189
Biochimica et Biophysica Acta, 3 8 9 ( 1 9 7 5 ) 1 8 9 - - 1 9 3 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA Report BBA 7 1 2 2 2
THE INDEPENDENCE OF MEMBRANE POTENTIAL AND POTASSIUM ACTIVATION OF THE SODIUM PUMP IN MUSCLE
L.A. B E A U G E * , R.A. SJODIN and O L G A O R T I Z
Department of Biophysics, Universityof Maryland School of Medicine, Baltimore, Md. 21201 (U.S.A.) (Received January 13th, 1975)
Summary The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K +] by equilibration in a 95 mM K +, 120 mM Na + Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E m sensitive to [K+]. In addition, in the presence of 10 mM K +, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10 --4 M ouabain. This suggests that the membrane potential does n o t play a major role in the potassium activation of the sodium pump in muscles.
The ionic requirements of the Na+-K + pump such as its activation by K +, are the same as those for an ATPase isolated from membrane fractions [ 1 ], leading to the postulate that K + activation of the pump is similar in nature to an enzyme-substrate reaction. In fresh frog skeletal muscle both K + and azide, when applied externally, activate ouabain-sensitive Na + efflux and also depolarize membrane potential (Era) [2, 3]. Since the relation between Na + efflux and transmembrane potential seemed to be the same in both instances, it was postulated t h a t K + activation of the Na + pump is due to its effects on the membrane potential, n o t to a catalytic action on the pump mechanism. On the other hand, in high Na + sartorius muscles, the addition of K + does n o t immediately depolarize the membrane close to the K÷ equilibrium potential (EK); rather, the potential is held at a higher value as a consequence of an electrogenic operation of the Na÷-K + pump [4, 5]. Under these conditions, a definite pump activation is observed with [K +] as low as 1 mM [6]. Since other excitable tissues like squid axon [7] and Anisodoris giant neurone [8], *Member of the Consejo Nacional de Investigationes Cientifieas y T~cnicas of Argentina.
190
as well as non-excitable human red blood cells [9], failed to show any membrane potential dependence of the Na÷-K + pump, it seemed worthwhile to reexamine the matter of the Na + pump activation in skeletal muscle. The idea was to work under conditions where the membrane potential and pump effects of K + could possibly be separated. This was done by taking advantage of the observation that the membrane potential of fibers which have been equilibrated in high K +, normal Na + solutions is insensitive to reduction o f K~ while the membrane itself behaves as a C1- electrode [10, 11]. In this way it was possible to study Na + fluxes at constant Era and variable K + . A basic experimental protocol and the constancy of membrane potential during the whole experiment are shown in Fig. 1. Fig. 2A summarizes all experiments performed to test the effect of [K+]o on Na ÷ efflux in muscles with membrane potentials clamped at about --23 mV. As can be seen, a [K÷]o as low as 1 mM was able to activate Na + efflux, while the shape of the curve indicates that the activation goes to saturation. As a comparison, a similar activation curve was studied in fresh muscles not subject to any treatment to clamp membrane potential, and this is shown in Fig. 2B. The characteristic of the activation curve in these conditions (e.g. membrane potential variable) was almost exactly the same as that observed when the membrane potential was 2.5 K 105 iIA 112,5 CL
95 K 120 ;JA 220 £L
