Na,K-ATPase: Reaction Mechanisms and Ion Translocating Steps

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 19

Na,K-ATPase: Reaction Mechanisms and Ion Translocating Steps PAUL DE WEER

Depanment of Physiology and Biophysics Washingion University School of Medicine St. Louis, Missouri

I.

INTRODUCTION

The purpose of t h i s c h a p t e r i s t o summarize b r i e f l y from t h e l i t e r a t u r e some selected f a c t s t h a t may have b e a r i n g on t h e q u e s t i o n : To what e x t e n t , and i n how much d e t a i l , can t h e " b i o p h y s i c a l " o b s e r v a t i o n s on t h e o p e r a t i o n of t h e sodium pump ( i . e . , i o n t r a n s p o r t and c u r r e n t measurements) be r e c o n c i l e d w i t h what i s known about t h e "biochemical" r e a c t i o n mechani s m of t h e Na,K-ATPase? T h i s s e l e c t i o n i s by n e c e s s i t y t o p i c a l s i n c e it i s mainly i n s p i r e d by t h e a r t i c l e s p r e s e n t e d d u r i n g t h i s meeting a t t h e s e s s i o n devoted t o t h e r e l a t i o n s h i p between enzyme mechanisms and i o n t r a n s l o c a t i o n . F l u x e s i n r e c o n s t i t u t e d liposomes w i l l n o t be reviewed a t l e n g t h h e r e s i n c e o t h e r s w i l l d i s c u s s them e l s e w h e r e i n t h i s volume.

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Copyright 0 1983 by Academic Reas, Inc. All rights of reproductionin any form mewed. ISBN 012-153319-0

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11.

DO CARDIOTONIC STEROIDS I N H I B I T Na,K-ATPase AND PUMP-MEDIATED FLUXES TO THE SAME EXTENT?

T h i s q u e s t i o n may appear s u r p r i s i n g , s i n c e t h e consensus i s t h a t c a r d i o t o n i c s t e r o i d s block pump f l u x e s by a r r e s t i n g t h e ATPase machinery. Y e t a c u r i ous o b s e r v a t i o n made on s q u i d g i a n t axon ( B r i n l e y and M u l l i n s , 1 9 6 8 ; Beaug6 and M u l l i n s , 1 9 7 6 ) had s u g g e s t e d t h a t s t r o p h a n t h i d i n , i n a d d i t i o n t o a r r e s t i n g t h e "norm a l " o p e r a t i o n o f t h e A T P a s e and i t s concomitant f l u x e s , could induce a n o v e l mode of i o n exchange through t h e pump. Consequently, k i n e t i c and s t o i c h i o metric c o n c l u s i o n s based on f l u x d i f f e r e n c e measurements might be i n e r r o r . R e c e n t e x p e r i m e n t s (Beaug6 and DiPolo, 1981a) show, however, t h a t t h e r e e x i s t s a strophanthidin-resistant, ATP-dependent sodium e f f l u x i n s q u i d axon which p r o b a b l y a c c o u n t s f o r t h e e a r l i e r observations.

111.

OVERALL PUMP STOICHIOMETRY

K i n e t i c o b s e r v a t i o n s a l o n e are n o t s u f f i c i e n t t o e s t a b l i s h s t o i c h i o m e t r i e s . I t may be g r a t i f y i n g t o find H i l l coefficients t h a t f i t one's expectations but, i f t h e y do n o t , c a u t i o n i s a d v i s e d when drawing conclus i o n s concerning t h e pump's s t o i c h i o m e t r y . A c t u a l f l u x and/or e l e c t r i c a l measurements m u s t be c a r r i e d o u t simultaneously w i t h ATP h y d r o l y s i s measurements. The commonly a c c e p t e d Na:K:ATP s t o i c h i o m e t r y of 3:2:1 f o r t h e "normal" mode of o p e r a t i o n of t h e sodium pump ( G a r rahan and Glynn, 1967d) remains unchallenged, and has r e c e i v e d s u p p o r t from experiments c a r r i e d o u t on p u r i f i e d Na,K-ATPase r e c o n s t i t u t e d i n t o liposomes (Goldin, 1977). The 3:2 s t o i c h i o m e t r y f o r Na:K exchange h a s rec e i v e d some s u p p o r t from i s o t o p e f l u x measurements i n o t h e r c e l l s , b u t it must be r e a l i z e d t h a t t h e operat i o n a l d e f i n i t i o n of "pump-mediated K+ f l u x " i s o f t e n a d i f f i c u l t m a t t e r f o r t h e f o l l o w i n g reason. I n every i n s t a n c e t e s t e d , i n c l u d i n g e r y t h r o c y t e s (Hoffman et al., 1 9 7 9 ) , t h e N a : K exchange pump has proved e l e c t r o g e n i c , and a r r e s t i n g t h e pump w i l l l e a d t o c e l l d e p o l a r i z a t i o n ; t h i s , i n t u r n , may modify p a s s i v e K+ i n f l u x i n a way determined by t h e r e c t i f y i n g p r o p e r t i e s of t h e c e l l memb r a n e ' s potassium conductance. A p e r h a p s more promising approach t o e s t a b l i s h i n g t h e Na:K s t o i c h i o m e t r y i s t h e

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s i m u l t a n e o u s measurement of e l e c t r o g e n i c pump c u r r e n t sodium e f f l u x (Cooke et a l . , 1 9 7 4 ) . Pump c u r r e n t can be c a l c u l a t e d i n d i r e c t l y from membrane pot e n t i a l changes and membrane conductance, o r measured d i r e c t l y a s t h e e x t r a c u r r e n t r e q u i r e d t o clamp memb r a n e p o t e n t i a l a t a g i v e n ( u s u a l l y r e s t i n g ) v a l u e when t h e pump i s e i t h e r s t i m u l a t e d o r i n h i b i t e d . Pump s t i m u l a t i o n h a s u s u a l l y been achieved i n t h e p a s t by p r e s s u r e - o r i o n t o p h o r e t i c i n j e c t i o n of sodium s a l t s b u t , u n l e s s t h e anion i s very c a r e f u l l y chosen, t h e r e i s a danger of a r t i f a c t s a r i s i n g from d i f f u s i o n potent i a l s c r e a t e d by t h e i n j e c t e d a n i o n ( f o r a c r i t i q u e , see D e Weer, 1 9 7 5 ) . N o such problem e x i s t s i f t h e pump c u r r e n t i s determined a s t h e clamp c u r r e n t r e q u i r e d t o compensate f o r sudden blockage of t h e pump ( D e Weer, 1 9 7 4 1 , provided t h e b l o c k i n g a g e n t ( e . g . , o u a b a i n ) h a s no e f f e c t on membrane conductance. The work of Nelson and Lederer ( t h i s volume) on t h e sodium pump of i n t e r n a l l y p e r f u s e d g i a n t b a r n a c l e muscle f i b e r s i l l u s t r a t e s t h i s procedure. A f e w y e a r s ago t h e n o t i o n a r o s e t h a t t h e pump's N a : K s t o i c h i o m e t r y might be v a r i a b l e depending on memb r a n e p o t e n t i a l and i o n i c c o n d i t i o n s . C o n s i d e r i n g t h e t e c h n i c a l d i f f i c u l t y of measuring s t o i c h i o m e t r i e s acc u r a t e l y , t h e i s s u e s h o u l d be c o n s i d e r e d u n r e s o l v e d . Even w i t h v e r y a c c u r a t e measurements of t r a c e r N a f l u x and pump c u r r e n t , it may b e v e r y d i f f i c u l t t o d i s t i n g u i s h between genuine " v a r i a b l e s t o i c h i o m e t r y " and t h e s i m u l t a n e o u s o c c u r r e n c e of v a r i a b l e p r o p o r t i o n s o f , s a y , e l e c t r o g e n i c N a : K exchange and (presumably e l e c t r o n e u t r a l ) Na:Na exchange. Another l i n e of r e s e a r c h w i t h b e a r i n g on t h e probl e m of i o n t r a n s l o c a t i o n s t o i c h i o m e t r y i s d i r e c t d e t e r m i n a t i o n of t h e number of i o n s bound t o t h e enzyme p e r c a t a l y t i c u n i t . Most s t u d i e s so f a r have examined e q u i l i b r i u m b i n d i n g s t o i c h i o m e t r i e s ( s e e H a s t i n g s and Skou, 1 9 8 0 , f o r a r e c e n t e x a m p l e ) , b u t bound i o n s w i l l n o t n e c e s s a r i l y be t r a n s l o c a t e d d u r i n g pump t u r n o v e r . The a t t e m p t s of Glynn and co-workers (Beauge and Glynn, 1979a; Glynn and R i c h a r d s , t h i s volume) t o d e t e r m i n e t h e number of "occluded" i o n s bound p e r t r a n s p o r t c y c l e , on t h e o t h e r hand, may h e l p e s t a b l i s h t h e a c t u a l t r a n s l o c a t i o n s t o i c h i o m e t r y s i n c e occluded ( i. e . , s l o w l y r e l e a s a b l e ) i o n s presumably a r e "on t h e i r way" from one s i d e of t h e membrane t o t h e o t h e r .

