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Stoichiometry of the Electrogenic Na Pump in Barnacle Muscle: Simultaneous Measurement of Na Efflux and Membrane Current
CURRENT TOPICS IN MEMBRANES ANDTRANSPORT. VOLUME 19
Stoichiometry of the Electrogenic Na Pump in Barnacle Muscle: Simultaneous Measurement of Na Efflux and Membrane Current M. T. NELSON'AND W. J . LEDERER Deparhnent of Physiology Universiryof Maryland School of Medicine Baltimore, Maryland
I.
INTRODUCTION
T h e Na,K-ATPase i s an enzyme l o c a t e d i n t h e plasma membrane of c e l l s and f u n c t i o n s t o t r a n s p o r t sodium o u t o f t h e c e l l and p o t a s s i u m i n t o t h e c e l l . F l u x s t u d i e s have i n d i c a t e d t h a t a p p r o x i m a t e l y 3 moles of Na i o n s are t r a n s p o r t e d o u t of r e d b l o o d c e l l s f o r e v e r y mole of ATP h y d r o l y z e d (Garrahan and Glynn, 1 9 6 7 ) . Garrahan and Glynn a l s o i n v e s t i g a t e d o u a b a i n - s e n s i t i v e N a e f f l u x and o u a b a i n - s e n s i t i v e K i n f l u x and d e m o n s t r a t e d t h a t t h e r a t i o of t h e s e two o u a b a i n - s e n s i t i v e f l u x e s w a s between 2:l and 3:2 f o r N a : K f o r t h e c o u n t e r t r a n s p o r t o f t h e s e two c a t i o n s . The s t o i c h i o m e t r i c i n e q u a l i t y o f N a + and
'Present a d d r e s s : F a k u l t X t f i r B i o l o g i e , U n i v e r s i t a t K o n s t a n z , 0-7750 K o n s t a n z , F e d e r a l R e p u b l i c of G e r m a n y . 2 A d d r e s s a l l c o r r e s p o n d e n c e and r e p r i n t r e q u e s t s t o W. J . L e d e r e r i n B a l t i m o r e . 707
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0
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M. T. NELSON AND W. J. LEDERER
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t r a n s p o r t h a s been s u p p o r t e d i n numerous s u b s e q u e n t f l u x s t u d i e s and many e l e c t r i c a l s t u d i e s . A h y p e r p o l a r i z a t i o n o f t h e c e l l membrane accompanies a n i n c r e a s e d t u r n o v e r r a t e of t h e enzyme, as would be e x p e c t e d from t h e above s t o i c h i o m e t r y . Such e l e c t r o g e n i c i t y of sodium and p o t a s s i u m t r a n s p o r t by t h e N a pump h a s been used r e c e n t l y t o estimate t h e s t o i c h i o m e t r y o f t h e N a pump by measuring s i m u l t a n e o u s l y t h e change i n c u r r e n t and t h e change i n i n t r a c e l l u l a r Na a c t i v i t y i n s n a i l n e u r o n s (Thomas, 1 9 6 9 ) and i n c a r d i a c P u r k i n j e f i b e r s ( E i s n e r e t a l . , 1981) when t h e N a pump a c t i v i t y h a s been a l t e r e d . Such s t u d i e s assume a f i x e d r e l a t i o n s h i p between t h e amount o f N a t r a n s p o r t e d and t h e measured i n t r a c e l l u l a r Na a c t i v i t y . The