The Influence of External Cations and Membrane Potential on Ca ...

T h e I n f l u e n c e o f External Cations and M e m b r a n e Potential on Ca-Activated Na Efflux in

Myxicola Giant

Axons

R. A. S J O D I N and R. F. A B E R C R O M B I E From the Department of Biophysics, University of Maryland School of Medicine, Baltimore, Maryland 21201

A B S T a A C T In microinjected Myxicola giant axons with elevated [Na],, Na efflux was sensitive to Ca 0 u n d e r some conditions. In Li seawater, sensitivity to Ca 0 was high whereas in Na seawater, sensitivity to Ca0 was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Ca o was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cap-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cap-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cap-activated Na efflux. Experiments in which the m e m b r a n e potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca-activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. T h e same experimental conditions that favored a large component of Cap-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 m M K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]0. Overall, the results suggest that the Cap-activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li > Li > Choline > Na. INTRODUCTION

I n Myxicola g i a n t a x o n s with e l e v a t e d [Na],, N a e f f l u x b e c o m e s sensitive to e x t e r n a l Ca in Li s e a w a t e r ( A b e r c r o m b i e a n d S j o d i n , 1977a). Sensitivity o f N a e f f l u x to Ca was n o t o b s e r v e d in n o r m a l Na s e a w a t e r , n o r was it o b s e r v e d i n Li s e a w a t e r at n o r m a l values o f [Na]~. A n o t h e r f e a t u r e o f this c o m p o n e n t o f N a e f f l u x in Myxicola g i a n t a x o n s is t h a t it is n o t i n h i b i t e d by o u a b a i n at c o n c e n t r a t i o n s t h a t m a x i m a l l y i n h i b i t the N a p u m p . T h e s e p r o p e r t i e s o f the C a - a c t i v a t e d c o m p o n e n t o f N a e f f l u x i n Myxicola g i a n t a x o n s are at least q u a l i t a t i v e l y s i m i l a r to those r e p o r t e d f o r t h e C a - a c t i v a t e d N a e f f l u x o b s e r v e d i n s q u i d g i a n t a x o n s ( B a k e r et al., 1969). T h e C a 0 - d e p e n d e n t N a e f f l u x i n s q u i d g i a n t a x o n s is a c c o m p a n i e d by a N a ~ - d e p e n d e n t Ca i n f l u x , a fact t h a t s t r o n g l y suggests t h e presence of a Na:Ca exchange mechanism. J. GE•. PHYSIOL.~) The Rockefeller University Press. 0022-1295/78/0401-045351.00

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T h e p u r p o s e o f t h e p r e s e n t w o r k is to s t u d y s o m e o f the p r o p e r t i e s o f Caactivated N a e f f l u x in Myxicola g i a n t a x o n s in g r e a t e r detail a n d to i n v e s t i g a t e Ca i n f l u x u n d e r s i m i l a r c o n d i t i o n s to see if a N a : C a e x c h a n g e process is also p r e s e n t in this p r e p a r a t i o n . T h e m a i n a r e a o f t h e i n v e s t i g a t i o n is t h e sensitivity o f C a - a c t i v a t e d N a e f f l u x to the e x t e r n a l cations Na, Li, Mg, La, K, a n d c h o l i n e . As a n y a c t i o n o f e x t e r n a l K c o u l d be d u e to e i t h e r a specific effect o f K o n m e m b r a n e sites i n v o l v e d in N a : C a e x c h a n g e o r to a s e c o n d a r y effect via m e m b r a n e d e p o l a r i z a t i o n , it was also n e c e s s a r y to i n v e s t i g a t e t h e possible i n f l u e n c e o f V m o n the C a - a c t i v a t e d c o m p o n e n t o f N a e f f l u x . A p r e l i m i n a r y r e p o r t o f this i n v e s t i g a t i o n has b e e n m a d e ( A b e r c r o m b i e a n d S j o d i n , 1977 b). MATERIALS

AND

METHODS

Myxicola were obtained from Marine Research Associates, New Brunswick, Canada. Handling of animals, dissection of giant axons, microinjection of 22Na for Na efflux determination, and counting of radioactivity in axoplasm and efflux samples were as previously described (Abercrombie and Sjodin, 1977a). Unless otherwise indicated, axons were microinjected with nonradioactive Na to achieve a final [Na]~ = 100 mM/kg for the reason described previously and in the present text. Also, unless otherwise stated, experiments were performed in the presence of 10-4 M ouabain for reasons previously discussed and also to avoid activation of the Na:K p u m p by K in the experiments performed at elevated [K]o. Efflux samples of 22Na were taken at 3-min intervals. Sample radioactive counts were back-added to the final radioactive counts remaining in the axoplasm at the end of the experiment. The fraction of radioactivity remaining in the axoplasm during the experiment was plotted semilogarithmically against time to obtain rate constants for 22Na efflux. The rate constants remained stable in a solution of constant composition except in cases in which the Cao-activated Na efflux was occurring at a very high rate as in (K + Li) seawater. In such cases a rate constant that declined at a moderate rate with time in a given solution was often observed (Fig. 6). The reason for the decline is not known. It is not due to axon deterioration, however, inasmuch as the decline was not observed in cases where the Ca0-activated Na efflux was occurring at a lower rate nor was it observed in the K-activated, ouabain-sensitive Na p u m p fraction of Na efflux.When a declining rate constant occurred, solution changes were always of the type A-B-A where the letters refer to solution composition. In such cases, the rate constant for a given time interval was taken as the average over the interval. The difference in rate constant between solutions A and B was then obtained using the average of the two rate constants in A for the initial and final time intervals.

