h e S k M 2 m R N A l e v e l w a s h ig h e s t ine a r ly d e v e l o p m e n t , w h e n T T X - r e s i s ta n t N a c h a n n e ls p r e d o m i n a t e , b u t d e c l i n e dr a p i d l y w i t h t h e a g e o f t h e a n i m a l . T h e c a r d i a c T T X - r e s i s t a n t N a c h a n n e l s h a v eb e e n p r o v e d t o b e i d e n t i c a l t o t h o s e i n d e n e r v a t e d s k e l e t a l m u s c l e s ( K a l l e n e t a l .1990 ; Whi t e e t a l . 1 9 9 1 ). I t i s p u zz l i n g t h a t N a ch an n e l s ex p r e s s ed i n X e n o p u so o c y t e s b y i nj ec ti ng m R N A f r o m m a m m a l ia n h e a r t c e lls , w h i ch a r e c o n s i d e r a b l yr e s is t a n t t o T T X , b e c a m e h i g h ly s e n si ti v e to T T X ( S u t t o n e t a L 1 9 8 8 ) an d t h a tt r an s f ec t i o n o f th e r a s o n c o g e n e i n t o a c e l l l i n e d e r i v e d f r o m a m o u s e a n t e r i o rp i t u it a r y t u m o r c o n v e r t e d T T X - r e s i s t a n t N a c h a n n e l s t o T T X - s e n s i t iv e o n e s( F l a m m e t a L 1990).

F U N C T I O N A L S I G N I F IC A N C E O F T H E I T X - R E S I S T A N T N a C H N N E L

O g a t a a n d T a t e b a y a s h i ( 19 9 2 c, 1 9 93 ) p r o p o s e d t h a t t h e s l o w i n a c ti v a ti o np r o c e s s o f T T X - r e s i s t a n t N a c h a n n e l s p l a y s a n i m p o r t a n t r o l e i n d e c i d i n gce l l u l a r f u n c t i o n , i . e ., t h e r eg u l a t i o n o f ce l l u la r l o n g - t e r m ex c i t ab i l i ty s u ch a s fi ri n gp a t t e r n a n d a d a p t a t i o n . E l l io t t a n d E l l io t t ( 19 9 3 ) s u g g e s t th a t T T X - r e s i s t a n t N a ch an n e l s r ap i d l y r eco v e r f r o m i n ac t i v a t i o n an d a r e s u i t ed t o f i r i n g l o n g t r a i n s o fac t i o n p o t en t i a l s i n r e s p o n s e t o a d ep o l a r i z i n g s t i mu l u s . Th u s , d i s t in c t t y p es o fN a c h a n n e l s a r e s u g g e s t e d t o c o n t r i b u t e t o d i f f e re n t t y p e s o f s e n s o r y i n f o r m a -t io n . W e s h o u l d , h o w e v e r , c o n s i d e r t h a t th e D R G c e ll is u n i p o l a r ( o r p s e u d o -u n i p o l a r ) a n d t h e c e ll b o d y d o e s n o t d i r e c t ly t a k e p a r t i n i m p u l s e c o n d u c t i o n .

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TTX-Resi stant Na Channels 235

I n d e e d , N a c h a n n e ls o f b o t h A a n d C n e r v e f ib e rs o f m o u s e D R G c e ll s h a v eb e e n s h o w n t o b e s e n s i t i v e t o T T X a n d s u i t a b l e f o r r a p i d i m p u l s e c o n d u c t i o n( Y o s h i d a a n d M a t s u d a , 1 9 7 9 ; Y a m a g u c h i , 1 9 9 2 ) .