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Fig. I . Effect of different i n c u b a t i o n s o l u t i o n s o n m e m b r a n e p o t e n t i a l in frog sartorius. O n c e ~ was d e t e r m i n e d in n o z m a l 2.S m M K+-Ringer, the clamping period w a s b e g u n b y incubating the m u s c l e s for 4+ 22 ÷ 3 h in a high K s o l u t i o n . F o r N a r e p l e t+i o n w i t h or w i÷t h o u t N a loading ( d e p e n d i n g o n the÷ experiment), the m u s c l e s w e r e i n c u b a t e d in K -free, hiRh N a s o l u t i o n for 2 h. T h e i n c u b a t i o n in K -free, 1 0 6 m M N a + c o r r e s p o n d s to the c o n t r o l Na + efflux in the absence o f external K+: external N a + w a s red u c e d t o the u s u a l c o n c e n t r a t i o n b y partial r e p l a c e m e n t w i t h Tris. In the n e x t step, the m u s c l e s w e r e in a s o l u t i o n w i t h 10 m M K +, [ N a +] w a s 1 0 5 mM a n d Tris w a s r e d u c e d in a c c o r d a n c e w i t h t h e K + a d d e & This gives the activation of N a + efflux b y external K + at c o n s t a n t E m. (In o t h e r e x p e r i m e n t s n o t s h o w n here, E m w a s also c o n s t a n t for different [K +] .) S u b s e q u e n t changes in s o l u t i o n s (sulfate for chloride and chan~!n_l [K+]) p r o d u c e d t h e e x p e c t e d changes In Era, s h o w I n g that the preperatiori w a s still viable. A t the e n d o f the e x p e r i m e n t , the m u s c l e s r e m a i n e d excitable. In the net flux e x p e r i m e n t s , N a + c o n t e n t was d e t e r m i n e d after the K+free, 1 0 5 m M N a + period (before r e c o v e r y ) and after the 1 0 m M K* p e r i o d (after recovery). In the labelled Na + e f f l u x e x p e r i m e n t s , N a + c o n t e n t w a s f o l l o w e d for 1 h in K+-free, 1 0 6 m M N a + and then for 0 . 5 h in K+-containing media. In s o m e eases o u a b a l n w a s a d d e d ( 1 0 - 4 M) imm e d i a t e l y thereafter and flux w a s f o l l o w e d for a further 0 . 5 h period. Each p o i n t is the m e a n ± S.E. of 1 6 - - 2 4 p u n c t u r e s in f o u r muscles. T e m p e r a t u r e w a s 20 °C and pH 7.4. E m w a s d e t e r m i n e d b y a microe l e c t r o d e technique.
191
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Fig. 2. A c t i v a t i o n c u r v e o f N a + e f f l u x as a f u n c t i o n o f [ K + ] o in f r o g s a r t o r i u s w i t h m e m b r a n e p o t e n t i a l c l a m p e d a t a b o u t - - 2 3 m Y . E a c h p o i n t is t h e m e a n + S.E. o f t h r e e m u s c l e s a n d c o r r e s p o n d s t o a n increm e n t in t h e r a t e c o n s t a n t f o r 22Na+ e f f l u x o v e r t h e v a l u e in K ~ f r e e c o n d i t i o n s . T h e a v e r a g e r a t e of N a + loss in t h e a b s e n c e of K + w a s 1.06 + 0 . 0 8 2 h - 1 (n = 21). 22Na+ e f f l u x w a s d e t e r m i n e d as d e s c r i b e d elsew h e r e [ 1 2 ] . See l e g e n d o f Fig. 1 f o r details. (B) A c t i v a t i o n c u r v e of N a + e f f l u x b y [ K + ] o in f r e s h s a r t o r i u s i n c u b a t e d in 1 0 5 m M N a +. A f t e r l o a d i n g w i t h 22Na+ b y s o a k i n g t h e m u s c l e s f o r 2 h in 5 m M K +, 1 1 4 m M N a + R i n g e r , t h e e f f l u x of labelled N a + w a s f o l l o w e d f o r 1 h in K+-free, 1 0 5 m M N a + a n d t h e n for 0.5 h in s o l u t i o n s o f vs.ried [K+]. T h e t o t a l o s r n o l a r i t y w a s m a i n t a i n e d c o n s t a n t w i t h Trls at 2 3 0 m o s M . P o i n t s are t h e m e a n + S.E. of t h r e e m u s c l e s , e x c e p t t h a t at 10 m M K + w h i c h i n c l u d e s t w o muscles. T h e r a t e o f 22Na+ loss in K + f r e e c o n d i t i o n s w a s 1.25 ± 0 . 0 9 h - 1 (n = 18). T e m p e r a t u r e w a s 20 °C.