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IV.

OVERALL PUMP K I N E T I C S

I t i s f a i r t o say t h a t the Albers-Post (Albers, 1 9 6 7 ; P o s t e t a l . , 1 9 6 9 ) k i n e t i c model o f t h e N a , K A T P a s e , w i t h minor v a r i a t i o n s , i s w i d e l y a c c e p t e d (see Glynn et a l . , 1979, f o r a n updated v e r s i o n ) . T h a t i s , t h e A T P a s e enzyme a p p e a r s t o be c o n s e c u t i v e l y phosp h o r y l a t e d by ATP i n a s o d i u m - r e q u i r i n g s t e p and dephosp h o r y l a t e d i n a p o t a s s i u m - c a t a l y z e d s t e p . I t would t h e n seem l o g i c a l t o e x p e c t a c o n s e c u t i v e mechanism f o r t h e i o n t r a n s l o c a t i o n c y c l e a s w e l l , w i t h sodium e x p o r t a l -

t e r n a t i n g w i t h p o t a s s i u m u p t a k e . I n f a c t , T r e v o r Shawls (1954) c o n s e c u t i v e model f o r t h e sodium pump l o n g a n t e d a t e s t h e b i o c h e m i c a l model f o r t h e N a , K - A T P a s e , and one may wonder t o what e x t e n t Shawls model h a s guided t h e b i o c h e m i s t s ' e x p e r i m e n t s . Much i n t e r e s t w a s a r o u s e d , ion transport t h e r e f o r e , when c a r e f u l e v a l u a t i o n of t h e k i n e t i c s i n r e d b l o o d c e l l s (Hoffman and T o s t e s o n , 1 9 7 1 ; Garay and Garrahan, 1973; C h i p p e r f i e l d and W h i t t a m , 1976; Sachs, 1 9 7 7 ) suggested a simultaneous t r a n s p o r t model, i n which t h e pump enzyme t u r n s o v e r w i t h b o t h sodium and p o t a s s i u m t r a n s l o c a t i o n s i t e s o c c u p i e d , r a t h e r t h a n a c o n s e c u t i v e model. I n g e n i o u s models c a p a b l e of s a t i s f y i n g b o t h t h e a p p a r e n t c o n s e c u t i v e k i n e t i c s of phosphorylation/dephosphorylation and t h e a p p a r e n t s i multaneous f l u x k i n e t i c s were proposed by S t e i n e t a l . (1973) and by Repke and Schbn (1973; see a l s o S t e i n , 1 9 7 9 ) . Such models r e l i e d on t h e t h e n p r e v a i l i n g not i o n t h a t t h e pump enzyme w a s a dimer of c a t a l y t i c subu n i t s ; t h e i n d i v i d u a l s u b u n i t s were assumed t o undergo phosphorylation/dephosphorylation s e q u e n c e s , b u t o u t o f phase s o t h a t p h o s p h o r y l a t i o n of one s u b u n i t i s concom i t a n t w i t h d e p h o s p h o r y l a t i o n of t h e o t h e r . J u s t when i t appeared t h a t b i o c h e m i c a l k i n e t i c s and f l u x k i n e t i c s c o u l d b e t h u s r e c o n c i l e d , Sachs (1979) reexamined t h e same d a t a a n a l y z e d p r e v i o u s l y ( S a c h s , 1977) and conc l u d e d t h a t , i f allowance i s made f o r some "uncoupled" sodium e f f l u x , i . e . , e f f l u x o f sodium w i t h o u t accompanyi n g K e n t r y (Garrahan and Glynn, 1967b; Lew et a l . , 1973; Glynn and K a r l i s h , 1 9 7 6 1 , t h e f l u x k i n e t i c s can be i n t e r p r e t e d , a f t e r a l l , on t h e b a s i s of a s t r i c t l y c o n s e c u t i v e model. O t h e r s (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1981) have shown t h a t i t i s p e r f e c t l y p o s s i b l e t o a c c o u n t f o r t h e h i g h and low a f f i n i t y of t h e N a / K pump f o r ATP w i t h o u t r e s o r t i n g t o a d i m e r i c model. The p r o s p e c t s f o r a s i n g l e u n i t a r y model t h a t a c c o u n t s f o r t h e b i o c h e m i c a l s t e p s as w e l l a s t h e known f l u x modes ( N a : K exchange; ADP-dependent N a : N a exchange; Pi-dependent K:K exchange, e t c . ) , look b r i g h t e r again.

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A s i f on c u e , c e r t a i n s t r u c t u r a l f i n d i n g s (see elsewhere i n t h i s volume) a r e b e g i n n i n g t o c a s t d o u b t on t h e n o t i o n t h a t t h e o p e r a t i o n a l u n i t o f t h e sodium pump i s an A T P a s e dimer.

V.

TRANSPORT CORRELATES O F Na-ATPase

O u a b a i n - s e n s i t i v e Na,K-ATPase w i l l h y d r o l y z e ATP w i t h o u t t h e b e n e f i t o f p o t a s s i u m i o n s (Czerwinski et a l . , 1967; Neufeld and Levy, 1969; B l o s t e i n , 1 9 7 0 ) . Unlike t h e Na,K-stimulated a c t i v i t y , which h a s b o t h a low and a h i g h a f f i n i t y f o r ATP, t h e N a - s t i m u l a t e d act i v i t y d i s p l a y s a s i n g l e , h i g h - a f f i n i t y (<1 P M ) K O , ~ f o r ATP. The sodium dependence of ATP h y d r o l y s i s i s q u i t e complex: The a c t i v i t y c u r v e rises r a p i d l y a t v e r y l o w N a c o n c e n t r a t i o n s , ~ 0 . 5< 1 m~ ( P o s t et a l . , 1972; M&rdh and P o s t , 1977; Beaug6 and Glynn, 197933; F l a s h n e r and Robinson, 19791, r e a c h e s a p l a t e a u around 5-10 mM N a ( P o s t et a l . , 1972; M&dh and P o s t , 1 9 7 7 ; Beauge and Glynn, 1979b1, t h e n c o n t i n u e s t o r i s e w i t h i n c r e a s i n g sodium c o n c e n t r a t i o n s . I t h a s been p o i n t e d o u t by Beauge and Glynn (1979b) t h a t a c u r v e w i t h a p l a t e a u o r i n f l e c t i o n p o i n t r e f l e c t s a t least t h r e e k i n d s of sites: h i g h - a f f i n i t y s t i m u l a t i o n , medium-affinity i n h i b i t i o n , and l o w - a f f i n i t y s e c o n d a r y s t i m u l a t i o n . A t low ATP l e v e l s , p o t a s s i u m i n h i b i t s ATP h y d r o l y s i s (Kanazawa et al., 1967; Czerwinski e t a l . , 1967) and t h i s i s m o s t s a t i s f a c t o r i l y a c c o u n t e d f o r by t h e model of P o s t e t a l . (1972) i n which t h e enzyme becomes t r a p p e d i n a n " o c c l u d e d p o t a s s i u m " form. I n t r a c e l l u l a r sodium i s r e q u i r e d f o r enzyme phosp h o r y l a t i o n , and t h e sodium a f f i n i t y f o r t h i s a c t i v i t y i s v e r y h i g h ( B l o s t e i n , 1 9 7 9 ) . T h i s , and o t h e r e v i d e n c e s u c h as K a p l a n ' s f i n d i n g , r e p o r t e d i n t h i s volume, t h a t ADP:ATP exchange i n e r y t h r o c y t e s s a t u r a t e s a t 2 mM N a i l makes i t v e r y l i k e l y t h a t t h e i n i t i a l , h i g h - a f f i n i t y s t i m u l a t i o n o f N a - A T P a s e a c t i v i t y r e p r e s e n t s an a c t i o n a t i n t r a c e l l u l a r sodium s i t e s . Granted t h a t i n t r a c e l l u l a r sodium s i t e s are s a t u r a t e d a t v e r y low "a], it i s c o n v e n i e n t t o examine t h e p o s s i b l e f l u x c o r r e l a t e s o f Na-ATPase under t h r e e e x p e r i m e n t a l c o n d i t i o n s : i n t h e a b s e n c e of e x t e r n a l sodium, and i n t h e p r e s e n c e o f low and h i g h e x t e r n a l sodium c o n c e n t r a t i o n s . Pump-mediated ATP h y d r o l y s i s i n t h e t o t a l a b s e n c e of e x t e r n a l p o t a s s i u m o r sodium h a s been i d e n t i f i e d (Glynn and K a r l i s h , 1976) w i t h ';uncoupled" sodium e f f l u x (Garrahan and Glynn, 196733). Glynn and K a r l i s h

