presence of ATP a similar change did not have an appreciable effect. ... derived from the coupled inward movement of several Na ions down their ... of internal Na is in the neighborhood of 1-4 mM under the condition NadNat = 440/0. ... in Table I. Solutions which required a nonstandard concentration of an electrolyte were.
Calcium Efflux from Squid Axons under Constant Sodium Electrochemical Gradient JAIME REQUENA From the Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Caracas 101, Venezuela
A B ST R AC T The effect of varying Nao and Nai on Ca efflux while maintaining the ratio Na0/Na i constant was explored in squid giant axons dialyzed with and without ATP. In the absence of ATP, the Ca efflux increased 3.4 - 0.2-fold when the Nao/Nal concentrations were reduced from 440/80 to l l0/20 mM. In the presence of ATP a similar change did not have an appreciable effect. The inhibition of Ca efflux produced by Nat was studied in the presence and in the absence of ATP. In the absence of ATP, inhibition is very marked and is reminiscent of a unimolecular noncompetitive reaction (inactivation constant [Kt] of 34 -+ 5 mM of Na0 whereas in the presence of ATP, the slight inhibition observed indicates that ATP probably increases the Kt to 200 raM. From the inhibition of the Ca efflux produced by Nai in the presence or absence of ATP a curve describing the dependence of Nai of the ATP-promoted fraction of Ca efflux was constructed. The effect of Nao on Ca efflux was studied as a function of [Na]l: at low Na, an activation constant (Ka) of 41 mM for Na 0 was obtained either in the presence or in the absence of ATP. As the intracellular Na is increased in the presence of ATP, Na, seems to have no effect on the apparent half-activation constant. However, in the absence of ATP, the Ka for activation increases along a sigmoid curve reaching a value of ll2 mM at 100 mM Nat. It is concluded that the Ca efflux system uses the energy of the Na electrochemical gradient. The action of Nat appears to be such that the interaction of a single Na + is sufficient to block Ca extrusion whereas several Naps externally are necessary to activate Ca extrusion. INTRODUCTION T h e mechanism responsible for maintaining the very low concentration o f ionized intracellular Ca observed in nerve cells (DiPolo et al., 1976) must reside in the cell m e m b r a n e ( H o d g k i n and Keynes, 1957). Much work has been d o n e in a t t e m p t i n g to characterize this mechanism and to d e t e r m i n e the n a t u r e o f its e n e r g y source which is capable o f s u p p o r t i n g the extrusion o f Ca against a large and inwardly directed electrochemical gradient (Baker, 1972, 1976; Blaustein, 1974, 1976; Mullins, 1976). Only three possibilities are t h o u g h t to be feasible for the e n e r g y source o f this transport process: the inwardly directed Na electrochemical gradient; the hydrolysis o f a high e n e r g y nucleotide such as A T P ; o r a combination o f both. T h e idea that the e n e r g y for e x t r u d i n g Ca out o f a cell is derived f r o m the coupled inward m o v e m e n t o f several Na ions d o w n their electrochemical g r a d i e n t was developed by Reuter and Seitz (1968) in their study o f Ca efflux in cardiac muscle, and by Blaustein a n d H o d g k i n (1969) in their J. Gz~. PMVS1OL.@ The Rockefeller University Press 90022-1295/78/1001-044351.00 Volume 72 October 1978 443-470
443
444
T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 7 2 " 1 9 7 8
d e m o n s t r a t i o n that most o f the Ca efflux f r o m squid a x o n d e p e n d e d on external Na (Nao). T h e intracellular dialysis technique, d e v e l o p e d by Brinley a n d Mullins (1967), m a d e possible the study o f the effect o f highly labile c o m p o u n d s such as A T P on t r a n s p o r t m e c h a n i s m s . DiPolo (1974) clearly showed that the addition o f A T P to the internal m e d i a in a dialyzed squid a x o n caused a trebling o f the Ca efflux level o b s e r v e d in the virtual absence o f the nucleotide. T h i s fraction o f Ca efflux stimulated by A T P was shown also to be d e p e n d e n t u p o n Nao a n d Cao. In injected axons a similar effect o f A T P has b e e n i n f e r r e d (Baker a n d Glitsch, 1973). A l t h o u g h the a b o v e - m e n t i o n e d evidence a n d the specificity o f the m e c h a n i s m for A T P (DiPolo, 1976, 1977) would a p p e a r to s t r e n g t h e n the hypothesis that A T P can energize the Ca efflux m e c h a n i s m , the hydrolysis o f A T P as a direct result o f Ca extrusion has not b e e n observed. In the p r e s e n t study, the effect on Ca efflux o f varying the concentrations o f external a n d internal Na, while m a i n t a i n i n g a constant electrochemical g r a d i e n t for Na, was studied in squid giant axons dialyzed with a n d without A T P . This study o f the effect o f the absolute concentrations o f Na on Ca efflux is c o m p l e m e n t e d by separate observations, in the p r e s e n c e a n d in the absence o f A T P , o f the effect on Ca efflux o f the concentration o f internal Na (Nai) at constant Nao a n d o f the c o n c e n t r a t i o n o f external Na at constant Na~. I n the discussion an e x p l a n a t i o n is d e v e l o p e d for the effect on the Ca efflux o f the absolute concentrations o f Na, at a constant electrochemical g r a d i e n t for Na. A p r e l i m i n a r y r e p o r t o f these findings has b e e n c o m m u n i c a t e d to the AsoVAC (Asociacfon Venezolana p a r a el Avance de la Ciencia) (Requena, 1976), a n d to the Biophysical Society (Requena, 1978). METHODS
The experiments reported here were performed on giant axons isolated from living specimens of the tropical squid Dorytheutis plei. The hindmost axon from the steUate ganglion was dissected and carefully cleaned of connective tissue under a dissecting microscope. Axon diameters were measured using a calibrated eyepiece and usually were of the order of 400 ~m. The dialysis chamber had provisions for stimulation and extracellular recording of action potentials. In all the experiments reported here, isotope was collected as long as the axon showed signs of electrical activity, provided the external solution permitted such a response. The axon was kept in the dialysis chamber at 18 + I~ under continuous solution flow. The apparatus and basic technique for internal dialysis of giant excitable cells have been described previously and were used in this study with minor modifications (Brinley and Mullins, 1967; Brinley et al., 1975; DiPolo, 1977). Plastic tubing, specially manufactured by Fabric Research, Ltd. (Needham, Mass.) and kindly supplied by Professors F. j. Brinley and L. J. Mullins, was used for dialysis capillaries. The tubing was 145 /xm OD x 95 /xm ID. It was stretched (DiPolo, 1977) and rendered porous by soaking for 24 h in 0.05 M NaOH, 0.005 M EDTA (ethylenediaminetetraacetic acid). The porosity of the capillaries was occasionally checked by measuring the amount of 45Ca which permeated through the porous wall. It should be mentioned that although some internal dialysis media contained no Na, this concentration is probably not the actual one at the inner side of the axolemma. For this reason the experimental condition of zero Nat is referred throughout as nominally zero Na~. Most probably the actual concentration of internal Na is in the neighborhood of 1-4 mM under the condition NadNat = 440/0.