more d i r e c t approach used by Cooke e t a l . (1974) h a s a l s o been used i n t h e e x p e r i ments p r e s e n t e d here. Cooke e t a l . i n j e c t e d N a (and 2 4 N a ) i o n t o p h o r e t i c a l l y and measured N a e f f l u x from a n A p l y s i a neuron w h i l e t h e c e l l w a s v o l t a g e clamped e i t h e r i n t h e p r e s e n c e or a b s e n c e o f s t r o p h a n t h i d i n . C o r r e l a t i n g t h e o u a b a i n - s e n s i t i v e c u r r e n t and f l u x c h a n g e s , t h e y e s t i m a t e d a N a pump s t o i c h i o m e t r y ( N a : K ) of 2 . 9 t o 1 . 7 . T h i s r a n g e o f v a l u e s i s much l a r g e r t h a n t h e r a n g e rep o r t e d by Thomas o f 1 . 5 t o 1 . 3 , whereas i t overlaps t h e h i g h e r v a l u e s r e p o r t e d by Garrahan and Glynn (1967) and E i s n e r et al. ( 1 9 8 1 ) . I n t h e p r e s e n t e x p e r i m e n t s on b a r n a c l e muscle s i n g l e c e l l s , w e have measured N a e f f l u x and membrane c u r r e n t s i m u l t a n e o u s l y i n t h e manner of Cooke e t a l . b u t have c o n t r o l l e d i n t r a c e l l u l a r c o n s t i t u e n t s by means of a p e r f u s i o n method. O u r r e s u l t s a r e i n agreement w i t h t h e s t o i c h i o m e t r y o f sodium and p o t a s sium t r a n s p o r t by t h e Na,K-ATPase determined by Garrahan and Glynn (1967) and E i s n e r e t a l . ( 1 9 8 1 ) . K
11.
METHODS A s i n g l e muscle c e l l from t h e g i a n t b a r n a c l e
was p e r f u s e d w i t h an i n t e r n a l s o l u t i o n c o n t a i n i n g (among o t h e r c o n s t i t u e n t s ) ATP, a n ATP reg e n e r a t i n g s y s t e m , and 2 2 N a as h a s been d e s c r i b e d by Nelson and B l a u s t e i n ( 1 9 8 0 ) . The s i g n i f i c a n t d i f f e r ence between t h e p r e p a r a t i o n a s d e s c r i b e d by Nelson and B l a u s t e i n (1980) and t h e methods used f o r t h e s e e x p e r i ments i s t h e i n t e r n a l , a x i a l p l a t i n i z e d - p l a t i n u m w i r e ( t o p a s s c u r r e n t ) and s t a n d a r d voltage-clamp e l e c t r o n ics. Balanus nubilis
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F i g . 1 . O u a b a i n - d e p e n d e n t current and Na e f f l u x i n b a r n a c l e A t a h o l d i n g p o t e n t i a l of -39.2 mV, o u a b a i n (10-4 M ) was a p p l i e d a s i n d i c a t e d b y the s o l i d b a r i n the f i g u r e . C o n d u c t a n c e ( 0 ) was m e a s u r e d b y a p p l y i n g 2.26-mV d e p o l a r i z i n g p u l s e s f o r 4 sec once every 30 sec. T h e o u a b a i n - d e p e n d e n t f a l l i n Na e f f l u x ( ) i s shown t o be a c c o m p a n i e d b y a f a l l i n o u t w a r d current ( X ) T e t r a e t h y l a m m o n i u m ( T E A ) was p r e s e n t i n the i n t e r n a l p e r f u s a t e (50 mM) and 22Na was u s e d a s the Na t r a c e r .
muscle.
.
111.