Ca Influx Measurement Axons were soaked for 30 min in seawaters of varying composition which also contained 45Ca of known specific activity. At the termination of influx, axoplasm samples were taken and prepared for radioactive counting as previously described. As before, radioactivity was determined using a Beckman low level beta counter (Beckman Instruments, Inc., Fullerton, Calif.). Influx was calculated from a knowledge of radioactivity, the specific activity of Ca in the loading solution, the weight of axoplasm and the radius of the fiber.

Solutions T h e experimental seawater formulations used are summarized in Table I. Solutions with higher K concentrations or different concentrations of Ca were made by replacing an

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455

osmotic equivalent o f the major cation. Na-free and full Na solutions were mixed to obtain the desired Na concentration. T h e p H was adjusted to 7.5 and osmolarity monitored at 950 mosM as previously described. Experiments were p e r f o r m e d at 11~

Membrane Potential Control In some experiments it was necessary to prevent change in resting potential (V,,) when [K] o was altered and also to alter Vm with [K]o held constant. This was accomplished by means o f a platinum electrode attached to the microinjection capillary and a DC current source. T h e experimental a r r a n g e m e n t is d i a g r a m m e d in Fig. 1. T h e Pt-blacked Pt electrode was positioned after microinjection of 2ZNa so as to include all o f the injected region made visible with low concentration o f a dye. T h e 50 V DC voltage source was used to pass current t h r o u g h a 106 1~ resistor providing a constant current controllable by a potentiometer which was adjusted manually. At constant [K]o, Vm could be altered to the desired new value in about 10 s. Current was monitored with a microammeter (not TABLE

I

ARTIFICIAL SEAWATER SOLUTIONS Solution

CaCI2

MgCII MgSO4

NaCI

LiCI

C h o C I Mannitol

Tris. HEPES

EDTA

5 5 5 5

0,5 0.5 0.5 0,5

ram

K-free (Na) K-free (Li) K-free (Cho) K-free (Mg-mannitol)

10 10 l0 10

25 25 25 164

25 25 25 25

430 430 430 450

shown). When [K]0 was altered and it was desired to maintain Vm constant, the potentiometer was adjusted to maintain a constant Vm d u r i n g the -~ l - m i n period required to change solutions and reach stable conditions. The value of Vm was read with an oscilloscope using a high impedance preamplifier to record from an intracellular electrode. RESULTS

The Effects of Nao, Lio, and Cao on Na Efflux in the Presence of Ouabain T h e e f f e c t o f v a r i a t i o n s in [Ca]0 o n t h e Cao-sensitive N a e f f l u x was d e t e r m i n e d in b o t h Li s e a w a t e r a n d N a s e a w a t e r . All e x p e r i m e n t s w e r e p e r f o r m e d w i t h e l e v a t e d [Na]i a n d in t h e p r e s e n c e o f 10 -4 M o u a b a i n to e n h a n c e r e s o l u t i o n o f f l u x c h a n g e s . M e a s u r e m e n t s w e r e m a d e in C a - f r e e s o l u t i o n s a n d in s o l u t i o n s c o n t a i n i n g d i f f e r e n t v a l u e s o f [Ca]o. T h e C a - s e n s i t i v e N a e f f l u x is d e f i n e d as t h e d i f f e r e n c e b e t w e e n e f f l u x m e a s u r e d in t h e p r e s e n c e o f a g i v e n v a l u e o f [Ca]o a n d t h a t m e a s u r e d in a C a - f r e e m e d i u m . T h e r e s u l t s a r e p l o t t e d in Fig. 2 as r a t e c o n s t a n t s f o r 22Na e f f l u x vs. [Ca]0. A c t u a l N a e f f l u x in p m o l / c m ~ . s is p r o p o r t i o n a l to t h e r a t e c o n s t a n t via a f a c t o r t h a t i n v o l v e s t h e v a l u e o f [Na]l a n d t h e d i a m e t e r f o r a n y g i v e n a x o n . As a n a p p r o x i m a t e c a l i b r a t i o n , a r a t e c o n s t a n t o f 1 x 10 -3 m i n -1 is e q u i v a l e n t to a n e f f l u x o f a b o u t 30 p m o l / c m 2 . s f o r t h e v a l u e s o f [Na]i h o l d i n g in t h e s e e x p e r i m e n t s (see T a b l e I I I ) . L o w e r i n g [Na]i r e d u c e d sensitivity o f N a e f f l u x to Cao, a n d at n o r m a l [Na], n o Cao-sensitive N a e f f l u x

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FIouaE 1.