REQUENA Ca Afflux and Na in Squid Axon
445
Solutions T h e composition of the external and internal solutions used in the experiments is listed in Table I. Solutions which required a nonstandard concentration o f an electrolyte were prepared by mixing appropriate amounts o f stock solutions. All o f the inorganic chemicals used in the preparation of the solutions were reagent grade and when possible they were chosen so as to have minimum quantities of contaminant Ca, Na, and Mg. T h o s e solutions described as free o f a given cation, usually showed a contamination in the micromolar range for that particular ion as determined with emission (Na) or atomic absorption spectrometry (Ca, Mg). T h e external solutions were prepared SO~-free to avoid complexation o f Ca and (or) Mg. T h e pH o f these solutions was buffered to 7.8 with 10 mM of Tris (tris (hydroxymethyl) aminomethane). T h e biochemical reagents used in this work were purchased from Sigma Chemical Co. (St. Louis, Mo.) with the exception o f Hepes (N-2-hydroxyethylpiperazine-N'2-ethaneTABLE
I
SOLUTIONS External
Constituents
ASW
O Na
Internal O Na O Ca
O Ca
ANa
ACh
HNa
HCh
340 100 5 * 0 0 110 0 0 1 330 300 10 7.3 990
340 0 5 * 100 0 110 0 0 1 330 300 10 7.3 990
240 200 5 1.01 0 0 210 0 0 1 230 300 10 7.3 990
240 0 5 1.01 200 0 210 0 0 I 230 300 10 7.3 990
ram
K Na Mg Ca Choline TRIS CI CNEDTA EGTA Aspartate Glycine Hepes pH mosmol/kg
10 440 50 10 0 10 575 1 0.1 0 0 0 0 7.8 1,010
10 0 50 10 440 10 575 1 0.1 0 0 0 0 7.8 1,010
10 0 50 0 460 10 575 1 0.1 0 0 0 0 7.8 1,010
10 440 60 0 0 10 575 1 0.1 0. t -7.8 1,010
* Variable (see text). sulfonic acid) which was purchased from Calbiochem (San Diego, Calif.). T h e p H o f the internal solutions was set to 7.3 with 10 mM o f H e p e s - K O H buffer mixture. Stock solutions o f 100 mM Tris or Mg A T P neutralized to p H 7.2 were stored at -20~ When necessary, aliquots of this work solution were added, before the experimental run, to the radioactive dialysis solutions. T o destroy the functional capacity o f mitochondria to produce A T P and (or) sequester calcium from the axoplasm, the external media was always made to contain 1 mM C N by addition o f an aliquot o f freshly made stock C N - solution (Na + or K + as required), while to the internal solution 0.1 ~l/mi o f a dimethylsulfoxide solution containing 2 and 5 /xg/ml o f the uncoupler FCCP and oligomycin was added. Carbonyl cyanide ptrifluoromethoxyphenylhydrazone (FCCP) was a kind gift from Dr. A. Scarpa. Although isethionate is a normal constituent o f the axoplasm, it was excluded from the dialysis media because the commercially available K salt (Eastman Organic Chemicals
446
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 " V O L U M E 7 c) " ~ 9 7 8
Div., Eastman Kodak Co., Rochester, N. Y.) is heavily contaminated with Na and Ca. The ionized calcium concentration of the internal solution was set at will by using EGTA (ethyleneglycol-bis-(~-aminoethyl ether) N,N' tetraacetic acid) as a calcium buffer system. The total calcium concentration required to give a desired ionized fraction was produced by adding given amounts of CaClz from stock solutions to the internal dialysis solution which always contained 1 mM EGTA. In the computation of the fraction of the total calcium which is ionized, the apparent dissociation constant of 0.15 wM was taken for the Ca-EGTA complex; this is the value computed by DiPolo et al. (1976) for the complex at physiological pH and ionic strength. The dialysis solution was made radioactive by the addition of a desired amount of 4~CaCL2 of the highest specific activity available (usually 20 mCi/mg) obtained from New England Nuclear (Boston, Mass.). As a visual tracer of the radioactive dialysis solution, an aliquot of stock solution of phenol red was added such as to give a final concentration of 0.5 raM. This solution had its final pH corrected to 7.3 + 0.1 because this is the value that has been obtained for the axoplasm (Boron and De Weer, 1976). The osmolality of the internal dialysis solutions was set to 990 mosmol/kg using a commercial psychrometer which compared the dewpoint of the solution with that of a standard solution of NaCI (5700A osmometer, Wescor Inc., Logan, Utah). The external solutions were adjusted to 1010 mosmol/kg by similar means. RESULTS