C o m p a r i so n o f C N S a n d P N S N e u r o n sD i f f e r e n c e s in i o n ic m e c h a n i s m s o f a c ti o n p o t e n t i a ls a r e o b s e r v e d i n C N S

a n d P N S n e u r o n s . A c t i o n p o t e n t i a ls o f s p in a l c o r d n e u r o n s a r e d e p e n d e n te x c l u s iv e l y o n T T X - s e n s i ti v e N a c h a n n e l s , w h e r e a s T T X - r e s i s t a n t N a a n d C a 2c h a n n e l s a l s o c o n t r i b u t e t o a c ti o n p o t e n ti a l s in D R G c e ll s o f t h e m o u s e ( R a n s o ma n d H o l z , 1 9 77 ; H e y e r a n d M a c D o n a l d , 1 98 2). T h e r e f o r e , C a 2+ e n t e r s d u r i n g a na c t io n p o t e n t i a l i n D R G c e ll s a n d t h e s l o w t i m e c o u r s e o f T I X - r e s i s t a n t N a ch an n e l s a i d s i n f lu x o f C a 2 b y ca u s i n g a s u s t a i n e d d ep o l a r i za t i o n o f t h em e m b r a n e ( F e d u l o v a e t a l . 1 99 1). I n t e r e s t i n g l y , i n fl u x o f C a 2 h as b ee n s h o w n t ob e t o x i c t o C N S n e u r o n s b u t n o t t o P N S n e u r o n s ( Y o s h i d a e t a l . 1979) . Indeed ,t h e c o n d u c t a n c e o f N a c h a n n e ls w a s r e d u c e d b y i n t ra c e l lu l a r C a 2 ( H e y e r a n dM a c D o n a l d , 1 9 82 ). I t i s p o s s i b l e th a t t h e f u n c t i o n o f C N S n e u r o n s is s o d e l ic a t e l yco n t r o l l ed b y t h e i n t r ace l lu l a r co n cen t r a t i o n o f C a 2 t h a t i t is n eces s a r y t o av o i dex p o s u r e t o a l a r g e C a 2 i n fl u x f r o m o u t s i d e .

K i n e ti c s o f T T X - r e s i s ta n t N a c h a n ne l s h a v e b e e n d e m o n s t r a t e d t o b es l o w e r th a n t h o s e o f T T X - s e n si ti v e o n e s i n P N S n e u r o n s ( K o s t y u k e t a l . 1981;F e d u l o v a e t a l . 1 9 9 1 ; O g a t a an d Ta t eb ay as h i , 1 9 9 3 ; E l l i o t t an d E l l i o t t , 1 9 9 3 ) .H o w e v e r , m o s t N a c h a n n e l s i n ce ll b o d i e s o f C N S n e u r o n s a r e s e n s i ti v e t o T T X ,an d b o t h TT X - s e n s i t i v e an d T - l X - r e s i s tan t N a ch an n e l s ex h i b i t s i m i l a r k i n e ti c si n C N S n e u r o n s ( O g a t a a n d T a t e b a y a s h i , 1 9 9 2 c ; W h i t e e t a L 1 9 9 3 ) . I t h a s b eens p e c u l a t e d t h a t t h e k i n e ti c s o f a ll N a c h a n n e ls m u s t b e f a s t in C N S n e u r o n s f o rr ap i d an d e f f ec t i v e i n f o r m a t i o n p r o ces s i n g .T h e a d v a n t a g e s o f u s in g N a c h a n n e l s , e s p e c i a ll y T T X - s e n s i ti v e N a c h a n n e l s w h i c h h a v e a h i g h s i n g l e - c h a n n e l c o n d u c t a n c e a n d t h e f a s t e s t k i n e t i c sa m o n g i o n ic c h a n n e ls , f o r p h y l o g e n e t i c a l ly h i g h e r a n i m a l s a n d o n t o g e n e t i c a l l yd i f f e r en t i a t ed ce l ls a r e ( 1 ) f a s t co n d u c t i o n o f ac t i o n p o t en t i a l s a l o n g t h e ax o n , ( 2 )r a p i d p r o c e s s i n g o f i n f o r m a t i o n in n e u r o n s , a n d ( 3) a c c u r a t e a n d u n d i s t u r b e di n f o r m a t i o n p r o c e s s in g , b e c a u s e u s i n g N a s e p a r a t e s t h e p r o c e s s f r o m o t h e r m a n yc e l lu l a r f u n c t io n s w h i c h a r e d e p e n d e n t o n C a 2 . T T X - c a r r y i n g a n i m a ls s e e m t ou t i l i z e t h e t o x i n f o r p r o t e c t i n g t h e m s e l v e s f r o m e n e m i e s w h o s e m o v e m e n t s a r ef a s t e r b u t a r e d e p e n d e n t o n t h e a c t i v it y o f T T X - s e n s i t i v e N a c h a n n e ls .