Diagram of experimental arrangement for altering and controlling

Vm. (a) Myxicola axon membrane, (b) internal current passing electrode, (c) external ground electrode, (d) internal potential measuring capillary, (e) calomel electrodes, (f) differential preamplifier, (g) recorder or oscilloscope, (h) outflow, (i) inflow.

1.5

Elevated [Na]i

O_

E

1.0

c tO

n," x

0.5

[Ca]o (raM) FIGURE 2. The effect on Na efflux of replacing Na seawater with Li seawater at different values of [Ca]o. All solutions were K-free and contained 10 -4 M ouabain. The vertical bars represent +-- 1 SE for four or more measurements.

Calcium-ActivatedSodium Efflux

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was observed even in Li seawater. T h e results show that increasing [Ca]o causes an increasing activation o f a c o m p o n e n t o f Na efflux that is considerably higher in Li seawater than in Na seawater. At the n o r m a l value o f [Ca]o - 10 mM, no Gao-sensitive Na efflux was observed in Na seawater in c o n f i r m a t i o n o f o u r previous work (Abercrombie and Sjodin, 1977a). T h e curve in Li seawater seems to a p p r o x i m a t e simple Michaelis kinetics with a Km for Ca o f ~ 8 mM. T h e curves would have a partial explanation if the binding o f an external Li ion at a m e m b r a n e site increased the affinity o f an external activation site for Ca ions above the value existing in Na seawater.

The Influence of Other Cations I n the next series o f experiments, [Ca]o was varied when Nao was replaced with substitutes o t h e r than Li. T h e results are illustrated in Fig. 3. In choline 1.5

[Na]i

Elevated

% 'TC

E fU

S S S S

1.0

# S

/

tO

}

o

a) o

0.5 uJ 0

z

o !

I

I

I

I

o

to

20

3o

40

[Co]0(mM)

FIGURE 3. The influence of substituting choline or Mg for Na in seawater on Na efflux in the absence and in the presence of different concentrations of Ca. All solutions were K-free and contained 10-4 M ouabain. The vertical bars represent -+ 1 SE for four or more measurements. seawater, an activation due to Cao was clearly a p p a r e n t , whereas in Mg seawater no activation o f Na efflux by Cao was observed at any value o f [Ca]o e m p l o y e d . In Fig. 3, the curve obtained in Li seawater is included as a reference. Above [Ca]o -- 10 mM, activation by Cao in choline seawater is intermediate between activation in Na a n d Li seawater until [Ca] = 40 mM, when activations in choline and in Na seawater are about the same. T h e results indicate that no activation o f Na efflux by Cao occurs when only divalent cations are present in the external m e d i u m . This could signify that monovalent cations are required externally for

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a c t i v a t i o n by Cao to o c c u r , o r a l t e r n a t i v e l y , t h a t h i g h c o n c e n t r a t i o n s o f M g i n h i b i t t h e a c t i v a t i o n d u e to Cao.

The Stimulating Effect of K o T h e i n f l u e n c e o f e x t e r n a l K ions o n C a - a c t i v a t e d N a e f f l u x was i n v e s t i g a t e d . R a i s i n g [K]o to a v a l u e o f 20 m M r e s u l t e d in a b o u t a t h r e e f o l d i n c r e a s e in t h e C a - a c t i v a t e d c o m p o n e n t o f N a e f f l u x o v e r t h e v a l u e o b s e r v e d in K - f r e e , Li s e a w a t e r . R a i s i n g [Na]o by s u b s t i t u t i o n f o r Li r e s u l t e d in a d i m i n u t i o n o f Caa c t i v a t e d N a e f f l u x b o t h in K - f r e e s e a w a t e r a n d in s e a w a t e r with [K]o = 20 m M . T h e r e s u l t s a r e s u m m a r i z e d in Fig. 4 w h e r e t h e C a - a c t i v a t e d c o m p o n e n t o f N a e f f l u x is p l o t t e d a g a i n s t [Na]o in t h e N a - Li s e a w a t e r m i x t u r e s . T h e i n f l u e n c e o f e x t e r n a l K is m o s t p r o n o u n c e d w h e n [Na]o -- 100 m M . W h e n [Na]o = 200 Elevated [Na]i

f.O

% M r K Io

x

E c-

0 0

c) 0 n~

Ld 0

0.5

E 0 0

o

.I 0 0 c~

mM [Na]o(LiSubstituted) FmURZ 4. T h e Ca-activated c o m p o n e n t o f Na efflux is plotted against [Na]o in Na-Li seawater mixtures for K-free media and for media in which [K] o = 20 mM. T h e ordinate axis refers to the difference in rate constant for 22Na efflux between that observed in the presence o f 10 mM Cao and that observed in Ca-free media. All solutions contained 10-4 M ouabain. T h e vertical bars represent + 1 SE for four or more measurements.