The Effect on Ca Efflux of the Concentration of Sodium at Constant Electrochemical Gradient for Na in the Absence of ATP D u r i n g the past few years the effect on Ca efflux o f varying the c o n c e n t r a t i o n o f Na b a t h i n g o n e face o f the a x o l e m m a while the Na concentration is k e p t constant in the o t h e r side o f it has b e e n studied in some detail (Blaustein et al., 1974; DiPolo, 1974; Brinley et al., 1975). T h e i n t e r p r e t a t i o n o f the results obtained, in t e r m s o f a m o d e l for the energetics o f Ca t r a n s p o r t , in which the Na electrochemical g r a d i e n t is t h o u g h t to be the e n e r g y source, is not unequivocal, however, i n a s m u c h as the effect o f a chemical interaction o f Na with the t r a n s p o r t m e c h a n i s m could not be easily s e p a r a t e d f r o m that o f changes in the e n e r g y supplied by the Na electrochemical gradient. In view o f this, it was t h o u g h t to be o f interest to study the effect on Ca efflux o f various Na concentrations at constant electrochemical gradient for Na. Fig. 1 shows a typical e x p e r i m e n t . T h e time-course for the Ca efflux f r o m an a x o n dialyzed with a m e d i u m free o f A T P and an ionized calcium level b u f f e r e d to 0.5 t~M was observed u n d e r several concentrations o f Nao a n d Na~, m a i n t a i n i n g the ratio Na0/Na~ constant. At the b e g i n n i n g o f the e x p e r i m e n t the external m e d i u m c o n t a i n e d 440 m M Nao a n d 80 m M Nat. At this level o f N a concentrations, called the control condition, this a x o n showed a steady-state Ca efflux o f 0.08 pmol.cm-S.s -~. W h e n the Na concentrations were halved to 220 m M Nao and 40 m M Nai the Ca efflux level increased to 0.19 pmol.cm-2.s -a, even t h o u g h these new concentrations o f Na had the same ratio as in the control condition. F u r t h e r reduction o f the absolute concentration o f Na to 165/30 mM N a d N a , raised the Ca efflux to a value o f 0.30 pmol.cm-~.s -~. W h e n the Na concentrations were m a d e o n e - f o u r t h that o f the control condition, 110 a n d 20 mM o f NadNa~, respectively, the calcium efflux r e a c h e d a p e a k value o f 0.43 pmol-cm-2-s -~. This level for the Ca efflux r e p r e s e n t s an almost f o u r f o l d increase w h e n
REQUENA Ca Efflux and Na in Squid Axon
447
c o m p a r e d with 0.12 pmol.cm-2.s -~ o b s e r v e d when the Na concentrations were r e t u r n e d to the control levels. Finally, a large fraction o f the Ca efflux level was d e p e n d e n t on the p r e s e n c e o f external Na and Ca, as shown at the e n d o f the experiment. T a b l e I I s u m m a r i z e s the results o f all the e x p e r i m e n t s d o n e in a fashion
OUT IN
I_, 1,01 i1 0tco 440
220
165
80
40
30
I10 20
440
0
80
0.5
R 0 40376 A e 5 5 0 pm Co~*0.5pM
0.4
7
E u o
0.3
E v X / 0.2 o
0.1
I
I I
I
I 2
I
I 3
I
I 4
TIME ( h o u r s )
l. The time-course of the Ca efflux under various concentrations of Nao/ Na~ at a constant electrochemical gradient for Na in an ATP-free dialyzed squid axon. FIGURE
similar to the o n e j u s t described. In all o f these e x p e r i m e n t s the intracellular ionized calcium concentration was b u f f e r e d to 0.5/zM while the extracellular Ca was kept constant at 10 m M . T h i s table lists the o b s e r v e d Ca efflux level for each test condition, the ratio o f e x t e r n a l / i n t e r n a l Na concentrations always b e i n g the same. In all cases listed, the Ca efflux level increases above that o f the control condition w h e n b o t h concentrations o f Na were p r o p o r t i o n a l l y r e d u c e d .
448
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 72 9 1978
THE jOURNAL
Fig. 2 shows relative Ca efflux values plotted as a function o f the external a n d internal concentrations o f Na at a constant electrochemical g r a d i e n t for this cation. For each e x p e r i m e n t listed in T a b l e I I , Ca efflux values were t a k e n a n d n o r m a l i z e d with respect to the level o b s e r v e d at 440/80 m M Na0/Nat, to which the value o f 1.0 was assigned. T h e line d r a w n in the figure is the s m o o t h curve that joins the m e a n for all o f the e x p e r i m e n t a l points for a given set o f Na concentrations. It can be seen that the reduction o f the absolute concentrations o f sodium at constant electrochemical g r a d i e n t for Na a n d in the absence o f A T P slowly increases the Ca efflux level to a m a x i m u m o f 3.4 +- 0.2 ( m e a n + SEM) at 110/20 m M o f Nao/Na~. F u r t h e r reduction o f the absolute concentrations o f sodium rapidly decreases the Ca efflux level as Nao a n d Na~ a p p r o a c h zero. TABLE SENSITIVITY CONSTANT
OF
Ca
EFFLUX
TO
ELECTROCHEMICAL
THE
II CONCENTRATION
GRADIENT
ABSENCE
OF
FOR
OF
THIS
SODIUM
CATION
IN
AT THE
ATP
External/internal sodium concentration
raM/raM Ax . . . .
ference
440
396
~30
275
220
165
rio
66
44
~
7-2
~
"50
~
~
~
]2
8
22
C a efflux
pmol.cm-2.s -t
R 290176
0.29
--
--
--
0 . 5 9
.
.
.
.
.
R 030276 A
0.14
-
-
-
0 . 3 9
.
.
.
.
.
R 030276 B
0.19
-
-
-
0.45
--
R 190276
0.16
0.17
0.17
-
0.35
.
.
.
.
.
R 240276 B
0.11
--
--
0.20
0.37
.
R 040376 B
0.08
-
-
-
0.19
0.30
R 230376 B
0.12
R 240376
0.22
R 260376 A
0.20
R 260376 B
0.29
R 270376
0.30
R 010476
0.40
R 020476
Cat buffered
0.56
--
--
0.21
-
-
-
--
.
.
.
.
0.43 --
B
--
--
--
O.87
--
--
--
--
0.89
--
--
--
--
0 . 9 2
--
--
--
m
--
--
--
--
0 . 8 8
-
--
m
m
--
--
--
1 . 0 0
--
--
0.30
0 . 4 3
--
0.67
--
--
--
0.18
--
--
--
0.61
0.48
0.48
m
to 0.5/zM.