RESULTS AND D I S C U S S I O N
The methods u s e d i n t h e s e e x p e r i m e n t s a l l o w t h r e e (1) p e r t i n e n t v a r i a b l e s t o be measured c o n t i n u o u s l y : N a e f f l u x , ( 2 ) membrane c u r r e n t a t a f i x e d h o l d i n g pot e n t i a l , and ( 3 ) membrane c o n d u c t a n c e . F i g u r e l shows
710
M. T. NELSON AND W. J. LEDERER
an e x p e r i m e n t d u r i n g which t h e membrane p o t e n t i a l w a s clamped t o -39 mV. Throughout t h e e x p e r i m e n t , conduct a n c e w a s measured c o n t i n u o u s l y by a p p l y i n g 2.26-mV dep o l a r i z a t i o n s f o r 4 sec e v e r y 30 sec. F i g u r e 1 shows t h a t upon a p p l y i n g 1 0 - 4 M o u a b a i n t o t h e s u p e r f u s i n g sol u t i o n , there w a s a decrease i n N a e f f l u x of about 42 pmoles/sec and a d e c r e a s e o f outward c u r r e n t o f a b o u t 2 VA. [Note t h a t I / F = ( 2 x 1 0 - 6 C / s e c ) / ( l 0 5 C/Eq) = 20 pEq/sec.] T h i s r e s u l t i n d i c a t e s t h a t a b o u t 0.48 of t h e N a e f f l u x t h a t i s o u a b a i n - s e n s i t i v e a p p e a r s t o move as uncompensated c h a r g e . The t o t a l membrane conductance was n o t d i f f e r e n t a t t h e end o f t h e e x p e r i m e n t compared t o i t s v a l u e a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . Between 35 min and 50 min t h e r e was a b o u t a 0 . 1 mS change i n conductance w i t h o u t any obvious e f f e c t on t h e N a e f f l u x . During t h e c o u r s e of t h i s e x p e r i m e n t , t h e i n t e r n a l p e r f u s a t e c o n t a i n e d 50 mM tetraethylammonium (TEA) t o r e d u c e qK. The p r e s e n c e of t h i s potassium-channel b l o c k e r had no d i s c e r n i b l e e f f e c t o n t h e c a l c u l a t e d e l e c t r o g e n i c f r a c t i o n of o u a b a i n - s e n s i t i v e N a e f f l u x when compared t o e x p e r i m e n t s performed i n t h e absence of i n t e r n a l TEA. I n f o u r e x p e r i m e n t s t h i s f r a c t i o n w a s 0 .3 9 f 0 . 0 4 . I n summary, t h e i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e c e l l h a s been used t o estimate t h e s t o i c h i o m e t r y of t h e N a , K - A T P a s e under c o n d i t i o n s of c o n t r o l l e d i n t e r n a l and e x t e r n a l environments o f t h e c e l l membrane. C o n t r o l l i n g f o r a l t e r a t i o n s i n t o t a l membrane conductance and potassium-channel c o n d u c t a n c e , w e h a v e found t h a t 1.5-2.0 N a i o n s are t r a n s p o r t e d o u t o f t h e c e l l f o r every K ion transported i n t o t h e cell. Besides confirming t h e r e s u l t s of earlier i n v e s t i g a t i o n s i n t o t h e stoichiome t r y of t h e Na pump and besides d e v e l o p i n g a method f o r a d d i t i o n a l q u a n t i t a t i v e f l u x and c u r r e n t s t u d i e s of t h e Na,K-ATPase, t h i s work d e m o n s t r a t e s t h e u t i l i t y o f f l u x and c u r r e n t s t u d i e s i n g e n e r a l u s i n g t h i s p r e p a r a t i o n (see L e d e r e r and Nelson, 1 9 8 1 ) .
ACKNOWLEDGMENTS We would l i k e t o thank M. P. B l a u s t e i n for h i s s u p p o r t , encouragement, and advice throughout t h e course of t h e s e experiments. D r . E . Santiago helped w i t h t h e d i s s e c t i o n s and w i t h trouble-shooting equipment problems. D r s . L. Goldman and L. Horn helped us prepare t h e p l a t i n i z e d - p l a t i n u m e l e c t r o d e s . This work has been supported by NSF and MDA g r a n t s to M. P. B l a u s t e i n . M. T . Nelson had a r e s e a r c h fellowship supported by t h e Maryland
STOICHIOMETRY OF ELECTROGENIC Na PUMP IN BARNACLES
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A f f i l i a t e o f t h e American Heart A s s o c i a t i o n . A d d i t i o n a l s u p p o r t h a s been p r o v i d e d by t h e N I H (HL-25675) and by a B a s i l O'Connor G r a n t o f t h e N a t i o n a l F o u n d a t i o n f o r t h e March o f D i m e s t o W. J. L e d e r e r , who i s a n e s t a b l i s h e d i n v e s t i g a t o r of t h e American Heart A s s o c i a t i o n and i t s Maryland S t a t e A f f i l i a t e .