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showed t h a t t h i s e f f l u x h a s a v e r low K0.5 f o r ATP ( < 1P M ) , i s i n h i b i t e d a t low [ATPY by e x t e r n a l p o t a s sium, and t r a n s p o r t s two t o t h r e e N a i o n s p e r ATP s p l i t . I f Nao- and KO- independent sodium e f f l u x i s t r u l y "unc o u p l e d , " it s h o u l d be e l e c t r o g e n i c and have d e t e c t a b l e e f f e c t s on membrane p o t e n t i a l . T h i s q u e s t i o n i s examined i n t h e p r e s e n t volume by D i s s i n g and Hoffman. These a u t h o r s show t h a t , c o n t r a r y t o e x p e c t a t i o n , "uncoupled" sodium e f f l u x i s e l e c t r o n e u t r a l , presumably because a n i o n s accompany t h e sodium i o n s on t h e i r p a t h through t h e pump. A o u a b a i n - s e n s i t i v e t r a n s p o r t of s u l f a t e , which o n l y p a r t l y a c c o u n t s f o r t h e pump's elect r o n e u t r a l i t y , i s found i n t h e absence of c h l o r i d e . I t i s n o t immediately a p p a r e n t how t h i s unexpected new act i v i t y can be f i t i n t o t h e c u r r e n t model f o r t h e sodium pump, b u t t h e f i n d i n g i s r e m i n i s c e n t of t h a t by Baker ( 1 9 6 4 ) of an Nai-dependent, o u a b a i n - s e n s i t i v e l o s s of g l u t a m a t e and a s p a r t a t e from c r a b n e r v e b a t h e d i n Nafree, K-free s o l u t i o n . KO-independent sodium e f f l u x i s i n h i b i t e d by low e x t e r n a l sodium c o n c e n t r a t i o n s (Garrahan and Glynn, T h i s phenomenon h a s been c o n v i n c i n g l y 1967a) ( F ' m - . i d e n t i f i e d by Glynn and K a r l i s h (1976) a s r e f l e c t i n g t h e i n h i b i t o r y a c t i o n of l o w "a] ( ~ m 5 ) on Na-ATPase a c t i v i t y r e s p o n s i b l e f o r t h e p l a t e a u i n t h e sodium a c t i v a t i o n c u r v e mentioned e a r l i e r . When Na-ATPase a c t i v i t y was p l o t t e d a g a i n s t e x t e r n a l "a] ( F i g . 1), o n l y i n h i b i t i o n ( 0 - 5 m ) followed by s t i m u l a t i o n ( > 5 mM) w a s s e e n (Glynn and K a r l i s h , 1 9 7 6 ; L e e and B l o s t e i n , 1 9 8 0 1 , confirming the notion t h a t t h e high-affinity stimulation by N a of Na-ATPase a c t i v i t y i n unsided p r e p a r a t i o n s ref l e c t s b i n d i n g of Na+ t o i n t e r n a l s i t e s . How Nao might i n h i b i t Na-ATPase (and concomitant sodium e f f l u x ) i s n o t immediately a p p a r e n t from t h e c u r r e n t r e a c t i o n schemes f o r Na,K-ATPase. Beau96 and Glynn (1979b) found t h a t 5 m~ Na s t r o n g l y i n h i b i t s t h e r a t e of dephosphoryl a t i o n of p h o s p h o r y l a t e d p i g kidney A T P a s e i n K-free media ( F i g . 1 1 , and have proposed t h i s as t h e mechanism whereby low [Na] i n h i b i t s Na-ATPase. High e x t e r n a l sodium l e v e l s , i n t h e nominal absence of potassium a n d P , promote pump-mediated Na:Na exchange --a s w e l l a s ATP h y d r o l y s i s ( F i g . 1) ( L e e and B l o s t e i n , 1 9 8 0 ; see a l s o B l o s t e i n i n t h i s volume). I t i s very l i k e l y t h a t t h i s l a t t e r a c t i v i t y r e p r e s e n t s t h e ( > 5 m ) arm of t h e Na-ATPase a c t i v a t i o n c u r v e . high-"a] What i s less c l e a r i s t h e p r e c i s e mechanism of t h i s ATP-hydrolyzing Na:Na exchanging mode of o p e r a t i o n of t h e sodium pump. Sodium could be pumped inward a s a congener of K + , f o l l o w i n g t h e u s u a l E2-P -+ E 2 + P i p a t h way; one would t h e n e x p e c t a 3:2 0utward:inward s t o l -

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NA-ATPASE (1) ADP:ATP EXCHANGE (2) NA EFFLUX; ADP PRESENT ( 3 )

E-P

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HYDROLYSIS

RATE (UNSIDED)

(6)

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EXTRACELLULAR SODIUM F i g . 1 . S c h e m a t i c summary o f the e f f e c t o f external s o d i u m ions on v a r i o u s a c t i v i t i e s c a t a l y z e d b y the Na,K-ATPase. T h e numbers i n p a r e n t h e s e s r e f e r t o t h e f o l l o w i n g l i t e r a t u r e s o u r c e s : ( 1 ) G l y n n and K a r l i s h , 1 9 7 6 ; ( 2 ) K a p l a n and H o l l i s , 1 9 8 0 ; ( 3 ) Garr a h a n and G l y n n , 1 9 6 7 a ; (4) L e e and B l o s t e i n , 1 9 8 0 ; (5) S a c h s , 1 9 7 2 a ; ( 6 ) Beau96 and G l y n n , 1 9 7 9 b . T h e sole p u r p o s e of the g r a p h i s t o c o m p a r e the s h a p e s o f the c u r v e s , not the a b s o l u t e m a g n i tudes. T h e f i r s t four a c t i v i t i e s a r e i n i t i a l l y i n h i b i t e d w i t h h i g h a f f i n i t y , and then s t i m u l a t e d w i t h l o w a f f i n i t y and w i t h o u t evidence o f saturation. T h e next t w o a c t i v i t i e s a r e not i n h i b i t e d , o n l y s t i m u l a t e d b y Na,. T h e l a s t a c t i v i t y (E-P b r e a k d o w n ) i s i n i t i a l l y s t r o n g l y i n h i b i t e d , then m o d e r a t e l y s t i m u l a t e d .

c h i o m e t r y . A l t e r n a t i v e l y , N a : N a exchange i n t h e abs e n c e of ADP c o u l d p r o c e e d v i a t h e E l - P pathway f o r ADP-ATP exchange, b u t w i t h s u b s t i t u t i n g € o r ADP;

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such mechanism would c a l l f o r an e l e c t r o n e u t r a l N a : N a exchange. S t i l l o t h e r r e t u r n pathways can be e n v i s i o n e d . W e w i l l r e t u r n t o t h i s q u e s t i o n when examining t h e ADP:ATP exchange phenomenon. A contribution t o t h e problem of t h e o u t : i n s t o i c h i o m e t r y of N a : N a exchange i s provided by Forgac and Chin ( t h i s volume). I t a p p e a r s t h a t t h e pump enzyme, r e c o n s t i t u t e d i n t o l i p o s o m e s , can g e n e r a t e a membrane p o t e n t i a l when pumping sodium i n t h e absence of K + , s u g g e s t i n g an o u t : i n N a : N a s t o i c h i o m e t r y g r e a t e r t h a n u n i t y . Moreover, t h e ( n e t t r a n s p o r t ) : (ATP h y d r o l y s i s ) s t o i c h i o m e t r y d e c l i n e d with i n c r e a s i n g N a c o n c e n t r a t i o n , s u g g e s t i n g t h a t , as "a] i s i n c r e a s e d , t h e sodium t r a n s p o r t mode s h i f t s from an e l e c t r o g e n i c mechanism ("uncoupled" and/or " N a : K-like"?) toward an e l e c t r o n e u t r a l one ( " c o n v e n t i o n a l " N a : N a exchange?) Whereas i n h i b i t i o n of Na-ATPase i n e r y t h r o c y t e s c o u l d be r a t i o n a l i z e d as r e s u l t i n g from a Na-induced r e d u c t i o n of E-P breakdown r a t e , a c o n v e r s e mechanism c a n n o t e a s i l y be invoked i n t h e case of h i g h [ N a ] , s i n c e E-P breakdown i s enhanced no more t h a n 2 - f o l d between 5 and 150 m N a ( P o s t et a l . , 1972; Beau96 and Glynn, 1 9 7 9 b ) , whereas N a e f f l u x and Na-ATPase are s t i m u l a t e d many-fold ( F i g . 1 ) . I t must b e remembered, however, t h a t E-P breakdown r a t e s were determined on u n s i d e d p r e p a r a t i o n s from p i g o r g u i n e a p i g k i d n e y , a t a much lower temperat u r e , and t h a t t h e p r e c i s e dependence o f s t e a d y - s t a t e E-P l e v e l s on [Na] w a s unknown. In sided erythrocyte t h a t high p r e p a r a t i o n s , L e e and B l o s t e i n (1980)nd [Nal0, i f a n y t h i n g , d e c r e a s e d s t e a d y - s t a t e E-P l e v e l s . I t i s s a f e t o conclude t h a t , i f t h e s i d e d n e s s o f t h e three-pron e d e f f e c t o f N a on ATP h y d r o l y s i s i n t h e abs e n c e of Kq i s u n d e r s t o o d , t h e p r e c i s e mechanisms i n volved are n o t .