T h e o b s e r v e d b e h a v i o r o f the Ca efflux level as a function o f the absolute concentrations o f Na, at constant electrochemical g r a d i e n t for Na, c a n n o t be e x p l a i n e d in t e r m s o f changes in the resting m e m b r a n e potential, because the threshold for excitation scarcely c h a n g e d t h r o u g h o u t the e x p e r i m e n t , n o r in t e r m s o f the electrochemical g r a d i e n t for Ca because that was k e p t constant. T h e r e f o r e , the effect described in Fig. 2 m u s t be associated with the absolute concentrations o f Na. T h e biphasic n a t u r e of the curve seen in this figure indicates that two processes m a y be o c c u r r i n g concomitantly with the r e m o v a l o f Na f r o m the extra- a n d intracellular media. T h e increase in Ca efflux level f r o m 0 u p to the m a x i m u m o f 3.4 (at 110/20 m M Nao/Na,) argues in favor o f the a p p e a r a n c e o f an activating factor, p r o b a b l y the external Na, that energizes the
REQUENA Ca Efflux and Na in SquidAxon
449
transport mechanism, whereas the decrease in the Ca efflux level f r o m 3.4 toward the r e f e r e n c e value o f 1.0 (at 440/80 mM Na0/Na0, argues in favor o f the action o f an inhibitory factor, probably internal Na. The Effect on Ca Efflux of the Concentration of Sodium at Constant Electrochemical Gradient for Na in the Presence of A T P Fig. 3 shows that time course o f the Ca efflux f r o m an axon dialyzed with Na0/ Na~ = 440/80 and Nao/Nai = 110/20, both o f which exhibit the ratio 5.5. T h e effects o f these Na concentrations were tested in the absence and in the presence o f A T P . T h e intracellular Ca concentration was b u f f e r e d to 0.5/xM while Cao 5.0 Q
w
"l.J
4.0
E u
-~
3.0
~9
2.0
1.0
OUT 1 INI
I
I
I
II0
220
330
I
440
I
I
t
I
20
40
60
80
Sodium
Concentrotion (raM)
FIGURE 2. The effect on Ca efflux of the absolute concentrations of NaJNa~ at constant electrochemical gradient for Na in the absence of ATP. Ca efflux values were normalized with respect to the level observed at 440/80 mM Nao/Na~. See text for further details. was kept constant at 10 mM. In the first half o f the e x p e r i m e n t the axon was exposed, in the absence o f internal A T P , to the control concentrations o f Nao/ Nat, i.e., 440/80 mM. Once the Ca efflux level reached a steady-state value o f 0.30 pmol.cm-2.s -1, both concentrations o f Na were r e d u c e d to o n e - f o u r t h that o f the control value. As shown previously, this e x p e r i m e n t a l condition p r o d u c e s an increase in Ca efflux c o m p a r e d to that level at the control concentrations o f Na. At this point the introduction o f 1 mM (Tris) A T P into the dialysis media increased Ca efflux f r o m 0.88 pmol.cm-2.s -1 to 1.48 pmol.cm-2.s -~. Finally, the restoration o f the control concentrations o f Na in the presence o f A T P , p r o d u c e d a reduction in the Ca efflux to a new steady state value o f 1.29 pmol.cm-2-s -a. It should be noticed that the change in Ca efflux due to the variation o f the concentrations o f Na f r o m 110/20 to 440/80 mM o f Nao/Na~
450
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 " V O L U M E
72" 1978
r e p r e s e n t e d a 1.15-fold increase in the level o f Ca efflux in the p r e s e n c e o f A T P , whereas a similar t r e a t m e n t d o n e at the b e g i n n i n g o f the e x p e r i m e n t , but in the absence o f A T P , caused r o u g h l y a threefold increase. T a b l e I I I s u m m a r i z e s the results o f all the e x p e r i m e n t s carried out following the protocol described above. Ca efflux values are given in the absence a n d in the presence o f A T P at each set o f test concentrations o f Nao/Nai. It can be concluded f r o m the data p r e s e n t e d in this table that a reduction o f the concentrations o f extra- a n d intracellular sodium f r o m the control condition o f
ou. 44o I IN 1 80
,,o 44oNo 20 80 No 1
0
[
1
(Tris) ATP
16
u~
'E o
1.2
0
E O. R 260376 ~ 4 4 0 prn Co"i* O. 5 p M
X .J
O.B W
8
0.4
I
I
I
]
L
2
I
I
I
3
TIME ( h 0 u r s )
FIGURE 3. The time-course of Ca efflux at two concentrations of Nao/Na~ which exhibit the same ratio, in the presence and in the absence of ATP.
440/80 m M to 110/20 mM p r o d u c e d an insignificant increase (5%) in the level o f Ca efflux in the presence o f A T P .
The Effect on Ca Efflux of lnternal Na in the Absence of ATP T o u n d e r s t a n d the effect on Ca efflux o f the concentrations o f external a n d internal Na, at constant electrochemical g r a d i e n t for Na, a series o f e x p e r i m e n t s was carried out in which the effect o f internal Na on the Ca efflux was studied at constant external Na (440 mM). T h e effect on Ca efflux of Na~ has previously
REQUENA Ca
45 l
E f f l u x a n d N a in Squid Axon
been e x a m i n e d by Blaustein a n d Russel (1975) a n d by Brinley et al., (1975) who o b s e r v e d an inhibition o f Ca efflux with increasing concentration o f Na,. In the e x p e r i m e n t plotted in Fig. 4 a, the time-course for the Ca efflux is shown for an a x o n dialyzed with various concentrations o f Nai in a m e d i u m free o f A T P a n d with an ionized Ca concentration set to 250/~M. At the b e g i n n i n g o f the e x p e r i m e n t , the axon was dialyzed with a solution containing 0 m M Na, all o f the internal Na having been replaced by choline. T h e external m e d i u m , which was kept constant almost u p to the end o f the e x p e r i m e n t , contained the n o r m a l c o n c e n t r a t i o n o f Na (440 raM) and Ca (10 mM). W h e n the Na~ concentration was m a d e 80 m M , the Ca efflux level decreased f r o m 3.1 pmol.cm-~-s -1 to 0.6 pmol.cm-2.s -~. This fivefold d r o p in Ca efflux was partially TABLE
III
SENSITIVITY OF Ca EFFLUX T O T H E CONCENTRATION OF SODIUM AT CONSTANT ELECTROCHEMICAL GRADIENT FOR T H I S CATION IN T H E PRESENCE OF ATP Externat/internal sodium concentrations raM~raM
0 ATP
A . . . . efe . . . . .