SJODIN

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459

Calcium-Aaivated Sodium Efflux

ABERCROMBIE

raM, no activation by Cao occurs either in the presence or absence o f K and when [Na]o = 0, the p e r c e n t a g e reduction in Ca-activated Na efflux o c c u r r i n g u p o n removal o f K o is less than when [Na]o = 100 mM. Measurements o f Na efflux were also made at various o t h e r values o f [K]o. T h e results are shown in Fig. 5. T h e e n h a n c i n g effect o f Ko was not complete at 20 mM because a greater Ca-sensitive fraction was observed at [K]o = 50 raM. Also, it should be observed that at a c o m m o n l y e m p l o y e d K concentration, [K]o = 10 mM, K is exerting a

b 1.5 K IC

IOmM C~-Li SW

E Z t...-

m 1.0 z o L) W I,.-

I'

X

0.5 1,1_ U,/ o Z

/I/i~CoF!~ -L SW

o

0

IN |

I

I

I

I

1

0

tO

20

30

40

50

[K]o (raM) FIGURE 5. The influence of [K]o on Na efflux in Li seawater with and without Ca. All solutions contained 10-4 M ouabain. All axons were subjected to experimental elevation of [Na]t. The points with vertical bars represent average values -+ 1 SE for more than four measurements. detectable influence on activation by Cao in Li seawater. T h e presence o f 5 mM La ions completely inhibited the Ca-activated Na efflux o c c u r r i n g in 20 mM K, Li seawater, as illustrated in Fig. 6. T h e Na efflux was r e d u c e d to the value observed in Ca-free 20 mM K, Li seawater, indicating that all o f the Ca-activated Na efflux u n d e r these conditions is blocked by La. Similar e x p e r i m e n t s were p e r f o r m e d in choline seawater. Addition o f K to choline seawater gave rise to increases in the Cao-activated Na efflux as in the case o f Li seawater. In contrast with results in Li seawater, however, addition o f Na to K-free choline seawater first increased the m a g n i t u d e o f the Cao-sensitive Na efflux as [Na]o was increased u p to 100 raM. When [NaJo was increased to values above 100 mM in choline/Na seawater mixtures, the Cao-sensitive Na efflux diminished. T h e effects o f K o are thus similar in Li a n d choline seawaters

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whereas the effects o f Nao differ in that values o f [Na]o < 100 mM e n h a n c e Caoactivated Na efflux in choline seawater but are inhibitory in Li seawater.

Separation of Effects of Vm and [K]o An interesting question arose regarding the effect o f 20 mM K on the Caactivated Na efflux. T h e e n h a n c i n g effect o f K 0 could be due to a secondary ,"'9

2.0

%

2 0 K / O No (Li) ASW ~--Co-Free----~

~SmM

Lo

_4

\ 0

"7 C

E E O

o O

hi O

z

\

AA

t

\ Z~Z~

0

o-"~'o

0.5

I A Z~

Eleveted [NoT i

I

|

I

3O

6O

9O

Minutes

FmuxE 6. The inhibitory action of La ions on Ca-activated Na efflux ([Ca]0 = 10 raM) in 20 mM K, Li seawater (ASW = artificial seawater). All solutions contained 10-4 M ouabain. action via m e m b r a n e depolarization. T o check this point, s o m e e x p e r i m e n t s were p e r f o r m e d in which the m e m b r a n e potential was varied i n d e p e n d e n t l y o f [K]0 by passing electrical currents f r o m a central wire attached to the potential m e a s u r i n g capillary. A typical e x p e r i m e n t a l protocol is illustrated in Fig. 7. First, Na efflux was m e a s u r e d in K-free, Li seawater with 10 mM Ca. Current was then passed to achieve a depolarization o f the m e m b r a n e about equivalent to that p r o d u c e d by [K]o = 20 raM. As is evident from the record, no change in

Calcium-Activated Sodium

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461

Efflux

Na efflux o c c u r r e d . A f t e r r e t u r n i n g to the initial m e m b r a n e potential, the solution was c h a n g e d to 20 m M [K]o, Li seawater. T h e m e m b r a n e d e p o l a r i z e d by a b o u t the a m o u n t that o c c u r r e d with c u r r e n t flow. Now, however, the depolarization was a c c o m p a n i e d by a large increase in Na efflux. F u r t h e r m o r e , a h y p e r p o l a r i z a t i o n by c u r r e n t flow d u r i n g the period o f high Na efflux failed to lower the flux. Subsequent r e m o v a l o f Cao p r o m p t l y r e d u c e d Na efflux to the value m e a s u r e d b e f o r e addition o f K indicating that all o f the increase in Na efflux b r o u g h t a b o u t by Ko was d e p e n d e n t on external Ca. T h e conclusion f r o m this e x p e r i m e n t a n d others similar to it is that the stimulating action o f Ko on I Li ASW+IO'4M OUABAIN > (-----OK-IOCa ~ 20K-IOCc ~'~-~20K-OCc~K-->20K- I0Co moo

,~~

9

,,-.