N a C H A N N E L S D U R I N G D E V E L O P M E N T

I o n i c c h a n n e l s c h a n g e t h e i r n u m b e r s a n d p r o p e r t i e s d u r i n g d e v e l o p m e n t . I nt h e m o u s e , a c t io n p o t e n t ia l s c o u l d b e e v o k e d i n o v a r ia n a n d o v u l a t e d o o c y t e sw h e n i n a c t iv a t io n o f io n i c c h a n n e l s w a s r e m o v e d b y h y p e r p o l a r iz i n g t h em e m b r a n e p o t e n t i a l ( O k a m o t o e t a l . 1977 ; Yosh ida 1982 , 1983a , b ) . Th i s ac t ion

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36 Yoshida

potential was dependent on the opening of Ca -* channels which were insensitiveto TTX. No Na channels were detected. However, Na could pass throughoocyte Ca 2 channels when the extracellular Ca 2 concentrat ion was very low.Similarly, action potentials were exclusively dependent on the activity of Ca 2channels in cleaving embryos of the mouse and the density of Ca 2 channels wasreduced with development (Yoshida, 1983b, 1985, 1986b; Mitani, 1985).

The next stages of development have been studied in dorsal root ganglion(DRG ) cells from fetal to adult mice. The cell body of the fetal mouse D RG hada high proportion of TTX-resistant Na channels. Na channels increased innumber and sensitivity to TTX with the maturation of cells and the myelination ofaxons in the adult mouse (Matsuda e t a l . 1978; Yoshida e t a I . 1978, 1980a). Thisreplacement of TI X-resis tant Na channels by TFX-sensitive ones has beenreported in various other tissues such as skeletal muscle cells (Gonoi e t a l . 1985;Weiss and Horn, 1986), human neuroblastoma cells (Weiss and Sidell, 1991), andmouse brain (Unsworth and Hafemann, 1975). In contrast, Ca2 channelsacquired sensitivity to an organic Ca -,+ channel blocker diltiazem (Yoshida,1986a, b) while reducing their number during development from oocytes to adults(Matsuda e t a l . 1978; Yoshida e t a l . 1978, 1980a; Yoshida, 1983b, 1985; Mitani,1985; Kostyuk e t a l . 1993). Interestingly, TTX-resistant Na + channels of rat DR Gcells were inhibited by Ca 2 channel blockers, but the selectivity of these channelsdid not significantly differ from TFX-sensitive Na channels (Kostyuk e t a l . 1981;Roy and Narahashi, 1992), indicating that the T-FX-resistant Na channelcombines some features of Ca 2 and Na channels. Also, TFX-resistant Na channels in embryonic chick heart share some characteristics with the Ca 2+channel and the T-FX-sensitive Na channel (Bkaily e t a l . 1991). In contrast,Ca 2 conducting Na + channels have been reported in rat hippocampal neurons(Akaike and Takahashi, 1992; Takahashi e t a l . 1992). This Ca 2 conducting Na channel was sensitive to TTX and blocked by Ca : antagonists. Also, theTTX-resistant Na channel in entorhinal cortex neurons of the rat was suppressedby inorganic Ca 2 channel blockers, although the channel kinetics were similar tothose of a T-I X-sensitive Na current (White e t a L 1993).

In contrast to the cell body, the axon of mouse DRG produced conductingspikes chiefly dependent on TTX-sensitive Na + channels (Yoshida and Matsuda,1979). Hence it is possible that (1) the axon develops faster than the cell bodywith respect to spike-generation mechanisms because impulse conduction occursalong the axon and the cell body does not directly take part in it, and (2) Na channels in the cell body whose axon participates in primitive information (e.g.,conveying dull pain along the unmyelinated C fiber) remains undifferentiated, i.e.,somatic Na + channels are resistant to TTX and slow in time course.