440 80-
110 ~-
1 mM ATP Efflux ratio ll0 d#~-~ 440
440 ~-
ca e/flux (r )
Mean
0.30 0.40 0.30
0.88 1.DO -
110 ~
ca efllux (ek)
#mol.cm-t.s-,
R 260376 B R 270376 R 280376
Efflux ratio ll0 O 440
pmol.cra- *.s - t
2.93 2.50 2.72
1.29 1.40 1.63
1.48 1.34 1.70
1.15 0.95 1.04 1.05 -+ 0.05
Cat buffered to 0.5 pM. reversed by lowering Na~ f r o m 80 m M to 40 m M as shown by the next step o f solution c h a n g e . A Ca efflux level o f 1.3 pmol.cm-Z.s -1 was increased to 2.3 p m o l . c m - 2 . s -1 w h e n Na~ in the dialysis fluid was lowered 20 m M . At 10 m M Nai, the Ca efflux was 2.5 pmol.cm-2.s -1 a n d , with the r e m o v a l o f the r e m a i n i n g Nal, r e a c h e d a value of 3 p m o l . c m - 2 . s -1. T h e e x p e r i m e n t e n d e d with the r e p l a c e m e n t o f all the external Na a n d Ca by choline, a p r o c e d u r e which d e c r e a s e d the Ca efflux to a negligible value (20 fmol.cm-2.s-~). This c h a n g e d e m o n s t r a t e s the existence o f a sodium-calcium c o u n t e r - t r a n s p o r t m e c h a n i s m in the nominal absence o f internal Na. I n Fig. 4 b a similar e x p e r i m e n t is shown. H e r e the internal ionized Ca c o n c e n t r a t i o n was b u f f e r e d to 100 nM. O n e observes that the addition o f 20 mM Na~ reduces the steady state Ca efflux level o f 13.1 fmol.cm-2.s -1 seen u n d e r the condition o f 0 Nat to 8.2 fmol.cm-2.s -~. S u b s e q u e n t addition o f a n o t h e r 20 m M o f internal Na r e d u c e d the level o f Ca efflux to 6.7 fmol.cm-2.s -~. When the Nat
452
THE jOURNAL OF GENERAL PHYSIOLOGY " VOLUME
72
- 1978
concentration was m a d e 100 m M , the Ca efflux r e a c h e d a value o f 2.4 fmol.cm-2-s -~. This level was raised to 5.4 fmol.cm-2.s -1, when 40 m M Nat was r e m o v e d f r o m the dialysis media. Finally, r e p l a c e m e n t o f the r e m a i n i n g internal Na by choline b r o u g h t the Ca efflux level to 13 fmol.cm-2.s -1 . T a b l e IV lists the results obtained fi)r the Ca efflux as a function o f the internal concentrations o f Na in axons dialyzed with two concentrations o f
,o
OUT IN
IocoI
440 0
l
80
i 40
I 20
[
I0
I
0
Ne NO
R 270776 e 4 2 0 .IJ m Ca T" 2 5 0 ~M
y
9
X ::::b _1 1.~ LM
I
1
1
I
I
I
2
TIME
1
1 3
"W~
I 4
(hours)
(a) FIGURE4. The time-course of Ca efflux at various concentrations of internal Na in axons dialyzed without ATP. (a) Ionized axoplasmic Ca concentration set at 250 ~M. (b) Ionized axoplasmie Ca concentration buffered to 0.1 ~M. ionized intracellular Ca, a high level o f 300 /xM a n d a lower level o f 100 nM which is a m o r e physiological figure for the ionized axoplasmic calcium. It can be n o t e d in this table that at either ionized Ca concentrations, the addition o f internal Na always r e d u c e d the Ca efflux level below that o b s e r v e d u n d e r the e x p e r i m e n t a l condition o f nominally zero Nat. T h e p a t t e r n for the inhibition p r o d u c e d on the Ca efflux by Nat is m o r e clearly shown in Fig. 5. In this figure, n o r m a l i z e d Ca efflux levels are plotted as a function o f the concentration o f internal Na. For each e x p e r i m e n t , the Ca efflux value obtained u n d e r the
453
REQUENA Ca Efflux and Na in Squid Axon
condition of nominally zero Nat is taken as 1.0, while the other Nai-dependent Ca efflux levels are normalized accordingly. Examination of the figure and a statistical analysis of the data shows that there is no significant difference between the extent of the inhibition produced by Nai at high or low concentrations of ionized intracellular calcium. This fact strongly argues in favor of the existence of a noncompetitive interaction in which a specific site in the transport
0
IN I
Asw
I 20
I 40
60
I
NoJ
0
No
R210477B -6.475 jJm Co~* 0,1 pM
20 f
1'001
,
/
15 fJ
E
I0 X ._I b_ LL laJ o
1 I
I
I
I
TIME
1
3
2
1
1
4
(hours)
(b) FIGURE 4
mechanism binds exclusively internal Na, a binding which results in an inhibition of the outward transport of Ca ions. The data summarized in Table IV, normalized as in Fig. 5 can be fitted, by the least squares method, to an Eadie-Haldane linear transformation (Eq. 1) in an attempt to describe the molecular nature of the inhibitory phenomena of Nat on Ca efflux. ] = 1
1 - lml~
{ K,
'~"
(1)
1 + \~-~]
In Eq. l, i represents the noninhibited fraction of Ca efflux at a given
454
T H E JOURNAL OF GENERAL P H Y S I O L O G Y ' V O L U M E 7 2 .
1978
concentration o f Nai, Imin is the m i n i m u m level o f this noninhibited fraction to be observed, at high Nat, Kt is the a p p a r e n t dissociation constant and n is the molecularity o f the inhibition p r o c e s s . T h e continuous line drawn in Fig. 5 represents the curve best fitted to the data using 1 for the molecularity o f the reaction, while the b r o k e n line c o r r e s p o n d s to a similar curve in which a value o f 2 was chosen for n. Table V lists the kinetic p a r a m e t e r s (K z and Imin) obtained by fitting the data to Eq. 1, for both uni- and bimolecular reactions. It also lists the predicted noninhibited fractions o f the Ca efflux for internal Na concentrations o f 100 and 200 mM for uni- and bimolecular reactions. As can be seen in the table, the magnitude o f the predicted fraction at 200 mM o f Na~ is different for each type TABLE
IV
EFFECT OF Nat ON Ca EFFLUX IN THE ABSENCE OF ATP Axon reference
Ca ++
Internal sodium concentration 0
10
20
30
-
-
-
4O Ca efflux
#A~ R 190876 A
0.10
Flux units
50
60
80
100
mM 18.1
11.6
fmol cm2.s
R 190876 B
0.10
18.0
.
R 120477
0.10
17.9
--
. 11.7
. -
.
.
R 150477
0.10
18.3
--
11.3
-
-
0.10
13.1
-
8.2
--
6.7
R 120477 A
0.25
142.0
.
R 270776
250
3.1
2.5
. 2.3
. -
2.5
-
-
6.3
4.5
7.4
-
5.5
5.4
-
5.4
-
5.6
R 210477 B
.
6.4 -
.
.