X

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f

,.,.,f

1.5

2 0 IO

jO00~ x ooo ~

.o

W 0 0.5 Z

9

9

SOso~~ Vrn,mV

L

iO0 Minutes

FIGURE 7. The effects of V m compared with the effect of [K]o on Ca-activated Na efflux in Li seawater (ASW). Numbers before constituents refer to mM concentrations. As before, [Na], was elevated to around 100 mM by microinjection of Na2SO4 before the experiment. the Ca-activated c o m p o n e n t o f Na efflux is a direct effect on m e m b r a n e sites at which Ca ions activate Na efflux r a t h e r t h a n an indirect effect via the m e m b r a n e potential. T h e m e m b r a n e potential p e r se, on the o t h e r h a n d , does not a p p e a r to have a r e g u l a t o r y effect on the Ca-activated c o m p o n e n t o f Na efflux. T o answer a possible criticism that d i f f e r e n t conclusions m i g h t have b e e n r e a c h e d had o u a b a i n not b e e n present, similar e x p e r i m e n t s were p e r f o r m e d in the absence o f ouabain. T h e results are shown in Fig. 8. T h e main d i f f e r e n c e in the results is that, in the absence o f ouabain, m e m b r a n e depolarization via c u r r e n t flow now has a detectable effect on Na efflux. Depolarizing the m e m b r a n e by an a m o u n t r o u g h l y equivalent to that p r o d u c e d by [K]o = I0 m M increased Na efflux in a K-free m e d i u m by a b o u t 10%. T h e increase is quite small, however, c o m p a r e d with the increase in Na efflux b r o u g h t a b o u t by [K]0 = 10 mM a n d a m o u n t s to only 15% o f the K - d e p e n d e n t increase. F u r t h e r m o r e ,

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the effect o f Vm on Na efflux in the absence o f o u a b a i n has an a d e q u a t e explanation that is i n d e p e n d e n t o f the action o f Cao. W h e n a depolarizing c u r r e n t is passed, a net efflux o f K ions occurs to carry the c u r r e n t . In a K-free m e d i u m with no external Na to inhibit activation o f the Na p u m p by K o, the outflowing K ions would be e x p e c t e d to have an activating effect on the Na p u m p in the absence o f ouabain but not in the p r e s e n c e o f ouabain. Also, the presence o f a d e q u a t e external K should r e m o v e or r e d u c e the effect. T h e results in Fig. 8 show that this prediction is b o r n e out e x p e r i m e n t a l l y .

Dependence of Ca Influx on [Na]o Ca influx was first m e a s u r e d on fresh, n o r m a l Myxicola giant axons (see Methods). A x o n s were t h e n microinjected with Na2SO4 to achieve elevated 20

Li .4SW

1r

OK=IOCc ~

b_

',OK-IOCc

)

~

"'..y%_.

x re:

OK-IOCo

15 9

i+,..., ~-_ ; ~

e" 0 (D

>

/-\..

I0 ,o

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II

0.5

/....

0 Z

r Od

i 30

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M Inures

FIGURE 8. ouabain.

I 90

l

so706~ VlTI' mV

J 120

The effect of V,, on Na efflux in Li seawater (ASW) in the absence of

[Na]~, a n d Ca influx was m e a s u r e d in d i f f e r e n t external media. F r o m a knowledge o f axon d i a m e t e r , length o f injection p a t h , injection v o l u m e , a n d concentration o f the Na2SO4 injected, the change in [Na]~ could be calculated for each axon. T h e n o r m a l [Na]~ for fresh axons was taken to be 20 mM, the average b e t w e e n previous results obtained by flame analysis in o u r l a b o r a t o r y and those r e p o r t e d by Gilbert (1975). T h e results are s u m m a r i z e d in T a b l e II. In Na seawater, a fivefold elevation o f [Na]i p r o d u c e d no significant c h a n g e in Ca influx. Elevation o f [Na]i a n d r e p l a c e m e n t o f e x t e r n a l Na by Li, however, p r o d u c e d a 3.5-fold increase in Ca influx. T h e value o f Ca influx m e a s u r e d in 20 mM K, Li seawater did not differ significantly f r o m that m e a s u r e d in 0 K, Li seawater. T h e p r e s e n c e o f 5 mM La inhibited Ca influx in Li seawater to values below those m e a s u r e d in Na seawater. T h e results clearly reveal a c o m p o n e n t o f Ca influx that is sensitive to [Na]. This c o m p o n e n t o f Ca influx is activated by the same conditions that favor a C a o - d e p e n d e n t Na efflux a n d inhibited by the