REFERENCES Cooke, I . M., L e b l a n c , G . , and Tauc, L. ( 1 9 7 4 ) . Sodium pump s t o i c h i o m e t r y i n A p l y s i a n e u r o n e s f r o m s i m u l t a n e o u s c u r r e n t and tracer measurements. Nature (London) 251, 254-256. E i s n e r , D. A., L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 1 ) . The dependence o f sodium pumping and t e n s i o n o n i n t r a c e l l u l a r sodium a c t i v i t y i n v o l t a g e - c l a m p e d s h e e p P u r k i n j e f i b r e s . J. P h y s i o l . (London) 317, 163-187. G a r r a h a n , P . , and Glynn, I. M. ( 1 9 6 7 ) . The s t o i c h i o m e t r y o f t h e sodium pump. J. P h y s i o l . (London) 1 9 2 , 217-235. L e d e r e r , W. J . , a n d N e l s o n , M. T . ( 1 9 8 1 ) . C u r r e n t a s s o c i a t e d w i t h Nao-dependent C a e f f l u x i n b a r n a c l e muscle c e l l s . J. P h y s i o l . (London) ( i n p r e s s ) N e l s o n , M. T . , and B l a u s t e i n , M. P. ( 1 9 8 0 ) . P r o p e r t i e s o f sodium pumps i n i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e f i b e r s . J. Gen. P h y s i o l . 7 5 , 183-206. Thomas, R. C . ( 1 9 6 9 ) . Membrane c u r r e n t and i n t r a c e l l u l a r sodium c h a n g e s i n a s n a i l neurone d u r i n g e x t r u s i o n of i n j e c t e d sodium. J. P h y s i o l . (London) 201, 495-514.
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Stoichiometry of the Electrogenic Na Pump in Barnacle Muscle: Simultaneous Measurement of Na Efflux and Membrane Current M. T. NELSON'AND W. J . LEDERER Deparhnent of Physiology Universiryof Maryland School of Medicine Baltimore, Maryland
I.
INTRODUCTION
T h e Na,K-ATPase i s an enzyme l o c a t e d i n t h e plasma membrane of c e l l s and f u n c t i o n s t o t r a n s p o r t sodium o u t o f t h e c e l l and p o t a s s i u m i n t o t h e c e l l . F l u x s t u d i e s have i n d i c a t e d t h a t a p p r o x i m a t e l y 3 moles of Na i o n s are t r a n s p o r t e d o u t of r e d b l o o d c e l l s f o r e v e r y mole of ATP h y d r o l y z e d (Garrahan and Glynn, 1 9 6 7 ) . Garrahan and Glynn a l s o i n v e s t i g a t e d o u a b a i n - s e n s i t i v e N a e f f l u x and o u a b a i n - s e n s i t i v e K i n f l u x and d e m o n s t r a t e d t h a t t h e r a t i o of t h e s e two o u a b a i n - s e n s i t i v e f l u x e s w a s between 2:l and 3:2 f o r N a : K f o r t h e c o u n t e r t r a n s p o r t o f t h e s e two c a t i o n s . The s t o i c h i o m e t r i c i n e q u a l i t y o f N a + and
'Present a d d r e s s : F a k u l t X t f i r B i o l o g i e , U n i v e r s i t a t K o n s t a n z , 0-7750 K o n s t a n z , F e d e r a l R e p u b l i c of G e r m a n y . 2 A d d r e s s a l l c o r r e s p o n d e n c e and r e p r i n t r e q u e s t s t o W. J . L e d e r e r i n B a l t i m o r e . 707