1.3
. -
2.4 26.3
-
0.6
--
pmol cm2.s
R 030876
300
7.5
-
-
4.5
-
-
3.0
2.5
R 050876
300
11.6
-
-
6.9
-
-
-
3.0
-
R 120876
300
8.7
6.7
6.5
-
4.4
-
3.3
-
-
R 260477
300
5.8
-
-
-
R 270477
300
6.6
.
.
.
.
.
.
.
3.4
-
2,8
-
-
1.4 2.7
R 120577
300
6.0
.
.
.
.
.
.
.
1.7
R 130577
300
6.3
.
.
.
.
.
.
.
1.7
R 070677 B
300
11.2
.
.
.
.
.
.
.
3.9
R 090677
300
11.0
.
.
.
.
.
.
.
2.0
o f reaction. T h e ratio o f the estimated noninhibited fraction present at 100 Nai to that observed at 200 Nat is calculated to be 1.60 if the reaction is unimolecular. I f the reaction is bimolecular the predicted ratio is 1.05. T h e s e ratios d i f f e r e n o u g h to p e r m i t an experimental distinction between the two processes. T o test this hypothesis, axons were dialyzed with media which contained u p to 200 mM Nai. T o do this, about 100 mM o f K / m u s t be replaced by choline or Na. U n d e r these conditions the axons were depolarized by some 10 mV f r o m their n o r m a l resting potential. An ionized Ca concentration o f 10 tzM was chosen for this e x p e r i m e n t . Fig. 6 shows the time-course for the Ca efflux in one o f the e x p e r i m e n t s in which the effect o f very high internal Na was tested. At the beginning o f the e x p e r i m e n t , in the nominal absence o f A T P and N a , a Ca efflux which increased and reached a steady value o f 6.1 pmol.cm-Z.s -~ was
REQUEtr
Ca Efflux and Na in Squid Axon
455
observed. T h i s efflux level d r o p p e d to 0.65 pmol-cm-*-s -~ when Nai was m a d e 200 raM. T h e r e p l a c e m e n t is the dialysis m e d i u m o f 100 m M Na by choline, increased the Ca efflux value to 1.3 p m o l . c m - * . s -~, a level which r e p r e s e n t s a d o u b l i n g o f the level previously o b s e r v e d u n d e r the condition o f 200 Na~. F u r t h e r r e d u c t i o n o f the internal Na to 30 m M b r o u g h t the Ca efflux level to 3.2 pmol.cm-Z.s -~, whereas r e t u r n to the initial e x p e r i m e n t a l condition o f nominally zero Na~, raised the CA efflux to its initial value. T a b l e VI lists the Ca
I.Oq
'\ \
e 3 0 0 pM oO.IpM
0.8
o
UA
E 0.6 o
0~4
g
0.2
o o
I
I
l
I
I
20
40
60
80
I00
[N0]i
( mM )
FIGURe 5. The effect on Ca efflux of internal Na at constant Naa (440 raM) in the absence of ATP. Ca efflux values were normalized in each experiment with reference to the Ca efflux level observed at zero Na~ to which the value of 1.0 was assigned. (0) Axoplasmic ionized Ca set at 300/zM; (O) ionized Ca buffered to 0.1 tzM. The continuous line corresponds to a unimolecular inhibition reaction; the broken line corresponds to a bimolecular reaction as given by Eq. 1. The kinetic parameters of the two curves are those given in Table V. For further details see text. efflux values o b t a i n e d at c o n c e n t r a t i o n s o f 200, 100, 30, a n d 0 m M Na~ f o r the two e x p e r i m e n t s p e r f o r m e d . As can be seen in the last c o l u m n o f this table, the ratio o f the o b s e r v e d Ca efflux level at 100 m M Nat to that o b s e r v e d at 200 m M Na~ was 2.0 in o n e e x p e r i m e n t a n d 1.6 in the o t h e r . T h e a g r e e m e n t between these ratios a n d the predicted ratio o f 1.6 f o r the u n i m o l e c u l a r reaction, strongly suggests that the inhibitory effect o f internal Na on the m e c h a n i s m that translocates Ca o u t w a r d is u n i m o l e c u l a r in n a t u r e .
456
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 72 9 1978
The Effect on Ca Efflux of Internal Na in the Presence of A T P T h e effect on Ca efflux o f the internal concentration o f Na, at a constant external Na c o n c e n t r a t i o n o f 440 m M , was e x p l o r e d in several axons dialyzed with A T P . Fig. 7 shows two records o f the time-course o f the Ca efflux u n d e r various levels o f internal Na. In Fig. 7 a the intracellular ionized Ca level was set to 300/zM whereas in Fig. 7 b it was b u f f e r e d to 100 nM. In Fig. 7 a a Ca efflux level o f 11.0 p m o l . cm -2. s -~ was o b s e r v e d at the b e g i n n i n g o f the e x p e r i m e n t in the n o m i n a l absence o f internal Na a n d A T P . T h i s level d r o p p e d to 2.0 p m o l . c m - 2 . s -~ w h e n the concentration o f Na in the a x o p l a s m was m a d e 100 m M . At that point, the addition o f 2 m M (Mg) A T P to the dialysis m e d i a stimulated Ca efflux which increased to a level o f 7.2 p m o l . cm -z. s -l. It should be n o t e d that this level o f Ca efflux o b s e r v e d in the p r e s e n c e o f A T P a n d 100 m M o f Nat is considerably lower t h a n that o f 11.0 p m o l . cm -2. s -I o b s e r v e d at the b e g i n n i n g o f the e x p e r i m e n t in the absence o f Nai a n d A T P . T h e r e m o v a l o f all o f the internal Na r e t u r n e d the Ca efflux to its initial level o f 11.0 p m o l . cm -z. s -I even t h o u g h the A T P c o n t e n t o f the fiber was u n c h a n g e d . T h e TABLE
V
KINETICS PARAMETERS OF T H E CURVES RELATING Ca EFFLUX AND Na~ I N H I B I T I O N AND EXTENT OF T H E PREDICTED I N H I B I T I O N AT H I G H Na~ Molecularity
Kt
Minimal noninhibited fraction ]mh.