clamped. On the ot her hand, and in opposition to the results of Horowicz and Gerber [2], [K+]o as low as 1 mM was able to produce a detectable and consistent increase in ouabain sensitive Na + efflux above the values observed in K +free conditions. The main conclusion that can be drawn from Figs 2A and 2B is t h a t the [K+]c ef f ect on Na + efflux in frog sartorius muscles is the same regardless of wh e t he r the m e m b r a n e potential is variable or constant. Since, b y definition, the Na + p u m p is a mechanism which produces n e t Na ÷ extrusion against an electrochemical gradient, as a furt her test, the effect of 10 mM [K÷]o on net Na + efflux against an electrochemical gradient in the
192
TABLE I N E T S O D I U M E X T R U S I O N IN E m C L A M P E D M U S C L E S ÷ . . . . . . . . Net Na extrusion against an electrochemical gradient in frog sartonus with membran e potential clamped a t a b o u t - - 2 3 m V a n d i n c u b a t e d i n 1 0 m M K ÷, 1 0 5 rnM N a ÷ R i n l e r in the presence and absence o f 1 0 - 4 M o u a b a l n . D e t e r m i n a t i o n s o f cell N a + w e r e a c c o m p l i s h e d b y t h e m e t h o d p r o p o ~ d by S j o d i n a n d Beaug~ [ 1 2 ] . T h e n u m b e r o f m u s c l e s is given in p a r e n t h e s i s . S e e l e g e n d o f Fig. 1 f o r details. T e m p e r a t u r e w a s 20 o C. Muscle N a + (#tool/g) Control
O u a b a i n (10 -4 M )
Before recovery
After recovery
ANa+ (1 h)
Before recovery
After recovery
ANa+ (1 h )
7.26* +0.13 (9)
5.23* +0.18 (9)
--2.04 +0.15 (9)
8.23** +0.50 (6)
9.95** ±0.36 (6)
1.88 ±0.26 (6)
*P ~ 0. 0 0 1 **P
Biochimica et Biophysica Acta, 3 8 9 ( 1 9 7 5 ) 1 8 9 - - 1 9 3 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA Report BBA 7 1 2 2 2
THE INDEPENDENCE OF MEMBRANE POTENTIAL AND POTASSIUM ACTIVATION OF THE SODIUM PUMP IN MUSCLE
L.A. B E A U G E * , R.A. SJODIN and O L G A O R T I Z
Department of Biophysics, Universityof Maryland School of Medicine, Baltimore, Md. 21201 (U.S.A.) (Received January 13th, 1975)
Summary The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K +] by equilibration in a 95 mM K +, 120 mM Na + Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E m sensitive to [K+]. In addition, in the presence of 10 mM K +, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10 --4 M ouabain. This suggests that the membrane potential does n o t play a major role in the potassium activation of the sodium pump in muscles.
The ionic requirements of the Na+-K + pump such as its activation by K +, are the same as those for an ATPase isolated from membrane fractions [ 1 ], leading to the postulate that K + activation of the pump is similar in nature to an enzyme-substrate reaction. In fresh frog skeletal muscle both K + and azide, when applied externally, activate ouabain-sensitive Na + efflux and also depolarize membrane potential (Era) [2, 3]. Since the relation between Na + efflux and transmembrane potential seemed to be the same in both instances, it was postulated t h a t K + activation of the Na + pump is due to its effects on the membrane potential, n o t to a catalytic action on the pump mechanism. On the other hand, in high Na + sartorius muscles, the addition of K + does n o t immediately depolarize the membrane close to the K÷ equilibrium potential (EK); rather, the potential is held at a higher value as a consequence of an electrogenic operation of the Na÷-K + pump [4, 5]. Under these conditions, a definite pump activation is observed with [K +] as low as 1 mM [6]. Since other excitable tissues like squid axon [7] and Anisodoris giant neurone [8], *Member of the Consejo Nacional de Investigationes Cientifieas y T~cnicas of Argentina.