SJODIN AND ABERCROMBIE

Calcium-Activated

Sodium

463

Efflux

same conditions that inhibit C a 0 - d e p e n d e n t Na efflux, n a m e l y high [Na]0 or La. F r o m a k n o w l e d g e o f the m e a s u r e d rate constants for Na efflux, the a x o n diameters, a n d the values o f [Na]t, Na efflux can be calculated in units o f p m o l / cm 2" s. T h e C a - d e p e n d e n t Na efflux can be d e t e r m i n e d by subtracting efflux in the absence o f Cao f r o m efflux in the p r e s e n c e o f Cao. T h e average values obtained for two conditions, K-free a n d [K]0 = 20 m M , are p r e s e n t e d in T a b l e I I I . T h e flux values r e p o r t e d in Tables I I a n d I I I can t h e n be used to arrive at estimates o f the stoichiometry o f the N a : C a interchanges (see Discussion). TABLE

II

Ca INFLUX FROM 10 mM Cao SOLUTIONS External

solution, mM

Observations*

[Na], 3= SE

n

mMIkg

pmollcm 2 .s

3 3 3 7 5

20---5 97--.17 111-+12 90-+5 128-+12

1.28+-0.43 1.57-+0.37 5.47-+1.52 6.71-+1.44 0.90-+0.19

0 K-Na SW 0 K-Na SW 0 K-Li SW 20 K - L i S W 20 K - L i S W + L a

Ca influx • SE

* Number of experimental observations (Ca influx determinations) on separate axons. TABLE

III

10 mM Cao-DEPENDENT Na EFFLUX IN Na-FREE (Li) ASW [K]o

Observations

Rate constant

Diam

Approx. Na~

Efflux • SE

mM

n

x l O-s rain -t

~

mM / kg

pmo[/cm I ' s

0 20

6 4

0.37-+0.08 1.0-----0.08

642-'-24 675•

114--+7 104 +-- 10

11 • 29~4.4

DISCUSSION

T h e results show that elevation o f [Na]i in Myxicola giant axons gives rise to a c o m p o n e n t o f Na efflux that is activated by Cao in a m a n n e r that d e p e n d s strongly on the cation composition o f the external m e d i u m . T h e Cao-activated Na efflux is large in Li seawater at n o r m a l values o f [Ca]o a n d is a c c o m p a n i e d by an increased influx o f Ca that is revealed e x p e r i m e n t a l l y in axons with elevated [Na]t w h e n e x t e r n a l Na ions are replaced by Li ions. T h e results are so similar to those o b s e r v e d in squid giant axons that it seems safe to conclude that the same general m e c h a n i s m for linked Na:Ca t r a n s p o r t is present in Myxicola giant as is present in squid axons (Baker et al., 1969). T h e Myxicola giant a x o n is thus a suitable p r e p a r a t i o n in which to study Na:Ca exchanges. T h e ratio o f the a v e r a g e C a o - d e p e n d e n t Na efflux to the a v e r a g e Nao-sensitive Ca influx in Myxicola giant axons is 2.8 - 40% f r o m the p r e s e n t data in K-free solutions. T h e r a n g e for this ratio observed in squid giant axons was 3-5 (Baker et al., 1969). T h e effects o f e x t e r n a l cations on the glycoside-insensitive Na efflux in squid giant axons has b e e n investigated (Baker et al., 1969; Beaug6 a n d Mullins, 1976). T h e cations K a n d Li had mainly an activating effect on glycoside-treated axons as did Na at concentrations below 200 mM. At higher concentrations, Na was inhibitory. Choline h a d no activating effect above the value o f efflux o b s e r v e d in Na seawater. In Mg seawater, h o w e v e r , Na efflux was below that o b s e r v e d in