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0
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M. T. NELSON AND W. J. LEDERER
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t r a n s p o r t h a s been s u p p o r t e d i n numerous s u b s e q u e n t f l u x s t u d i e s and many e l e c t r i c a l s t u d i e s . A h y p e r p o l a r i z a t i o n o f t h e c e l l membrane accompanies a n i n c r e a s e d t u r n o v e r r a t e of t h e enzyme, as would be e x p e c t e d from t h e above s t o i c h i o m e t r y . Such e l e c t r o g e n i c i t y of sodium and p o t a s s i u m t r a n s p o r t by t h e N a pump h a s been used r e c e n t l y t o estimate t h e s t o i c h i o m e t r y o f t h e N a pump by measuring s i m u l t a n e o u s l y t h e change i n c u r r e n t and t h e change i n i n t r a c e l l u l a r Na a c t i v i t y i n s n a i l n e u r o n s (Thomas, 1 9 6 9 ) and i n c a r d i a c P u r k i n j e f i b e r s ( E i s n e r e t a l . , 1981) when t h e N a pump a c t i v i t y h a s been a l t e r e d . Such s t u d i e s assume a f i x e d r e l a t i o n s h i p between t h e amount o f N a t r a n s p o r t e d and t h e measured i n t r a c e l l u l a r Na a c t i v i t y . The more d i r e c t approach used by Cooke e t a l . (1974) h a s a l s o been used i n t h e e x p e r i ments p r e s e n t e d here. Cooke e t a l . i n j e c t e d N a (and 2 4 N a ) i o n t o p h o r e t i c a l l y and measured N a e f f l u x from a n A p l y s i a neuron w h i l e t h e c e l l w a s v o l t a g e clamped e i t h e r i n t h e p r e s e n c e or a b s e n c e o f s t r o p h a n t h i d i n . C o r r e l a t i n g t h e o u a b a i n - s e n s i t i v e c u r r e n t and f l u x c h a n g e s , t h e y e s t i m a t e d a N a pump s t o i c h i o m e t r y ( N a : K ) of 2 . 9 t o 1 . 7 . T h i s r a n g e o f v a l u e s i s much l a r g e r t h a n t h e r a n g e rep o r t e d by Thomas o f 1 . 5 t o 1 . 3 , whereas i t overlaps t h e h i g h e r v a l u e s r e p o r t e d by Garrahan and Glynn (1967) and E i s n e r et al. ( 1 9 8 1 ) . I n t h e p r e s e n t e x p e r i m e n t s on b a r n a c l e muscle s i n g l e c e l l s , w e have measured N a e f f l u x and membrane c u r r e n t s i m u l t a n e o u s l y i n t h e manner of Cooke e t a l . b u t have c o n t r o l l e d i n t r a c e l l u l a r c o n s t i t u e n t s by means of a p e r f u s i o n method. O u r r e s u l t s a r e i n agreement w i t h t h e s t o i c h i o m e t r y o f sodium and p o t a s sium t r a n s p o r t by t h e Na,K-ATPase determined by Garrahan and Glynn (1967) and E i s n e r e t a l . ( 1 9 8 1 ) . K
11.