.

VI. A.

Na:Na EXCHANGE AND ADP:ATP EXCHANGE C H A R A C T E R I S T I C S OF N a : N a E X C H A N G E

The sodium pump c a n engage i n N a : N a exchange (Caldw e l l et a l . , 1960; Garrahan and Glynn, 1967a; Baker et a l . , 1 9 6 9 ; D e Weer, 1 9 7 0 ; Kennedy and D e Weer, 1 9 7 6 ) . T h i s exchange a p p e a r s t o be one-for-one (Garrahan and Glynn , 1967a) and e l e c t r o n e u t r a l (Abercrombie and D e Weer, 1978)--not n e c e s s a r i l y a redundant s t a t e m e n t , c o n s i d e r i n g t h e f i n d i n g s of D i s s i n g and Hoffman ( t h i s volume). I t i s i n h i b i t e d by e x t e r n a l K+, half-maximal i n h i b i t i o n o c c u r r i n g a t t h e ~ 0 . 5f o r a c t i v a t i o n of po-

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tassium i n f l u x ( G a r r a h a n and Glynn, 1 9 6 7 ~ ) . I t req u i r e s ADP ( D e Weer, 1970; Glynn a n d Hoffman, 1971) a n d a l s o ATP ( C a v i e r e s a n d Glynn, 1979) , b u t t h e l a t t e r i s n o t h y d r o l y z e d ( G a r r a h a n a n d Glynn, 1 9 6 7 d ) . Kennedy et a l . (see e l s e w h e r e i n t h i s volume) f o u n d a ~ 0 . 5f o r ADP o f a b o u t 350 U M , i n d e p e n d e n t o f [ATP] i n t h e r a n g e 0.4-1 mM. The exchange i s s t i m u l a t e d by N a o w i t h l o w a f f i n i t y a n d by N a i w i t h h i g h a f f i n i t y ( K ~ 3 m ~ ) ,a n d i s n o t i n h i b i t e d by h i g h [ N a ] i (Garay a n d G a r r a h a n , 1973) a l t h o u g h e a r l i e r o b s e r v a t i o n s had s u g g e s t e d t h e contrary. I n t r a c e l l u l a r K s t i m u l a t e s Na:Na e x c h a n g e w i t h l o w a f f i n i t y (Garay and G a r r a h a n , 1973; S a c h s , 1 9 8 1 b ) . I n t r a c e l l u l a r Mg s t i m u l a t e s N a : N a e x c h a n g e w i t h h i g h a f f i n i t y ( ~ 0 . 5 9 U M ) and h a s a n i n h i b i t o r y e f f e c t a t c o n c e n t r a t i o n s above m i l l i m o l a r ( F l a t m a n and Lew, 1981; and see e l s e w h e r e i n t h i s volume). Oligomyc i n a l s o i n h i b i t s N a : N a e x c h a n g e ( G a r r a h a n and Glynn, 1967d) Q,

Q,

.

CHARACTERISTICS

B.

OF ADP : A T P EXCHANGE

I n view o f i t s r e q u i r e m e n t f o r ADP and o t h e r c h a r a c t e r i s t i c s , i t h a s l o n g a p p e a r e d a t t r a c t i v e t o assume ( D e Weer, 1970; Glynn a n d Hoffman, 1971) t h a t Na:Na e x c h a n g e i s t h e f l u x correlate o f sodium-dependent ADP:ATP e x c h a n g e , a well-documented r e a c t i o n c a t a l y z e d by t h e Na,K-ATPase (Skou, 1 9 6 0 ; Fahn et a l . , 1 9 6 6 a ; S t a h l , 1 9 6 8 ) , and which presumably r e p r e s e n t s t h e rev e r s a l of t h e f i r s t p a r t o f t h e A l b e r s - P o s t r e a c t i o n scheme ( A l b e r s , 1967; P o s t e t a l . , 1 9 6 9 ) : ATP N a i

ADP Nao +

J . J .

El

, -

L

E1-ATP-Na + E

I

.

1=P-ADP*Na

The ATP r e q u i r e m e n t of t h i s r e a c t i o n u n d o u b t e d l y ref l e c t s t h e need f o r p h o s p h o r y l a t e d enzyme i n t h e backward d i r e c t i o n . T r e a t m e n t o f t h e enzyme w i t h N-ethylmaleimide or o l i g o m y c i n , b o t h o f which i n h i b i t t h e ATPase a c t i v i t y , leaves t h e ADP:ATP e x c h a n g e a c t i v i t y unharmed o r e n h a n c e d (Fahn e t a l . , 1 9 6 6 a , b ; B l o s t e i n , 1970). Of p a r t i c u l a r i n t e r e s t f o r o u r p r e s e n t p u r p o s e are t h e e f f e c t s o f N a + , K + , a n d Mg2+ on t h e e x c h a n g e react i o n . Magnesium i o n s s t i m u l a t e a t l o w (micromolar f r e e ) and i n h i b i t t h e r e a c t i o n a t h i g h ( m i l l i m o l a r ) co n cen t rat i o n s (Fahn et al., 1966a; Robinson, 1976; Yamaguchi

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and Tonomura, 1 9 7 7 ; Beauge and Glynn, 197933). The act i v a t i n g e f f e c t undoubtedly r e f l e c t s t h e h i g h a f f i n i t y f o r Mg i n t h e enzyme p h o s p h o r y l a t i o n s t e p ( P o s t e t al., 1 9 6 5 ) , b u t t h e i n h i b i t o r y s t e p i s somewhat more c o n t r o v e r s i a l . O r i g i n a l l y , i t was t h o u g h t ( A l b e r s , 1 9 6 7 ; P o s t et al., 1 9 6 9 ) t h a t m i l l i m o l a r c o n c e n t r a t i o n s of Mg2+ drove t h e E l - P + E2-P e q u i l i b r i u m t o t h e r i g h t , b u t experiments of Klodos and Skou (1975) have r e n d e r e d t h a t t h e s i s u n t e n a b l e . Robinson ( 1 9 7 6 ) has proposed a l t e r n a t i v e schemes based on a d i m e r i c enzyme model. Within t h e framework of t h e Albers-Post scheme, howe v e r , it a p p e a r s (see D e Weer e t al. elsewhere i n t h i s volume) t h a t Mg2+ could s t i m u l a t e and i n h i b i t by a c t i n g a t a s i n g l e s i t e , provided ATP and Mg add t o t h e enzyme in t h a t o r d e r , a r e q u i r e m e n t f o r which t h e r e i s some e v i d e n c e m d h and P o s t , 1 9 7 7 ) :

El

ATP

Mg

J.

J.

, -

L

E1*ATP

E1*MgATP

* *

A t l o w c o n c e n t r a t i o n s , Mg w i l l a l l o w t h e f o r m a t i o n of phosphoenzyme. A t h i g h c o n c e n t r a t i o n s , Mg w i l l r e t a r d t h e r e l e a s e of l a b e l e d ATP formed from l a b e l e d ADP. A second i o n of i n t e r e s t i s potassium. Stimulat i o n o f ADP:ATP exchange by K has been d e s c r i b e d (Banerjee and Wong, 1 9 7 2 ; Robinson, 1 9 7 7 ) ; such stimu-

l a t i o n only o c c u r s i n t h e p r e s e n c e of sodium and under c o n d i t i o n s where Mg i s i n h i b i t o r y . A p o s s i b l e mechanism f o r t h i s a c t i o n i s t h e d i s p l a c e m e n t , by K , of t h e E1OATP + E z - A T P e q u i l i b r i u m t o t h e r i g h t ( K a r l i s h e t al., 1 9 7 8 ; Beauge and Glynn, 1980; Jldrgens e n and K a r l i s h , 1 9 8 0 ) , where ATP i s weakly bound and l a b e l , presumably, more r e a d i l y r e l e a s e d . A s f o r t h e e f f e c t s of sodium on ADP:ATP exchange, t h e s i t u a t i o n seems a s complex as t h a t f o r Na-ATPase. There i s a h i g h - a f f i n i t y ( ~ 0 . 5= 1 - 2 mM) s t i m u l a t i o n (Fahn e t al., 1966a; Wildes e t al., 1973; Beauge and Glynn, 1 9 7 9 b ) , followed by a d i p a t 5-10 mM Na, f o l l o w e d a g a i n by l o w - a f f i n i t y s t i m u l a t i o n (BeaugB and Glynn, 197933; Kaplan and H o l l i s , 1 9 8 0 ) . A curve t h a t rises, d i p s , and rises a g a i n c l e a r l y r e f l e c t s a t l e a s t t h r e e e f f e c t s : h i g h - a f f i n i t y s t i m u l a t i o n , medium-affinity i n h i b i t i o n , and l o w - a f f i n i t y s t i m u l a t i o n . The s i m i l a r i t y between t h e sodium a c t i v a t i o n c u r v e s f o r Na-ATPase and ADP:ATP exchange i s s t r i k i n g . Here a l s o , a t t e m p t s have been made t o a s s e s s t h e s i d e d n e s s of t h e t h r e e sodium e f f e c t s on ADP:ATP exchange j u s t mentioned.