190
as well as non-excitable human red blood cells [9], failed to show any membrane potential dependence of the Na÷-K + pump, it seemed worthwhile to reexamine the matter of the Na + pump activation in skeletal muscle. The idea was to work under conditions where the membrane potential and pump effects of K + could possibly be separated. This was done by taking advantage of the observation that the membrane potential of fibers which have been equilibrated in high K +, normal Na + solutions is insensitive to reduction o f K~ while the membrane itself behaves as a C1- electrode [10, 11]. In this way it was possible to study Na + fluxes at constant Era and variable K + . A basic experimental protocol and the constancy of membrane potential during the whole experiment are shown in Fig. 1. Fig. 2A summarizes all experiments performed to test the effect of [K+]o on Na ÷ efflux in muscles with membrane potentials clamped at about --23 mV. As can be seen, a [K÷]o as low as 1 mM was able to activate Na + efflux, while the shape of the curve indicates that the activation goes to saturation. As a comparison, a similar activation curve was studied in fresh muscles not subject to any treatment to clamp membrane potential, and this is shown in Fig. 2B. The characteristic of the activation curve in these conditions (e.g. membrane potential variable) was almost exactly the same as that observed when the membrane potential was 2.5 K 105 iIA 112,5 CL
95 K 120 ;JA 220 £L
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0 K 10 K 105 :IA 105 [IA 220 CL 22Q CL
+40
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,~
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50
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;
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Fig. I . Effect of different i n c u b a t i o n s o l u t i o n s o n m e m b r a n e p o t e n t i a l in frog sartorius. O n c e ~ was d e t e r m i n e d in n o z m a l 2.S m M K+-Ringer, the clamping period w a s b e g u n b y incubating the m u s c l e s for 4+ 22 ÷ 3 h in a high K s o l u t i o n . F o r N a r e p l e t+i o n w i t h or w i÷t h o u t N a loading ( d e p e n d i n g o n the÷ experiment), the m u s c l e s w e r e i n c u b a t e d in K -free, hiRh N a s o l u t i o n for 2 h. T h e i n c u b a t i o n in K -free, 1 0 6 m M N a + c o r r e s p o n d s to the c o n t r o l Na + efflux in the absence o f external K+: external N a + w a s red u c e d t o the u s u a l c o n c e n t r a t i o n b y partial r e p l a c e m e n t w i t h Tris. In the n e x t step, the m u s c l e s w e r e in a s o l u t i o n w i t h 10 m M K +, [ N a +] w a s 1 0 5 mM a n d Tris w a s r e d u c e d in a c c o r d a n c e w i t h t h e K + a d d e & This gives the activation of N a + efflux b y external K + at c o n s t a n t E m. (In o t h e r e x p e r i m e n t s n o t s h o w n here, E m w a s also c o n s t a n t for different [K +] .) S u b s e q u e n t changes in s o l u t i o n s (sulfate for chloride and chan~!n_l [K+]) p r o d u c e d t h e e x p e c t e d changes In Era, s h o w I n g that the preperatiori w a s still viable. A t the e n d o f the e x p e r i m e n t , the m u s c l e s r e m a i n e d excitable. In the net flux e x p e r i m e n t s , N a + c o n t e n t was d e t e r m i n e d after the K+free, 1 0 5 m M N a + period (before r e c o v e r y ) and after the 1 0 m M K* p e r i o d (after recovery). In the labelled Na + e f f l u x e x p e r i m e n t s , N a + c o n t e n t w a s f o l l o w e d for 1 h in K+-free, 1 0 6 m M N a + and then for 0 . 5 h in K+-containing media. In s o m e eases o u a b a l n w a s a d d e d ( 1 0 - 4 M) imm e d i a t e l y thereafter and flux w a s f o l l o w e d for a further 0 . 5 h period. Each p o i n t is the m e a n ± S.E. of 1 6 - - 2 4 p u n c t u r e s in f o u r muscles. T e m p e r a t u r e w a s 20 °C and pH 7.4. E m w a s d e t e r m i n e d b y a microe l e c t r o d e technique.