464

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y 9 V O L U M E

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9 1978

either choline or Na seawater. In addition, little or no Ca sensitivity o f Na efflux was observed in Mg seawater (Beaug~ and Mullins, 1976). T h e i r work shows that one m u s t exercise caution in ascribing effects o f external cations in glycoside-treated axons to the Ca-sensitive c o m p o n e n t o f Na efflux, as the cations can induce an i n c r e m e n t in Na efflux a p a r t f r o m the action o f Ca. T h e previous results with K ions in squid giant axons especially suffers f r o m this difficulty; it is not clear how m u c h activation is an e n h a n c e m e n t o f activation by Ca and how m u c h activation is simply a K-induced Na efflux. Also, the role o f m e m b r a n e depolarization at elevated values o f [K]o is u n k n o w n in the previous work. In the p r e s e n t work, care has been taken to delineate the Ca0-sensitive c o m p o n e n t o f Na efflux a n d to d e t e r m i n e the possible role o f the m e m b r a n e potential. T h e main conclusions a b o u t the effects o f external cations on the Cao-sensitive Na efflux in Myxicola giant axons are that the external m o n o v a l e n t cations tested vary in their ability to s u p p o r t activation by Ca a n d that the only divalent cation used as a Na r e p l a c e m e n t , Mg, failed to s u p p o r t any detectable activation by Ca. T h e action o f Nao is interesting because some evidence for a dual effect on activation by Ca 0 is present. W h e n [Ca]o is raised to 40 m M , e x t e r n a l Na clearly s u p p o r t s activation by Ca o (Fig. 2). W h e n Na is a d d e d to Li seawater, h o w e v e r , only decreases in the Ca0-activated Na effiux occurred. This could be partially due to the r e d u c t i o n in [Li] that occurs in Na-Li seawater mixtures. It is unlikely that this is the sole explanation, however, as in K-free m e d i a the Cao-activated Na efflux was r e d u c e d by two-thirds w h e n [Na]o = 100 mM a n d [Li]o was still as high as 330 m M . W h e n Na 0 was increased in e x p e r i m e n t s such as those shown in Fig. 4 using choline instead o f Li as the substitute cation, m o r e c o m p l e x kinetics were observed. At low concentrations, Nao increased the Cao-activated Na efflux whereas at h i g h e r concentrations an inhibition o c c u r r e d in a g r e e m e n t with results r e p o r t e d for squid giant axons (Baker et al., 1969). H o w e v e r , the Ca0-activated Na effluxes o b s e r v e d in Myxicola axons using choline/Na or choline/Na + K seawater m i x t u r e s were not as r e p r o d u c i b l e as those in Li seawater mixtures. T h e r a t h e r large e n h a n c i n g effect o f K o on activation o f Na efflux by Ca is clearly in addition to any e n h a n c e m e n t due to Li alone. Also, the e n h a n c i n g effect o f K 0 is i n d e p e n d e n t o f its action in depolarizing the m e m b r a n e . T h e influence o f K o is interesting because Ca-activated Na efflux was increased with little or no effect on N a t - d e p e n d e n t Ca influx. T a k e n at face value, this would indicate that elevating [K]0 alters the stoichiometric ratio o f Na to Ca in Myxicola giant axons. Calculations indicate that the ratio is increased to a value o f 5.6 -+ 33% in Li seawater with 20 mM K. Some caution is, however, w a r r a n t e d in this i n t e r p r e t a t i o n because o f the m a g n i t u d e o f the i n h e r e n t e r r o r s involved a n d because the axons used for Na efflux and Ca u p t a k e in the two conditions (i.e., 0 m M K [Li] and 20 m M K [Li]) h a d slightly d i f f e r e n t concentrations o f Nat (Tables I I a n d I I I ) , a n d Ca fluxes are k n o w n to d e p e n d strongly on Nay T h o u g h a completely satisfactory explanation for these results a n d the previously discussed results on squid axons does not yet exist, it seems clear that Ca ions act at external m e m b r a n e sites to activate a portion o f Na efflux a n d