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Attempts t o measure pump-mediated ADP:ATP exchange i n a " s i d e d " p r e p a r a t i o n such a s r e s e a l e d e r y t h r o c y t e g h o s t s have long been f r u s t r a t e d by t h e p r e s e n c e of o t h e r enzymes t h a t c a t a l y z e t h e same r e a c t i o n , and by h y d r o l y s i s o f ATP d u r i n g p r e p a r a t i o n of t h e c e l l s . These problems have been overcome w i t h more r e f i n e d methods f o r p r e p a r i n g " c l e a n " r e s e a l e d g h o s t s and t h e u s e of i n h i b i t o r s ( C a v i e r e s and Glynn, 1 9 7 9 ; Kaplan and H o l l i s , 1 9 8 0 ) ; r e a c t i o n s were i n g e n i o u s l y i n i t i a t e d e i t h e r by suddenly a l l o w i n g Mg2+ i n t o t h e p r e v i o u s l y magnesium-free g h o s t s ( C a v i e r e s , 1 9 8 0 ; and e l s e w h e r e i n t h i s volume) or by p h o t o l y s i s of "caged" ATP (Kaplan and H o l l i s , 1 9 8 0 ; a l s o s e e Kaplan e l s e w h e r e i n t h i s volume). The f i n d i n g s from K a p l a n ' s l a b o r a t o r y can be summarized a s f o l l o w s : Nai s t i m u l a t e s w i t h v e r y h i g h a f f i n i t y ( ~ 0 . 5 < 2 m M ) and does n o t i n h i b i t ; Nao i n h i b i t s between 0 and 5 m M and t h e n s t i m u l a t e s i n app r o x i m a t e l y l i n e a r f a s h i o n ( F i g . 1) and KO i n h i b i t s w i t h an a f f i n i t y s i m i l a r t o t h a t of i t s i n h i b i t o r y act i o n on Na:Na exchange. The h i g h - a f f i n i t y s t i m u l a t i o n by Nai undoubtedly r e f l e c t s Nai r e q u i r e m e n t f o r enzyme p h o s p h o r y l a t i o n , and KO presumably i n h i b i t s ADP:ATP exchange by s h i f t i n g t h e E l - P * E2-P e q u i l i b r i u m t o t h e r i g h t . The mode of a c t i o n of e x t e r n a l sodium i s , a s i t w a s i n t h e case of Na-ATPase, l e s s c l e a r . S t i m u l a t i o n of ADP:ATP exchange by h i g h can be r a t i o n a l i z e d a s r e s u l t i n g from a sodium-induced s h i f t of t h e El-P =+ E2-P e q u i l i b r i u m t o t h e l e f t (Taniguchi and P o s t , 1975; K u r i k i and Racker, 1 9 7 6 ; Hara and Nakao, 1 9 8 1 ) , b u t it i s d i f f i c u l t t o f i t i n h i b i t i o n of ADP:ATP exchange by l o w i n t o the current schemes w i t h o u t a d d i t i o n a l a d h o c assumptions. C.

R E L A T I O N S H I P BETWEEN N a r N a EXCHANGE AND A D P : A T P EXCHANGE

The p a r a l l e l s between N a : N a exchange and ADP:ATP exchange a r e s t r i k i n g . Both r e q u i r e ADP as w e l l a s ATP; a r e i n h i b i t e d by low e x t e r n a l [K] and s t i m u l a t e d by h i g h (presumably i n t e r n a l ) [ K ] ; are s t i m u l a t e d w i t h h i g h a f f i n i t y by Nai which is n o t i n h i b i t o r y a t h i g h concent r a t i o n s ; and are s t i m u l a t e d by high Nao c o n c e n t r a t i o n s . These s i m i l a r i t i e s a r e s t r o n g s u p p o r t f o r t h e view t h a t t h e two a c t i v i t i e s s h a r e a common p a t h . Some of t h e d i s s i m i l a r i t i e s can a l s o be accommodated w i t h o u t t o o much d i f f i c u l t y . For example, oligomycin i n h i b i t s Na:Na exchange i n e r y t h r o c y t e s , b u t n o t ADP:ATP exchange; t h i s would be e x p e c t e d i f ADP and Nao were r e l e a s e d i n t h a t o r d e r , and oligomycin a c t e d between t h e two s t e p s (Glynn and Hoffman, 1 9 7 1 ) :

610

PAUL DE WEER

( A c o r o l l a r y of t h i s o r d e r i n g i s t h a t s i f f i c i e n t l y h i g h l e v e l s of ADP s h o u l d i n h i b i t N a : N a exchange w i t h o u t i n h i b i t i n g ADP:ATP exchange; t h i s has n o t y e t been observed e x p e r i m e n t a l l y . ) S i m i l a r l y , f r e e Mg2+ concent r a t i o n s o v e r a f e w t e n s micromolar i n h i b i t ADP:ATP exchange b u t n o t Na:Na exchange: b u t a g a i n , p r o p e r o r d e r i n g of A T P , Mg, and N a i a d d i t i o n w i l l a c c o u n t f o r these o b s e r v a t i o n s :

( A c o r o l l a r y of t h i s o r d e r i n g i s t h a t s u f f i c i e n t l y h i g h l e v e l s o f N a i s h o u l d i n h i b i t ADP:ATP exchange w i t h o u t i n h i b i t i n g Na:Na exchange.) I f t h e o r d e r i n g s d i s c u s s e d above are c o r r e c t , i t i s o b v i o u s t h a t t h e r e i s no r e a s o n t o e x p e c t a f i x e d s t o i c h i o m e t r i c r e l a t i o n s h i p between Na:Na exchange and ADP:ATP exchange as measured w i t h i s o t o p e s . I n t h i s c o n t e x t , it i s worth p o i n t i n g o u t t h a t i n s q u i d axon no o u a b a i n - s e n s i t i v e ADP:ATP exchange w a s d e t e c t e d w h i l e Na:Na exchange w a s t a k i n g p l a c e ( D e Weer e t a l . , t h i s volume), whereas i n e r y t h r o c y t e g h o s t s c o n s i d e r a b l e ADP:ATP exchange a c t i v i t y w a s measured i n t h e a b s e n c e of e x t e r n a l Na, where obvio u s l y no m e a s u r a b l e N a : N a exchange can t a k e place (Kaplan and H o l l i s , 1 9 8 0 ; Kaplan, t h i s volume).

VII.

RELATIONSHIP BETWEEN Na-ATPase AND ADP:ATP EXCHANGE

Although t h e r e a r e s e v e r a l d i f f e r e n c e s between t h e e f f e c t s of v a r i o u s a g e n t s ( e . g . , i n t e r n a l K ; i n t e r n a l Mg; o l i g o m y c i n ) on Na-ATPase and ADP:ATP exchange, t h e r e i s a s t r i k i n g s i m i l a r i t y between t h e sodium a c t i v a t i o n c u r v e f o r Na-ATPase a c t i v i t y and t h a t f o r ADP:ATP exchange a c t i v i t y (BeaugB and Glynn, 197933). Both c u r v e s d i s p l a y a h i g h - a f f i n i t y s t i m u l a t i n g a c t i o n of N a i , and h i g h - a f f i n i t y i n h i b i t o r y and l o w - a f f i n i t y s t i m u l a t i n g a c t i o n s of Nao. There a p p e a r s t o be no problem i n