191
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Fig. 2. A c t i v a t i o n c u r v e o f N a + e f f l u x as a f u n c t i o n o f [ K + ] o in f r o g s a r t o r i u s w i t h m e m b r a n e p o t e n t i a l c l a m p e d a t a b o u t - - 2 3 m Y . E a c h p o i n t is t h e m e a n + S.E. o f t h r e e m u s c l e s a n d c o r r e s p o n d s t o a n increm e n t in t h e r a t e c o n s t a n t f o r 22Na+ e f f l u x o v e r t h e v a l u e in K ~ f r e e c o n d i t i o n s . T h e a v e r a g e r a t e of N a + loss in t h e a b s e n c e of K + w a s 1.06 + 0 . 0 8 2 h - 1 (n = 21). 22Na+ e f f l u x w a s d e t e r m i n e d as d e s c r i b e d elsew h e r e [ 1 2 ] . See l e g e n d o f Fig. 1 f o r details. (B) A c t i v a t i o n c u r v e of N a + e f f l u x b y [ K + ] o in f r e s h s a r t o r i u s i n c u b a t e d in 1 0 5 m M N a +. A f t e r l o a d i n g w i t h 22Na+ b y s o a k i n g t h e m u s c l e s f o r 2 h in 5 m M K +, 1 1 4 m M N a + R i n g e r , t h e e f f l u x of labelled N a + w a s f o l l o w e d f o r 1 h in K+-free, 1 0 5 m M N a + a n d t h e n for 0.5 h in s o l u t i o n s o f vs.ried [K+]. T h e t o t a l o s r n o l a r i t y w a s m a i n t a i n e d c o n s t a n t w i t h Trls at 2 3 0 m o s M . P o i n t s are t h e m e a n + S.E. of t h r e e m u s c l e s , e x c e p t t h a t at 10 m M K + w h i c h i n c l u d e s t w o muscles. T h e r a t e o f 22Na+ loss in K + f r e e c o n d i t i o n s w a s 1.25 ± 0 . 0 9 h - 1 (n = 18). T e m p e r a t u r e w a s 20 °C.
clamped. On the ot her hand, and in opposition to the results of Horowicz and Gerber [2], [K+]o as low as 1 mM was able to produce a detectable and consistent increase in ouabain sensitive Na + efflux above the values observed in K +free conditions. The main conclusion that can be drawn from Figs 2A and 2B is t h a t the [K+]c ef f ect on Na + efflux in frog sartorius muscles is the same regardless of wh e t he r the m e m b r a n e potential is variable or constant. Since, b y definition, the Na + p u m p is a mechanism which produces n e t Na ÷ extrusion against an electrochemical gradient, as a furt her test, the effect of 10 mM [K÷]o on net Na + efflux against an electrochemical gradient in the
192
TABLE I N E T S O D I U M E X T R U S I O N IN E m C L A M P E D M U S C L E S ÷ . . . . . . . . Net Na extrusion against an electrochemical gradient in frog sartonus with membran e potential clamped a t a b o u t - - 2 3 m V a n d i n c u b a t e d i n 1 0 m M K ÷, 1 0 5 rnM N a ÷ R i n l e r in the presence and absence o f 1 0 - 4 M o u a b a l n . D e t e r m i n a t i o n s o f cell N a + w e r e a c c o m p l i s h e d b y t h e m e t h o d p r o p o ~ d by S j o d i n a n d Beaug~ [ 1 2 ] . T h e n u m b e r o f m u s c l e s is given in p a r e n t h e s i s . S e e l e g e n d o f Fig. 1 f o r details. T e m p e r a t u r e w a s 20 o C. Muscle N a + (#tool/g) Control
O u a b a i n (10 -4 M )
Before recovery
After recovery
ANa+ (1 h)
Before recovery
After recovery
ANa+ (1 h )
7.26* +0.13 (9)
5.23* +0.18 (9)
--2.04 +0.15 (9)
8.23** +0.50 (6)
9.95** ±0.36 (6)
1.88 ±0.26 (6)
*P ~ 0. 0 0 1 **P