mM
Noninhibited fraction []]~.h
100 mM
200 mM
Ratio
[I] t00Naj
% n= l n=2
34.0 ~-5.0 17.6 -+6.3
3.5 -+6.1 30.8 -+2.5
28.0 -+0.4 32.9 -+6.4
17.6 +--0.7 31.4 ---3.1
1.60 -+0.07 1.05 ---0.23
addition o f 50 m M Nat b r o u g h t the Ca efflux d o w n to 9.5 p m o l . cm -z. s -~, w h e n the c o n c e n t r a t i o n o f Na in the dialysis m e d i a was again m a d e 100 m M . In this last solution c h a n g e , the concentration o f A T P was d o u b l e d to 4 raM. T h i s increase in the c o n c e n t r a t i o n o f intracellular A T P was accomplished in o r d e r to see w h e t h e r the c o n c e n t r a t i o n o f 2 m M o f A T P used d u r i n g m o s t o f the e x p e r i m e n t s was s u p r a m a x i m a l . T h i s point is considered p r o v e d because the Ca efflux level o f 7.2 p m o l - c m - ~ . s -1 o b t a i n e d in the middle o f the e x p e r i m e n t , u n d e r the conditions o f 100 m M Na~ a n d 2 m M A T P , was very similar to the Ca efflux value o f 6.6 p m o l . cm -z. s -I obtained at the e n d o f the e x p e r i m e n t at an identical level o f Na~ but at 4 m M A T P . Fig. 7 b shows an e x p e r i m e n t similar to the o n e j u s t described except that the ionized intracellular calcium level was b u f f e r e d to 100 nM. At the b e g i n n i n g o f the e x p e r i m e n t 3 m M (Mg) A T P was a d d e d to the dialysis fluid which c o n t a i n e d 100 m M choline instead o f Na~. A steady state Ca efflux o f 36.4 f m o l . cm -2. s -I was o b s e r v e d u n d e r these conditions. T h e addition o f 50 m M Na to the internal m e d i a b r o u g h t Ca efflux d o w n to 26.1 f m o l . cm -2- s-I; the level h a d risen to 33
457
REqUEN^ Ca Efflux and Na in Squid Axon
=If
t zoo
I ~oo [ 3a
L o
240
R 260777A "0"450 pm Ca~*lO IJM
7
E
o0
o
/~ro~
t.t
E
o. w
X ,-J It. It.
S I I
I
I 2
1
I
I
3
TIME ( h 0 u r s ) FIGURE 6. The time-course of Ca efflux at high concentrations of internal Na in an axon dialyzed with 240 mM of Kl and zero ATP.
f m o l . cm -z- s -~ w h e n 30 m M Nat was r e m o v e d f r o m the dialysis fluid. T h e initial value o f 36.4 f m o l . c m - 2 . s -~ was r e s t o r e d when the r e m a i n i n g 20 m M o f Nat were replaced by choline. At this point in the e x p e r i m e n t , all o f the choline a n d the A T P were r e m o v e d f r o m the dialysis m e d i u m a n d 100 m M o f Na was i n t r o d u c e d into the fiber. T h i s t r e a t m e n t p r o d u c e d a large d r o p in the Ca e f f l u x level which was partially r e v e r s e d , h o w e v e r , w h e n A T P was r e i n t r o d u c e d into the dialysis fluid.
458
T H E .JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 7 2 9 1 9 7 8
Fig. 8 shows n o r m a l i z e d Ca efflux values, obtained f r o m all the e x p e r i m e n t s p e r f o r m e d following the above described protocol, plotted as a function o f the concentration o f Na in the dialysis fluid. T h e value o f 1.0 was assigned to the level o f Ca efflux seen u n d e r the condition of nominally zero Nai. As observed in axons d e p l e t e d o f A T P , there was no significant d i f f e r e n c e between the inhibition p r o d u c e d by Nai at low or high levels o f intracellular ionized Ca. H o w e v e r , the extent o f the inhibition o f Ca efflux seen in the p r e s e n c e o f A T P is m a r k e d l y d i f f e r e n t f r o m that o b s e r v e d in axons dialyzed without A T P . At 100 mM Nai some 73% o f the Ca efflux m e c h a n i s m is inhibited in axons with no A T P whereas only a 25% inhibition is o b s e r v e d in A T P - f u e l e d axons. It should be noted that, in axons fueled with A T P , Ca efflux was i n d e p e n d e n t o f the absolute c o n c e n t r a t i o n o f Na if the N a j N a 0 ratio was held constant in the r a n g e o f 440/80 to 110/20 m M , whereas Na~ h a d little inhibitory action in that r a n g e . In axons d e p l e t e d o f A T P , however, raising Nai caused a large inhibition o f Ca efflux, while the p e a k level o f Ca efflux observed at 110/20 m M o f N a d N a i was TABLE
VI
EFFECT OF H I G H INTERNAL Na ON T H E Ca EFFLUX IN T H E ABSENCE OF ATP Axon reference
CA~"+
Internal sodium concentrations
uM
mM
0
30
100
200
Ratio
(EffluxhooN~ (Efflux) ~ooN~,
Ca effltlx
pmol.cm-~.s t
R 26077 A R 26077 B
10 10
6.1 5.0
3.2 2.0
1.3 0.6
0.65 0.38
2.0 1.6 Mean 1.8
Axons dialyzed with Kt = 240 mM. significantly r e d u c e d by raising the absolute concentration o f Na, the electrochemical g r a d i e n t for Na being kept constant. T a b l e V I I s u m m a r i z e s the absolute values for Ca efflux observed at various internal concentrations o f Na in the p r e s e n c e o f A T P a n d at two concentrations o f ionized axoplasmic Ca; Fig. 8 was constructed f r o m these values.