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61 1

a s s i g n i n g t h e h i g h - a f f i n i t y N a i e f f e c t on b o t h ATPase and exchange t o t h e enzyme p h o s p h o r y l a t i o n s t e p r e q u i r e d by b o t h a c t i v i t i e s . The s i m i l a r i t y of t h e l o w - a f f i n i t y s t i m u l a t i o n of b o t h Na-ATPase and ADP:ATP exchange by N a o c o u l d b e a c o i n c i d e n c e s i n c e t h e s e e x p e r i m e n t s have n e v e r been done under e x a c t l y i d e n t i c a l c o n d i t i o n s ( e . g . , ADP i s p r e s e n t i n t h e exchange e x p e r i m e n t s ) . I f it i s n o t a c o i n c i d e n c e , and t h e s i m i l a r i t y r e f l e c t s a s i n g l e k i n e t i c e v e n t , and i f one a t t e m p t s t o f i t t h i s o b s e r v a t i o n w i t h i n t h e A l b e r s - P o s t framework i n c l u d i n g t h e documented e f f e c t s of N a o on t h e E l - P / E 2 - P e q u i l i b r i u m , t h e concluvia s i o n seems i n e s c a p a b l e t h a t Na-ATPase m u s t p r o c e e d d i r e c t E l - P breakdown r a t h e r t h a n v i a E2-P w i t h N a o act i v a t i n g i n a Ko-like manner. ADP:ATP exchange and NaA T P a s e would t h e n r e p r e s e n t a l t e r n a t e r e t u r n pathways f o r E l - P + E l , t h e p h o s p h a t e a c c e p t o r b e i n g ADP i n one c a s e and H 2 0 i n t h e o t h e r . I t w i l l be r e c a l l e d t h a t ADP:ATP exchange can t a k e p l a c e w i t h and w i t h o u t sodium i n t h e e x t e r n a l medium and t h a t Na-ATPase a c t i v i t y can o c c u r w i t h o r w i t h o u t e x t e r n a l sodium p r e s e n t . Taken t o g e t h e r , t h e s e o b s e r v a t i o n s s u g g e s t t h a t E l - P c a n rev e r t t o El w i t h or w i t h o u t ADP, and w i t h o r w i t h o u t Nao, (1) "conl e a d i n g t o f o u r p o s s i b l e modes o f o p e r a t i o n : v e n t i o n a l " (ADP-requiring) Na:Na exchange w i t h o u t hyd r o l y s i s : ( 2 ) " u n c o u p l e d " e f f l u x w i t h o u t ATP h y d r o l y s i s : ( 3 ) Na:Na exchange w i t h ATP h y d r o l y s i s : and ( 4 ) " u n c o u p l e d " e f f l u x w i t h ATP h y d r o l y s i s . Only t h e s e c o n d mode h a s n o t been e x p l i c i t l y d e s c r i b e d , b u t it c o u l d have been p r e s e n t i n " u n c o u p l e d " f l u x e x p e r i m e n t s where ADP w a s p r e s e n t , s u c h as t h o s e o f G a r r a h a n and Glynn ( 1 9 6 7 a ) . A r e q u i r e m e n t of t h i s model i s t h a t oligomyc i n , i n a d d i t i o n t o p r e v e n t i n g N a r e l e a s e on t h e o u t s i d e , b e assumed t o b l o c k d e p h o s p h o r y l a t i o n o f E l - P by H 2 0 , b u t n o t by ADP. A s f o r t h e h i g h - a f f i n i t y i n h i b i t i o n by Nao of NaATPase, ADP:ATP exchange, and sodium e f f l u x i n t o K-free media, t h e r e i s no o b v i o u s p r o v i s i o n f o r s u c h a mechani s m i n t h e c u r r e n t schemes. I n a d d i t i o n t o t h e e v i d e n c e a l r e a d y q u o t e d , C a v i e r e s and E l l o r y (1975) a l s o have d e s c r i b e d e x p e r i m e n t s t h a t l e d them t o p o s t u l a t e an ext e r n a l h i g h - a f f i n i t y i n h i b i t o r y s i t e f o r sodium. P o s t e t a ] . ( 1 9 7 2 ) have d e s c r i b e d , i n u n s i d e d k i d n e y ATPase, a s t i m u l a t i n g e f f e c t ( i n a d d i t i o n t o t h e more f a m i l i a r i n h i b i t o r y o n e ) o f K+ on N a , K - A T P a s e a t low ATP c o n c e n t r a t i o n s , and Beauge et a l . ( 1 9 7 9 ) h a v e i d e n t i f i e d t h a t e f f e c t a s due t o e x t e r n a l K d i s p l a c i n g SOdium from an i n h i b i t o r y s i t e . T h e r e i s a c l e a r n e e d , t h e n , f o r a h i g h - a f f i n i t y a c t i o n of sodium i n any r e a l i s t i c model of t h e sodium pump. E i t h e r an a d d i -

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t i o n a l s i t e f o r sodium must be p o s t u l a t e d , o r t h e c u r r e n t schemes must be adapted t o accommodate t h e o b s e r v a t i o n s . The f o l l o w i n g i s a t e n t a t i v e p r o p o s a l f o r a

mechanism by which t h e c u r r e n t Albers-Post scheme could e x h i b i t h i g h - a f f i n i t y i n h i b i t i o n by e x t e r n a l sodium. L e t t h e E l - P * E2-P e q u i l i b r i u m , i n t h e absence of e x t e r n a l sodium o r potassium, be v e r y much t o t h e r i g h t . Nao s t a b i l i z e s t h e E l - P form by b i n d i n g t o it w i t h low a f f i n i t y . Now assume t h a t t h e r a t e c o e f f i c i e n t of d e p h o s p h o r y l a t i o n (by H 2 0 o r ADP) of i o n f r e e E l - P i s much h i g h e r t h a n t h a t of E l - P f u l l y s a t u r a t e d w i t h sodium: t h e s m a l l amount of i o n - f r e e E l - P t h a t e x i s t s i n t h e absence of Nao and KO w i l l s u s t a i n an a p p r e c i a b l e d e p h o s p h o r y l a t i o n r a t e ( i . e . , ATPase a c t i v i t y o r ADP :ATP exchange , and concomitant Na e f f l u x ) . E l e v a t i o n of Nao w i l l p r o g r e s s i v e l y remove t h e l a b i l e i o n - f r e e E l - P from t h e p o o l and e v e n t u a l l y r e p l a c e it w i t h a much l a r g e r p o p u l a t i o n of f u l l y ( t r i p l y ? ) s o d i u m - s a t u r a t e d , more s l o w l y dephosphorylati n g El-P-Na3, l e a d i n g a g a i n t o an a p p r e c i a b l e dephosphorylation r a t e . (For t h i s mechanism t o d i s p l a y higha f f i n i t y i n h i b i t i o n followed by l o w - a f f i n i t y s t i m u l a t i o n , it i s obvious t h a t v a n i s h i n g d e p h o s p h o r y l a t i o n r a t e c o e f f i c i e n t s must b e a s s i g n e d t o E l - P - N a and E l - P -Na2. )

-

VIII.

RELATIONSHIP BETWEEN Na:Na EXCHANGE AND

Na:K EXCHANGE

E x t e r n a l potassium i n h i b i t s N a : N a exchange (Garrahan and Glynn, 1967c) and ADP:ATP exchange (Kaplan, t h i s volume) a s i t s t i m u l a t e s Na:K exchange. These e f f e c t s are e a s i l y r a t i o n a l i z e d w i t h i n t h e Albers-Post framework a s r e f l e c t i n g t h e e f f e c t s of ext e r n a l sodium and potassium on t h e E l - P -+ E2-P e q u i l i b r i u m . I n t r a c e l l u l a r ADP, on t h e o t h e r hand, w i l l induce Na:Na exchange ( p r o v i d e d KO i s n o t s a t u r a t i n g ) w i t h o u t much e f f e c t on ongoing Na:K exchange, a t l e a s t when ATP i s i n t h e m i l l i m o l a r range (Kennedy and D e Weer, 1 9 7 7 ; D e Weer e t al., 1 9 7 9 ) . A t lower [ATP], t h e e f f e c t of ADP i s t o induce Na:Na exchange and t o i n h i b i t Na:K exchange (Kennedy et al., t h i s volume). The r e l e v a n t p o r t i o n of t h e pump c y c l e i s a s f o l l o w s :

BIOPHYSICAUBIOCHEMICALCORRELATIONS

E1*ATP

613

Nai

ADP

J.

4

E1-ATP*Na+E

1=P .ADP.Na

4

Ki + I ATP

I I

- + I

I I

pi + II

E1-P-Na

11 +

NaO

E2-P

I1

c

KO

E2-P*K

At high ATP levels, the hydrolysis of E2-P (lower line) is probably rate-limiting. For ADP to have little effect on the population of enzyme molecules in the El-P and E2-P forms, it appears that, under those conditions, the steady-state distribution of enzyme forms in the upper line must remain biased toward the right, so that a relatively large increase in the traffic from E1-P.Na to E1.ATP (i.e., Na influx) has little effect on the absoltte amounts.of enzyme present in the El-P .Na , E2-P, and E2-P*K forms.

IX.