L o w e r n i m a l s

Ionic channel types appear to be different in lower animals. Na + channels arepresent as early as in oocytes in the tunicate (Okamoto e t a l . 1976), starfish

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TFX-Resistant Na Channels 237Miyazaki e t a l . 1975), flatworm Miyazawa e t a l . 1987), and frog Schlichter,1983). These Na channels are all resistant to TTX. Also, in the blastomere of

the tunicate embryo, Na channels in differentiating neurons were highly resistantto TTX and the channel subtypes showed distinct kinetic properties at thesingle-channel level Okamura and Shidara, 1990).

M a j o r D e v e l o p m e n t a l o u r s es o f I o n ic h a n n e lsIn summary, deve lopmental changes of major ionic channels contributing to

action potentials under physiological conditions seems to take either one of thetwo courses. One course is that the inward current, which is responsible forgenerat ing action potentials, is initially dependent on Ca 2+ channels, then on Na +and Ca 2+ channels, and finally, on Na + channels, as was observed in mouse DR Gcells Matsuda e t a L 1978; Yoshida e t a L 1978, 1980a; Yoshida, 1985). Therehave been similar findings in chick Kano, 1975) and rat Kidokoro , 1975) skeletalmuscle cells and in amphibian neurons Spitzer, 1979). The alternat ive course isthat the number of Ca 2+ channels increases, whereas that of the Na channelsdecreases with age as seen in phylogenetically lower animals, e.g., tunicate musclecells Miyazaki e t a l . 1974).

EVOLUT ION OF Na CHANNELS

E v o l u t i o n o f I o n i c h a n n e l s S e e n a t t h e G e n e L e v e lThe homology of amino acid sequences of Na channels in various tissues

and in animals is high, suggesting that these Na channels are derived from thesame gene. Moreover, the structures of voltage-gated Ca 2 and K channels havebeen shown to be similar to the voltage-gated Na channel Catterall, 1988;Trimmer and Agnew, 1989; Neumcke, 1990; Mandel, 1992). This suggeststherefore, that these voltage-gated channels, i.e., Na , Ca 2 and K channels, alldifferentiated from the same ancestor gene encoding a prototype of thesechannels during development. It is to be noted that replacement of only oneamino acid of the Na channel structure has been demonstrated to change itsionic selectivity such that not only Na but also K and Ca 2 can easily passthrough the Na channel, i.e., the Na channel became like the nonselectivecation channel. In addition, when two amino acids were replaced, the Na channel became more permeable to Ca 2 than to Na Heinemann e t a l . 1992).These observations indicate that Na , Ca 2, and K channels utilize a verysimilar structure for their base, suggesting that these types of channels derivedfrom a prototype channel. Furthermore, it should be noted that TTX-resistantNa channels resemble Ca 2 channels in their slow time course, TTX insensitivityand sensitivity to inorganic divalent cations, and organic Ca 2 channel blockersKostyuk e t a L 1981; Roy and Narahashi, 1992). These findings suggest a close

developmental relationship between Na and Ca2 channels.

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38

% lS t0m .= l )Fig. 1. Sche ma ticdiagram o f presumed evolution of N a channels.Na +, C a2+, and nonselective cation channels probably derived fromthe same stem channel. Ca2 channels in the circle and squaredenote that Ca 2 channe ls are d iverse in type in various typ es ofcells and animal species. In contrast, Na channels are much thesame. Therefore, the primitive Na channel must have appearedlate r tha n the primitive C a ~- ch an ne l. Both Tl'X-sensitive andTTX -insensitive Na channels, which originated from the primitiveNa channel, are present in the tissu es named in the diagram.Dashed line s indicate the speculative connections. CNS and PNS,central and peripheral nervous systems.

Yoshida

E v o l u t i o n a l H y p o t h e s i s o f I o n i c C h a n n e l sD e v e l o p m e n t o f io n ic c h a n n e ls s h o u l d b e c o n s i d e r e d n o t o n l y o n t o g e n e t ic a l ly

b u t a l s o p h y l o g e n e t i c a l l y , a n d h y p o t h e s e s o n e v o l u t i o n o f c h a n n e l s h a v e b e e np r o p o s e d ( s ee r e v i e w b y H i l le , 1 99 2 ). I h a v e p r o p o s e d h e r e a n e v o l u t i o n a lh y p o t h e s i s o f i o n ic c h a n n e l s o n t h e b a s i s o f o u r r e s u l t s o n t h e m o u s e a n d t h e d a t ao b t a i n e d b y o t h e r r e s e a r c h e r s ( s e e F ig . 1) .