METHODS A s i n g l e muscle c e l l from t h e g i a n t b a r n a c l e
was p e r f u s e d w i t h an i n t e r n a l s o l u t i o n c o n t a i n i n g (among o t h e r c o n s t i t u e n t s ) ATP, a n ATP reg e n e r a t i n g s y s t e m , and 2 2 N a as h a s been d e s c r i b e d by Nelson and B l a u s t e i n ( 1 9 8 0 ) . The s i g n i f i c a n t d i f f e r ence between t h e p r e p a r a t i o n a s d e s c r i b e d by Nelson and B l a u s t e i n (1980) and t h e methods used f o r t h e s e e x p e r i ments i s t h e i n t e r n a l , a x i a l p l a t i n i z e d - p l a t i n u m w i r e ( t o p a s s c u r r e n t ) and s t a n d a r d voltage-clamp e l e c t r o n ics. Balanus nubilis
STOICHIOMETRY OF ELECTROGENIC Na PUMP IN BARNACLES
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F i g . 1 . O u a b a i n - d e p e n d e n t current and Na e f f l u x i n b a r n a c l e A t a h o l d i n g p o t e n t i a l of -39.2 mV, o u a b a i n (10-4 M ) was a p p l i e d a s i n d i c a t e d b y the s o l i d b a r i n the f i g u r e . C o n d u c t a n c e ( 0 ) was m e a s u r e d b y a p p l y i n g 2.26-mV d e p o l a r i z i n g p u l s e s f o r 4 sec once every 30 sec. T h e o u a b a i n - d e p e n d e n t f a l l i n Na e f f l u x ( ) i s shown t o be a c c o m p a n i e d b y a f a l l i n o u t w a r d current ( X ) T e t r a e t h y l a m m o n i u m ( T E A ) was p r e s e n t i n the i n t e r n a l p e r f u s a t e (50 mM) and 22Na was u s e d a s the Na t r a c e r .
muscle.
.
111.
RESULTS AND D I S C U S S I O N
The methods u s e d i n t h e s e e x p e r i m e n t s a l l o w t h r e e (1) p e r t i n e n t v a r i a b l e s t o be measured c o n t i n u o u s l y : N a e f f l u x , ( 2 ) membrane c u r r e n t a t a f i x e d h o l d i n g pot e n t i a l , and ( 3 ) membrane c o n d u c t a n c e . F i g u r e l shows
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an e x p e r i m e n t d u r i n g which t h e membrane p o t e n t i a l w a s clamped t o -39 mV. Throughout t h e e x p e r i m e n t , conduct a n c e w a s measured c o n t i n u o u s l y by a p p l y i n g 2.26-mV dep o l a r i z a t i o n s f o r 4 sec e v e r y 30 sec. F i g u r e 1 shows t h a t upon a p p l y i n g 1 0 - 4 M o u a b a i n t o t h e s u p e r f u s i n g sol u t i o n , there w a s a decrease i n N a e f f l u x of about 42 pmoles/sec and a d e c r e a s e o f outward c u r r e n t o f a b o u t 2 VA. [Note t h a t I / F = ( 2 x 1 0 - 6 C / s e c ) / ( l 0 5 C/Eq) = 20 pEq/sec.] T h i s r e s u l t i n d i c a t e s t h a t a b o u t 0.48 of t h e N a e f f l u x t h a t i s o u a b a i n - s e n s i t i v e a p p e a r s t o move as uncompensated c h a r g e . The t o t a l membrane conductance was n o t d i f f e r e n t a t t h e end o f t h e e x p e r i m e n t compared t o i t s v a l u e a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . Between 35 min and 50 min t h e r e was a b o u t a 0 . 1 mS change i n conductance w i t h o u t any obvious e f f e c t on t h e N a e f f l u x . During t h e c o u r s e of t h i s e x p e r i m e n t , t h e i n t e r n a l p e r f u s a t e c o n t a i n e d 50 mM tetraethylammonium (TEA) t o r e d u c e qK. The p r e s e n c e of t h i s potassium-channel b l o c k e r had no d i s c e r n i b l e e f f e c t o n t h e c a l c u l a t e d e l e c t r o g e n i c f r a c t i o n of o u a b a i n - s e n s i t i v e N a e f f l u x when compared t o e x p e r i m e n t s performed i n t h e absence of i n t e r n a l TEA. I n f o u r e x p e r i m e n t s t h i s f r a c t i o n w a s 0 .3 9 f 0 . 0 4 . I n summary, t h e i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e c e l l h a s been used t o estimate t h e s t o i c h i o m e t r y of t h e N a , K - A T P a s e under c o n d i t i o n s of c o n t r o l l e d i n t e r n a l and e x t e r n a l environments o f t h e c e l l membrane. C o n t r o l l i n g f o r a l t e r a t i o n s i n t o t a l membrane conductance and potassium-channel c o n d u c t a n c e , w e h a v e found t h a t 1.5-2.0 N a i o n s are t r a n s p o r t e d o u t o f t h e c e l l f o r every K ion transported i n t o t h e cell. Besides confirming t h e r e s u l t s of earlier i n v e s t i g a t i o n s i n t o t h e stoichiome t r y of t h e Na pump and besides d e v e l o p i n g a method f o r a d d i t i o n a l q u a n t i t a t i v e f l u x and c u r r e n t s t u d i e s of t h e Na,K-ATPase, t h i s work d e m o n s t r a t e s t h e u t i l i t y o f f l u x and c u r r e n t s t u d i e s i n g e n e r a l u s i n g t h i s p r e p a r a t i o n (see L e d e r e r and Nelson, 1 9 8 1 ) .