"OCCLUDED" FORMS AND I O N TRANSPORT

Experiments published in 1972 by Post and collaborators showed that the rate of rephosphorylation of freshly dephosphorylated Na,K-ATPase depended on the nature of the cation (K+,Rb+, Li+) that had catalyzed the dephosphorylation. These authors concluded that K+ and its congeners remain "occluded" in the enzyme for some time after dephosphorylation, the rate of deocclusion depending on the nature of the cation. They also concluded that ATP accelerates (with low affinity) the release of the occluded cation. This model has several verifiable "biophysical" consequences. One of them is that physical "trapping" of K congeners should be measurable if sufficiently long-lived; Glynn and Richards review this question elsewhere in this volume. Other "flux" consequences are kinetic. At high [ATP], deocclusion is not rate-limiting, and the overall transport velocity will be rate-limited by the saturation of E2-P with K+ or its congeners. At low [ATP], overall transport velocity could be rate-limited by the

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d e o c c l u s i o n v e l o c i t y . There i s no a p r i o r i r e a s o n t o e x p e c t any c o r r e l a t i o n between t h e a f f i n i t y of K+ and congeners f o r t h e e x t e r n a l E2-P s i t e , and t h e tendency of t h e s e i o n s t o remain occluded. Beauge and DiPolo ( 1 9 7 8 ; 1981b; and e l s e w h e r e i n t h i s volume) have shown t h a t i n s q u i d axon t h e o r d e r of e f f e c t i v e n e s s of K+ and congeners a s a c t i v a t o r s o f t h e sodium pump indeed d i f f e r s depending on whether [ATP] i s 3 m~ o r 30-50 U M . Also, a t low [ATP], s i n c e t h e r a t e - l i m i t i n g s t e p i s deo c c l u s i o n , t h e a p p a r e n t a f f i n i t y of K+ and congeners a t t h e e x t e r n a l s i t e w i l l i n c r e a s e . T h i s w a s found t o be t h e c a s e f o r K+ i n s q u i d axons (Beaug6 and DiPolo, 1979, 1981b; and t h i s volume) and f o r Rb+ i n e r y t h r o c y t e s ( E i s n e r and R i c h a r d s , 1 9 8 0 , 1 9 8 1 b ) . Conversely, low [KIo, by making d e p h o s p h o r y l a t i o n r a t e - l i m i t i n g , w i l l i n c r e a s e t h e a p p a r e n t a f f i n i t y f o r ATP, a p r e d i c t i o n made and v e r i f i e d by E i s n e r and R i c h a r d s (1981b). F i n a l l y , i n view of t h e r e c i p r o c a l e f f e c t s of K+ and ATP on t h e e q u i l i b r i u m between t h e dephosphoenzyme forms E l + E 2 (see Glynn et al., 1 9 7 9 ; K a r l i s h , 1 9 7 9 ; Beauge and Glynn, 1 9 8 0 ) , one might e x p e c t h i g h K t l e v e l s t o reduce t h e pump t u r n o v e r r a t e a t l o w [ATP? b u t n o t a t h i g h [ATP]. T h i s was v e r i f i e d i n s q u i d axon by Beaug6 and DiPolo ( 1 9 8 1 b ) .

X.

BIOCHEMICAL CORRELATES O F K:K EXCHANGE

The a b i l i t y of t h e sodium pump t o engage i n Na:Na exchange i n t h e absence of potassium, and i n K:K exchange i n t h e absence of sodium, h a s been a s t r o n g a r gument i n s u p p o r t of " c o n s e c u t i v e " models of t h e pump. Y e t , j u s t a s a s t r a i g h t f o r w a r d a p p l i c a t i o n of t h e ext a n t v e r s i o n (Glynn et a l . , 1 9 7 9 ) of t h e Albers-Post model does n o t y e t accommodate a l l c h a r a c t e r i s t i c s of Na:Na and ADP:ATP exchange, it i s f a i r t o s a y t h a t n o t a l l p r e d i c t i o n s of t h e c o n s e c u t i v e model w i t h r e s p e c t t o K:K exchange have been unambiguously v e r i f i e d e i t h e r . O u a b a i n - s e n s i t i v e K:K exchange h a s been w e l l s t u d i e d i n human e r y t h r o c y t e s (Glynn, 1957; P o s t and Sen, 1965; Glynn et al., 1 9 7 0 ; Sachs, 1972b, 1980, 1981a; Simons, 1 9 7 4 , 1975; E i s n e r and R i c h a r d s , 1 9 8 1 a ) . The exchange depends on t h e p r e s e n c e of i n t r a c e l l u l a r orthophosphate ( P o s t and Sen, 1965; Glynn et al., 1 9 7 0 ) w i t h a ~ 0 . 5= 1-2 mM (Simons, 1 9 7 4 ; Sachs, 1981a1, and a l s o on t h e p r e s e n c e of ATP, which i s n o t hydrolyzed (Simons, 1 9 7 4 ) , w i t h a ~ 0 . 55 1 0 0 P M (Simons, 1 9 7 4 ; Sachs, 1 9 8 1 a ) . ATP can be r e p l a c e d w i t h nonhydrolyzable

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615

a n a l o g s (Simons, 19751, s u g g e s t i n g t h a t enzyme phosp h o r y l a t i o n by ATP i s n o t r e q u i r e d f o r t h e r e a c t i o n . E x t e r n a l K+ a c t s w i t h h i g h a f f i n i t y : ~ 0 . 5< 0 . 1 mM i n t h e absence of e x t e r n a l sodium ( S a c h s , 1 9 8 1 a ) ; and i n t e r n a l K+ w i t h low a f f i n i t y : ~ 0 . 5% 1 0 mM (Simons, 1 9 7 4 ) . I n t e r n a l sodium i s a s t r o n g i n h i b i t o r (Simons, 1974). The q u e s t i o n a t hand i s whether t h e s e o b s e r v a t i o n s f i t t h e Albers-Post model, and what o t h e r v e r i f i a b l e p r e d i c t i o n s t h e model makes f o r K:K exchange. The requirement f o r P i i s r e g a r d e d a s an e x p r e s s i o n of t h e r e v e r s a l of K-catalyzed enzyme d e p h o s p h o r y l a t i o n (Glynn et al., 1 9 7 0 ) . The requirement f o r ATP was s u r p r i s i n g a t f i r s t , b u t i n t e r p r e t e d by Simons ( 1 9 7 4 ) a s p o s s i b l y r e f l e c t i n g t h e ATP-induced a c c e l e r a t i o n of K+ release from t h e lloccludedl' potassium-E2 form d e s c r i b e d by P o s t e t al. ( 1 9 7 2 ) . These f e a t u r e s w e r e e x p l i c i t l y i n c l u d e d by K a r l i s h e t al. (1978) i n t h e i r modified v e r s i o n of t h e Albers-Post c y c l e . The r e l e v a n t p a r t of t h e c y c l e ( i n t h e p r e s e n c e of [ATP] over a few micromolar) f o l l o w s : Ki

-

J.

E1.ATP

EIK-ATP $E2 (K) -ATP

m

+

+ E2 (K)

'i

KO

J.

1.

E2=P.K

E2=P

-

*

S t e i n ( 1 9 7 9 ) h a s p o i n t e d o u t t h a t such a model p r e d i c t s n o t o n l y t h e observed dependencies of K:K exchange on ATP and P i , b u t a l s o secondary i n h i b i t o r y e f f e c t s a s i n c r e a s i n g c o n c e n t r a t i o n s of ATP o r Pi f o r c e t h e enzyme p o p u l a t i o n i n t o one o r t h e o t h e r " c o r n e r . " A t t h e t i m e of S t e i n ' s a n a l y s i s , no such i n h i b i t i o n s had been d e s c r i b e d . I n s p e c t i o n of t h e model shows (see S t e i n , 1 9 7 9 , f o r a d e t a i l e d a n a l y s i s ) t h a t any i n h i b i t o r y e f f e c t o f , s a y , ATP would depend on t h e p r e v a i l i n g l e v e l s of P i , K i , and KO. ATP and P i s h o u l d a l s o a f f e c t t h e a p p a r e n t a f f i n i t i e s f o r K i and K O , i n o p p o s i t e d i r e c t i o n s (ATP i n c r e a s i n g Km f o r KO and d e c r e a s i n g t h a t f o r K i , and v i c e v e r s a i n t h e case o f P i ) . Some of t h e s e q u e s t i o n s have r e c e n t l y been s p e c i f i c a l l y i n v e s t i g a t e d . Sachs ( 1 9 8 1 a ) , working w i t h r e d blood c e l l s , found no i n h i b i t o r y e f f e c t of P i on K : K exchange up t o [ P i ] = 6 0 mN and no i n h i b i t o r y e f f e c t of ATP a t c o n c e n t r a t i o n s a s high a s 6 . 2 m ~ and , h a s concluded t h a t s i m u l t a n e o u s b i n d i n g of ATP and orthophosphate i s required t o account f o r t h i s observation. Since " t h e r e a p p e a r s t o be no e v i d e n c e e i t h e r f o r o r a g a i n s t t h e e x i s t e n c e " of c a t a l y t i c s u b u n i t s t h a t can b i n d ATP and o r t h o p h o s p h a t e s i m u l t a n e o u s l y , Sachs (1981a) prop o s e s a model i n which K:K t r a n s l o c a t i o n i s c a r r i e d o u t