SJODIN AND

ABERCROMBIE Calcium-ActivatedSodiumEfflux

465

that the ability o f Ca ions to activate at these sites d e p e n d s strongly on external cations. Either the cations affect the affinity for Ca, they control the p r o p o r t i o n o f the sites that are in a suitable c o n f o r m a t i o n to react with Ca, or they themselves e x e r t a catalytic effect on the translocation step in the presence o f Cao. A theory for similar results in squid giant axons has been p r o p o s e d (Baker et al., 1969). T w o externally directed kinds o f carrier sites are postulated, one that can bind either Ca or two m o n o v a l e n t cations and a n o t h e r that binds only monovalent cations. When the sites that bind either Ca or two m o n o v a l e n t cations are occupied by Ca and the monovalent cation sites are occupied, transport is activated. Inhibitory effects according to this model are ascribed to displacement o f Ca f r o m the Ca site by some monovalent cations. None o f the present results are inconsistent with this view and most o f t h e m could be explained on the basis that the Ca site has a relatively low affinity for Li and choline and a m u c h higher affinity for Na. T h e action o f K ions is less clear according to this model inasmuch as addition o f a relatively small concentration o f K (20 mM) to a m u c h larger concentration o f Li gave about a t h r e e f o l d e n h a n c e m e n t o f the Cao-activated Na efflux. It is possible that the m o n o v a l e n t site is not saturated even when [Li]o = 430 mM and that these sites have a considerably h i g h e r affinity for K. It is also possible that the model is incomplete, at least in Myxicola giant axons, and that a third carrier site is present that binds K with high affinity to modulate either the affinities o f the o t h e r sites for ions or the velocity o f t u r n o v e r o f the carrier. F u r t h e r investigation would be required to address these points. Because o f the fact that monovalent cations have the potential to both catalyze transport by the carrier and displace Ca f r o m the carrier, it is not possible to assign very precise affinities or rank o r d e r s o f effectiveness in the absence o f m u c h more kinetic data. T h e most reasonable m o d e l - i n d e p e n d e n t statement that can be made is that at a total external monovalent cation concentration o f 430 mM, the o r d e r o f effectiveness in s u p p o r t i n g carrier activation by Ca in Myxicola giant axons is K + Li > Li > choline > Na. T h e lack o f an effect o f m e m b r a n e potential alone on Ca-activated Na efflux in Myxicola giant axons deserves some c o m m e n t . It is widely accepted that the Ca-activated Na efflux is a part o f a more general mechanism for t r a n s p o r t i n g Ca and Na ions in either direction across excitable cell m e m b r a n e s (Reuter and Seitz, 1968; Baker et al., 1969; Blaustein and Hodgkin, 1969; Brinley et al., 1975; Blaustein and Russell, 1975; Mullins and Brinley, 1975). At low values o f [Na]~, the Na:Ca exchange system operates to move Ca out o f the cell via the large inward gradient for Na. Mullins and Brinley (1975) f o u n d Ca efflux in squid giant axons to have the sort o f d e p e n d e n c e on Vm that would be e x p e c t e d if changes in Vm acted by altering the electrochemical gradient for Na. For this reason, one might have expected V,, to influence the Ca-activated Na efflux as well, assuming that it is due to essentially the same system o p e r a t i n g in a reversed direction. An explanation for the lack o f effect o f Vra in the present study might be that [Ca]t was h i g h e r than in the e x p e r i m e n t s on squid axons in which Nao-dependent Ca effiux was f o u n d to be a function o f Vm. In squid axons, sensitivity o f Ca efflux to Vm was greatly r e d u c e d when [Ca]t was

466

THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 71 9 1978

e l e v a t e d . A l t h o u g h f r e s h Myxicola a x o n s r e m a i n e d in Li s e a w a t e r f o r o n l y 15 m i n b e f o r e a l t e r i n g V m, a s i g n i f i c a n t i n c r e a s e in [Ca]i c o u l d h a v e o c c u r r e d . C l e a r l y f u r t h e r i n v e s t i g a t i o n w o u l d b e u s e f u l in Myxicola g i a n t a x o n s in w h i c h [Ca]t c o u l d b e c o n t r o l l e d a n d a l t e r e d d u r i n g t h e e x p e r i m e n t by m e a n s o f i n t e r n a l dialysis. The authors wish to acknowledge the competent technical assistance of Mr. George Bury. This work was supported by grants NS 07626 from the National Institutes of Health, U. S. Public Health Service, and 74 12343 from the National Science Foundation, Bureau of Medical Sciences.

Receivedfor publication 3 November 1977. REFERENCES

ABERCROMBIE, R. F., and R. A. S J O D I N . 1977a. Sodium efflux in Myxicola giant axons.J. Gen. Physiol. 69:765-778. ABERCROMBIE, R. F., and R. A. SjODIN. 1977b. Calcium-dependent sodium efflux in Myxicola giant axons. Biophys. J. 17(No. 2):155a. (Abstr.) BAKER, P. F., M. P. BLAUSTEIN, A. L. HOBGKIN, and R. A. STEINHARDT. 1969. T h e influence of calcium on sodium efflux in squid axons. J. Physiol. (Lond.). 200:431-458. BEAUG~, L. A., and L. J. MULLINS. 1976. Strophanthidin-induced sodium efflux. Proc. R. Soc. Lond. B Biol. Sci. 194:279-284. BLAUSTEIN, M. P., and A. L. HOOGKIN. 1969. T h e effect of cyanide on the efflux o f calcium from squid axons. J. Physiol: (Lond.). 20@:497-527. BLAUSTEIN, M. P., and J. M. RUSSELL. 1975. Sodium-calcium exchange and calciumcalcium exchange in internally dialyzed squid giant axons.J. Membr. Biol. 22:285-312. BRINLEY, F. J., JR., S. G. SPANGLER, and L. J. MULLINS. 1975. Calcium and E D T A fluxes in dialyzed squid axons. J. Gen. Physiol. 66:223-250. GILBERT, D. S. 1975. Axoplasm chemical composition in Myxicola and solubility properties of its structural proteins.J. Physiol. (Lond.). 253:303-319. M U L L I N S , L . J . , and F. J. BRINLEY,JR. 1975. T h e sensitivity o f calcium efflux from squid axons to changes in membrane potential. J. Gen. Physiol. 65:135-152. REUTER, H., and N. SEITZ. 1968. T h e d e p e n d e n c e of calcium effiux from cardiac muscle on t e m p e r a t u r e and external ion composition. J. Physiol. (Lond.). 195:451-470.