ACKNOWLEDGMENTS We would l i k e t o thank M. P. B l a u s t e i n for h i s s u p p o r t , encouragement, and advice throughout t h e course of t h e s e experiments. D r . E . Santiago helped w i t h t h e d i s s e c t i o n s and w i t h trouble-shooting equipment problems. D r s . L. Goldman and L. Horn helped us prepare t h e p l a t i n i z e d - p l a t i n u m e l e c t r o d e s . This work has been supported by NSF and MDA g r a n t s to M. P. B l a u s t e i n . M. T . Nelson had a r e s e a r c h fellowship supported by t h e Maryland
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A f f i l i a t e o f t h e American Heart A s s o c i a t i o n . A d d i t i o n a l s u p p o r t h a s been p r o v i d e d by t h e N I H (HL-25675) and by a B a s i l O'Connor G r a n t o f t h e N a t i o n a l F o u n d a t i o n f o r t h e March o f D i m e s t o W. J. L e d e r e r , who i s a n e s t a b l i s h e d i n v e s t i g a t o r of t h e American Heart A s s o c i a t i o n and i t s Maryland S t a t e A f f i l i a t e .
REFERENCES Cooke, I . M., L e b l a n c , G . , and Tauc, L. ( 1 9 7 4 ) . Sodium pump s t o i c h i o m e t r y i n A p l y s i a n e u r o n e s f r o m s i m u l t a n e o u s c u r r e n t and tracer measurements. Nature (London) 251, 254-256. E i s n e r , D. A., L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 1 ) . The dependence o f sodium pumping and t e n s i o n o n i n t r a c e l l u l a r sodium a c t i v i t y i n v o l t a g e - c l a m p e d s h e e p P u r k i n j e f i b r e s . J. P h y s i o l . (London) 317, 163-187. G a r r a h a n , P . , and Glynn, I. M. ( 1 9 6 7 ) . The s t o i c h i o m e t r y o f t h e sodium pump. J. P h y s i o l . (London) 1 9 2 , 217-235. L e d e r e r , W. J . , a n d N e l s o n , M. T . ( 1 9 8 1 ) . C u r r e n t a s s o c i a t e d w i t h Nao-dependent C a e f f l u x i n b a r n a c l e muscle c e l l s . J. P h y s i o l . (London) ( i n p r e s s ) N e l s o n , M. T . , and B l a u s t e i n , M. P. ( 1 9 8 0 ) . P r o p e r t i e s o f sodium pumps i n i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e f i b e r s . J. Gen. P h y s i o l . 7 5 , 183-206. Thomas, R. C . ( 1 9 6 9 ) . Membrane c u r r e n t and i n t r a c e l l u l a r sodium c h a n g e s i n a s n a i l neurone d u r i n g e x t r u s i o n of i n j e c t e d sodium. J. P h y s i o l . (London) 201, 495-514.
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