V o l t a g e - g a t e d C a 2 c h a n n e l s a r e p r e s e n t i n v a r i o u s t y p e s o f c el ls i n m a n ya n i m a l s , e s p e c i a l ly a t e a r l y s t a g e s o f d e v e l o p m e n t . A l s o , p r o p e r t i e s o f C a 2c h a n n e l s a r e c o n s i d e r a b l y d i v e r s e b e t w e e n c e ll ty p e s a n d a n i m a l s p e c i es . T h u s ,C a 2 c h a n n e l s m a y b e a s s u m e d t o b e b o t h o n t o g e n e t i c a l l y a n d p h y l o g e n e t i c a l l yo l d a n d t o h a v e u n d e r g o n e a l t e r a t io n s d u r i n g a l o n g p e r i o d o f e v o l u t i o n . I nc o n t r a s t , N a c h a n n e l s s e e m t o b e i n c o r p o r a t e d i n to m u s c l e s , n e u r o n s , g l i al c e ll se t c . a t la t e r s t a g e s o f o n t o g e n e s i s a n d p h y l o g e n e s i s ( F i g . 1) . T h e r e f o r e , t h ep r o p e r t i e s o f N a c h a n n e ls a r e m o r e c o n s e r v e d a m o n g d i ff e re n t t i s su e s a n da n i m a l s .

A s m e n t i o n e d e a r l i e r , T T X - s e n s i t i v e N a c h a n n e l s w i th f a s t k i n e ti c s a r em o r e f r e q u e n t l y o b s e r v e d i n d i f f e r e n ti a t e d c e ll s a n d in h i g h e r a n i m a l s . I f w e t a k et h e s e n s i ti v i t y o f N a c h a n n e l s t o T I ' X a s a n i n d i c a t io n o f a h i g h e r e v o l u t i o n a ls t a g e , w e c a n c o n c l u d e t h a t a x o n s a r e m o r e d e v e l o p e d t h a n c e ll b o d i e s o f

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l 'TX -R e s i s t an t Na + C ha n ne l s 239

neurons, CNS neurons more developed than PNS neurons, and skeletal musclecells more developed than cardiac and smooth muscle cells (Fig. 1). The fact thatNa channels in axons are resistant to ITX in lower animals such as snail (Wald,1972), clams and shellfish (Twarog e t a l . 1972) is a phylogenetical example of lessdeveloped Na channels.Ionic selectivity of channels should also be considered here. Ca 2 channelscould be made permeable to Na when the concentration of Ca2 was low(Yoshida, 1982, 1983a, 1985). In addit ion, TI'X-res istant Na channels could beinhibited by Ca2 channel blockers but the ionic selectivity was not significantlydifferent from that of TrX-sensitive Na channels (Kostyuk e t a L 1981; Roy andNarahashi, 1992; White e t a l . 1993). Hence TrX-resis tant Na channels combinefeatures of both Ca 2 and Na channels. Furthermore, Ca2 conducting Na channels which are sensitive to TI'X but are blocked by Ca 2 antagonists havebeen reported (Akaike and Takahashi, 1992; Takahashi e t a l . 1992).When ionic channels are seen from a molecular aspect, it is possible thatthere is a ste m channel from which Ca 2 and Na channels derived (Hille,1992). This stem channel may explain the evolution of ionic channels, i.e.,nonselective cation channels and primitive forms of Ca 2 and Na channels mayhave originated from stem channels (Fig. 1). As indicated by dashed lines in Fig.1, the evolutional interactions of these channels still remain to be elucidated.

CKNOWLEDGMENT S

I appreciate Dr. S. Macmillan for her suggestions on the manuscript,Professors M. Niwa, Y. Matsuda, K. Taniyama, and H. Oka for theirencouragement.

REFERENCE S

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