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by an ATPase dimer, one s u b u n i t of which i s phosphoryla t e d , and t h e o t h e r b i n d s ATP. A s h o r t n o t e of E i s n e r and Richards ( 1 9 8 1 a ) , on t h e o t h e r hand, c l a i m s t h a t i n h i b i t i o n by ATP can be s e e n provided [Xi] i s s u f f i c i e n t l y low, and i n h i b i t i o n by P i provided [ATP] i s s u f f i c i e n t l y low. Recent work by K a r l i s h et a l . (1982) on ATP- and Pi-stimulated Rb:Rb exchange by Na,K-ATPase r e c o n s t i t u t e d i n t o liposomes may reconcile t h e s e f i n d i n g s . K a r l i s h et a l . ( 1 9 8 2 ) f i n d t h a t , a t low f i x e d [ATP] o r [ P i ] , Rb:Rb i s f i r s t s t i m u l a t e d and t h e n i n h i b i t e d by i n c r e a s i n g c o n c e n t r a t i o n s of P i o r ATP, r e s p e c t i v e l y . A t s u f f i c i e n t l y high f i x e d l e v e l s of one, i n c r e a s i n g t h e o t h e r s u b s t r a t e o n l y s t i m u l a t e s exchange. These k i n e t i c s a r e i n t e r p r e t e d on t h e b a s i s of a model t h a t a l l o w s s e v e r a l a l t e r n a t e pathways €or K:K exchange, one of v h i c h r e q u i r e s t h a t b o t h ATP and P i be bound t o t h e enzyme. Unlike Sachs ( m a ) , who p o s t u l a t e s a dimer, K a r l i s h e t a l . (1982) p o s t u l a t e independent and s i m u l t a n e o u s b i n d i n g of ATP and P i t o (presumably) a s i n g l e c a t a l y t i c u n i t . T h e i r model i s q u i t e g e n e r a l and comp l e x , b u t i t s e s s e n t i a l f e a t u r e w i t h r e g a r d t o ATP- and P i - s t i m u l a t e d K : K exchange i n i n t a c t c e l l s can be reduced t o t h e f o l l o w i n g :

I

L

The lower p a t h r e p r e s e n t s t h e " c o n v e n t i o n a l " Albers-Post pathway , which p r e d i c t s i n h i b i t i o n of K:K exchange by h i g h c o n c e n t r a t i o n s of e i t h e r ATP o r P i ( S t e i n , 1 9 7 9 ) . The upper pathway a l l o w s f o r t h e s i m u l t a n e o u s b i n d i n g of ATP and o r t h o p h o s p h a t e , and p r e d i c t s no i n h i b i t i o n of K:K exchange when b o t h [ATP] and [ P i ] are h i g h . Obvio u s l y , more work w i l l be r e q u i r e d t o apply t h e v a r i o u s tests t h a t w i l l v a l i d a t e o r i n v a l i d a t e competing models.

XI.

CONCLUSION

I t i s obvious t h a t t h e Albers-Post model, expanded t o i n c l u d e t h e l o w - a f f i n i t y s t i m u l a t i o n of potassium deo c c l u s i o n by ATP, has been extremely u s e f u l both i n t h e

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617

i n t e r p r e t a t i o n of e x p e r i m e n t a l " f l u x " f i n d i n g s and i n t h e d e s i g n of c r i t i c a l t e s t s . The a p p a r e n t c o n t r a d i c t i o n between o v e r a l l ATPase k i n e t i c s and Na/K t r a n s p o r t k i n e t i c s a p p e a r s t o be r e s o l v a b l e w i t h o u t t h e need t o invoke h a l f - o f - t h e - s i t e s b e h a v i o r . Both h i g h and low a f f i n i t i e s f o r ATP can be accommodated by a s i n g l e - u n i t model. Na:Na exchange, ADP:ATP exchange, and K : K exchange a r e p r e d i c t e d by t h e model. Many of t h e v a r i o u s i o n and n u c l e o t i d e i n t e r a c t i o n s a r e q u a l i t a tively interpretable. Problems remain, however. There i s no unambiguous scheme f o r Na-ATPase a c t i v i t y ( E l - P breakdown or E2-P breakdown, o r b o t h ? ) . The model l a c k s an obvious l o c u s €or t h e h i g h - a f f i n i t y i n h i b i t o r y e f f e c t of e x t e r n a l sodium on ATPase, sodium e f f l u x , and ADP:ATP exchange. The s t r i k i n g s i m i l a r i t i e s between t h e k i n e t i c s of NaATPase and t h o s e of ADP:ATP exchange remain p u z z l i n g . A s f o r t h e p r e c i s e mechanism of K:K exchange and i t s requirement f o r ATP and o r t h o p h o s p h a t e , t h e f i n a l v e r d i c t i s n o t i n . None of t h e s e unanswered q u e s t i o n s , however, a p p e a r t o c a l l f o r a complete o v e r h a u l of t h e c u r r e n t way of t h i n k i n g about t h e sodium pump. A more d i s c o n c e r t i n g prospect i s t h e p o s s i b i l i t y t h a t anion movements might t a k e p l a c e v i a t h e pump machinery. a posteriori

ACKNOWLEDGMENT

The a u t h o r ' s r e s e a r c h i s s u p p o r t e d by N I H g r a n t N S 11223.

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J d r g e n s e n , P. L . , and K a r l i s h , S. J. D. ( 1 9 8 0 ) . D e f e c t i v e c o n f o r m a t i o n a l r e s p o n s e i n a s e l e c t i v e l y t r y p s i n i z e d Na,K-ATPase Biochim. Biophys. s t u d i e d with tryptophan fluorescence. A c t a 597, 305-317. Kanazawa, T . , S a i t o , M., and Tonomura, Y . ( 1 9 6 7 ) . P r o p e r t i e s o f a p h o s p h o r y l a t e d p r o t e i n as a r e a c t i o n i n t e r m e d i a t e of t h e N a + K s e n s i t i v e ATPase. J. B i o c h e m . ( T o k y o ) 6 1 , 555-566. ( 1 9 8 0 ) . E x t e r n a l N a dependence Kaplan, J . H . , and H o l l i s , R . o f o u a b a i n - s e n s i t i v e ATP:ADP exchange i n i t i a t e d by photol y s i s of i n t r a c e l l u l a r caged-ATP i n human red c e l l g h o s t s . N a t u r e (London) 288, 587-589, K a r l i s h , S. J . D. ( 1 9 7 9 ) . C a t i o n i n d u c e d c o n f o r m a t i o n a l s t a t e s o f Na,K-ATPase s t u d i e d w i t h f l u o r e s c e n t probes. I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ndrby, eds.) , pp. 115-128. Academic P r e s s , New York. K a r l i s h , S . J. D . , Yates, D. W . , a n d Glynn, I. M. ( 1 9 7 8 ) . Conform a t i o n a l t r a n s i t i o n s between Na+-bound and K+-bound forms o f (Na+-K+) -ATPase, s t u d i e d w i t h formycin n u c l e o t i d e s . Biochim. B i o p h y s . A c t a 525, 252-264. K a r l i s h , S. J. D . , L i e b , W. R . , and S t e i n , W. D. ( 1 9 8 2 ) . Combined e f f e c t s of ATP and p h o s p h a t e o n Rb-Rb exchange mediated by J. (Na,K)ATPase r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s . P h y s i o l . (London) 328, 333-350. Kennedy, B G . , a n d D e Weer, P. ( 1 9 7 6 ) . Strophanthidin-sensitive sodium f l u x e s i n m e t a b o l i c a l l y p o i s o n e d f r o g s k e l e t a l m u s c l e . J. G e n . P h y s i o l . 68, 405-520. Kennedy, B. G . , and D e Weer, P. ( 1 9 7 7 ) . R e l a t i o n s h i p between N a : K and N a : N a exchange by the sodium pump of s k e l e t a l muscle. N a t u r e (London) 268, 165-167. Klodos, I. , and Skou, J. C. ( 1 9 7 5 ) . The e f f e c t o f Mg2+ a n d c h e l a t i n g a g e n t s on i n t e r m e d i a r y s t e p s o f t h e r e a c t i o n o f N a + , K+a c t i v a t e d ATPase. B i o c h i m . B i o p h y s . A c t a 3 9 1 , 474-485. K u r i k i , Y . , and R a c k e r , E. ( 1 9 7 6 ) . I n h i b i t i o n o f ( N a + , K + ) adenos i n e t r i p h o s p h a t a s e a n d i t s p a r t i a l r e a c t i o n s by q u e r c e t i n . B i o c h e m i s t r y 1 5 , 4951-4956. L e e , K. H . , a n d B l o s t e i n , R . ( 1 9 8 0 ) . Red c e l l sodium f l u x e s catal y s e d by t h e sodium pump i n t h e a b s e n c e o f K+ and ADP. N a t u r e (London) 285, 338-339. Lew, V. L . , Hardy, M. A . , J r . , and E l l o r y , J. C . ( 1 9 7 3 ) . The unc o u p l e d e x t r u s i o n o f N a + t h r o u g h t h e N a + pump. B i o c h i m . B i o p h y s . A c t a 232, 251-266. M z r d h , S. , and P o s t , R . L. ( 1 9 7 7 ) . P h o s p h o r y l a t i o n from a d e n o s i n e t r i p h o s p h a t e o f sodium- a n d p o t a s s i u m - a c t i v a t e d a d e n o s i n e triphosphatase. J. B i o l . C h e m . 252, 633-638. Moczydlowski, E . G . , a n d F o r t e s , P. A. G . ( 1 9 8 1 ) . I n h i b i t i o n of sodium and p o t a s s i u m a d e n o s i n e t r i p h o s p h a t a s e by 2 ' , 3 ' - 0 (2,4,6-trinitrocyclohexadienylidine)adenine n u c l e o t i d e s . I m p l i c a t i o n s f o r t h e s t r u c t u r e and mechanism of t h e N a : K pump. J . B i o l . C h e m . 256, 2357-2366.

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