The Effect of Internal Na on the ATP-Sensitive Fraction of the Ca Efflux T h e r e is an aspect o f Fig. 7 a which should be e m p h a s i z e d . It concerns the effect o f A T P on the Ca efflux level o b s e r v e d in the n o m i n a l absence.of internal Na. At the b e g i n n i n g o f the e x p e r i m e n t a Ca efflux level o f 11.0 p m o l . cm -2. s was o b s e r v e d in the absence o f A T P a n d internal Na. 3 h later a Ca efflux level o f 11.0 p m o l . c m - 2 . s -1 was seen, this time in the p r e s e n c e o f A T P a n d , as earlier, in the absence o f internal Na. In two o t h e r e x p e r i m e n t s , a similar result was obtained. T a b l e V I I I lists the Ca efflux values o b s e r v e d in the p r e s e n c e or absence o f A T P in axons dialyzed with a high internal level o f ionized calcium a n d nominally zero Na~. F r o m these values and f r o m a similar observation m a d e by DiPolo (1976) in an a x o n dialyzed with an ionized Ca level b u f f e r e d to 0.6
REQUENA Ca Efflux and Na in Squid Axon
459
g-M, it can be concluded that in axons in which the internal Na has been r e m o v e d by dialysis, the level o f Ca efflux observed in the absence o f A T P is not affected by the addition o f A T P , at least within the specified range o f internal calcium concentrations o f 0.6-300 g-M. In Fig. 9 curve c (taken from Fig. 8) describes the inhibitory action o f Nat on Ca efflux in the presence o f A T P , and curve b (taken f r o m Fig. 5) describes the inhibitory action o f Nat on Ca efflux in the absence o f A T P . Subtracting curve b from curve a we obtain curve a which represents the d e p e n d e n c e on internal Na o f the fraction o f Ca efflux p r o m o t e d by A T P . T o construct this figure, curve c, which relates Ca efflux to internal Na in the presence o f A T P , was extrapolated linearly f r o m 100 mM, the highest concentration o f Nat used in the presence o f A T P , to 200 mM, the highest concentration o f Nai used in the absence o f A T P . Fig. 9 shows that A T P is capable o f relieving part o f the inhibition o f Ca efflux p r o d u c e d by Nat in the physiological range o f Na concentration. Fig. 9 can be also i n t e r p r e t e d as if the effect o f Nat on Ca efflux in the presence o f A T P is the result o f two simultaneous processes: (a) a c o m p o n e n t o f Ca efflux present in the absence o f ATP; and (b) an A T P d e p e n d e n t c o m p o n e n t o f Ca efflux. The Effect o f External N a on Ca E f f l u x at Low Internal N a
T h e activating effect o f external Na on Ca efflux has been extensively studied. In axons subjected to internal dialysis, DiPolo (1974) showed that the a p p a r e n t half-dissociation constant (Ka) for the process is 144 mM in axons dialyzed with out A T P and is 80 mM Nao in axons dialyzed with A T P . In those e x p e r i m e n t s , the internal Na concentration was kept at 72 mM while the internal ionized Ca was b u f f e r e d at 0.3 g-M. Blaustein et al. (1974) observed, in the absence o f A T P , a strong d e p e n d e n c e o f Ca efflux on Nao with a K A o f 125 mM at 50 mM Nat. More recently, Blaustein (1977) obtained values o f 50 and 120 mM o f Nao for axons dialyzed with and without A T P . A similar shift o f the Ka for Nao has been observed in injected axons. Baker and Glitsch (1973) showed that A T P shifts the curve relating Ca efflux to Na0. More recently, Baker and M c N a u g h t o n (1976) observed that in intact axons the value o f Nao that causes half-maximal activation o f Ca efflux changes f r o m 50 mM to 300 mM as cyanide poisoning proceeds. This change in Ka was accompanied by a change in the curve relating Ca efflux to the concentration o f Nao f r o m a rectangular hyperbola to a clearly sigmoid curve. In some o f the e x p e r i m e n t s r e p o r t e d elsewhere, especially those d o n e on injected axons, the concentration o f internal Na is u n d e t e r m i n e d . Inasmuch as the effect o f A T P on Ca efflux seems to be related to Na~, we studied the effect o f Nao on calcium efflux in axons dialyzed with various levels on Nat with or without A T P . Fig. 10 shows a typical e x p e r i m e n t . An axon, in which the ionized Ca concentration was b u f f e r e d with 2 mM o f E G T A to 0.33 g.M, was dialyzed with a solution containing 5 mM o f internal Na and 95 mM choline. T h r o u g h o u t this e x p e r i m e n t the external solution was Ca-free and its concentration o f Na was varied. At the beginning o f the e x p e r i m e n t Ca efflux was about 0.7 p m o l . cm 2. -1 with a Nao o f 441 raM. Replacement o f all o f Nao by 441 mM o f choline d r o p p e d the Ca efflux to 0.033 pmol. cm -2. s -~ . This level went u p to 0.436 pmol. cm -2. s -~
460
TIlE
uT I
I
o
I
JOURNAL
OF
,oo
o
GENERAL
1o,
[
PHYSIOLOGY
o
9 VOLUME
72 9 1978
,oo.
z
4 (M 9) ATPI
R090677 90. 5 0 0 )J m
c=7 3oo.M
,o (,
8
m '7,'
E
(3.
X t.L Lz_ L,~
3
1
_
t
|
I
~
l
,
2
|
3
TIME
I
I
1
4
(hours)
(o) FIGURE 7. The time-course of Ca efflux at various concentrations of internal Na in axons dialyzed with ATP. (a) Ionized axoplasmic Ca concentration set to 300 ~M. (b) Ionized axoplasmic Ca concentration buffered to 0.1 ~M. when Na0 was raised to 45 mM. At 89 at the normal concentration o f Nao When Na0 was lowered to 23 mM the this point 2 mM o f (Mg) A T P was
mM Na0 the Ca efflux was 0.586 whereas the efflux rose to 0.601 p m o l . c m - 2 . s -~. Ca efflux fell to 0.221 p m o l . c m - 2 . s -l. At added to the dialysis fluid, which still
REQUENA
461
Ca Efflux and Na in Squid Axon
R230677
e!.75.r~
Co i 0,1 pM
40
9
9
QO 9
O0
30
7
E
0
E
20
x J w
IO
I
I I
I
I
I
2
I 3
[
I 4
TIME ( h o u r s )
(b) FIGIJRE 7
contained 5 m M o f internal Na a n d Ca efflux rose to 0.350 p m o l . c m - 2 . s -~. Raising Nao f r o m 23 to 45 m M increased the efflux to 0.686 p m o l . cm -2. s -a. A r e t u r n to the n o r m a l Na concentration o f 441 in millimolar p r o d u c e d a transient r e b o u n d in Ca e f f l u x which eventually stabilized at 1.06 p m o l - c m -z- s -1 . At this point all o f the e x t e r n a l Na was replaced by 441 m M choline a n d the Ca efflux fell to 0.270 p m o l . cm -2. s -~. R e i n t r o d u c t i o n o f 89 m M o f Na0 raised the efflux to 0.888 p m o l . c m -2. s-1. Finally, Na0 was r e t u r n e d to the level at which A T P was originally a d d e d , i.e., 23 m M a n d the Ca efflux r e t u r n e d to the s a m e value
462
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
- VOLUME
72 - 1978
seen when the axon was first exposed to 23 mM Nao and A T P , i.e., 0.22 pmol. cm -2- s -~, F r o m this kind o f e x p e r i m e n t , the a p p a r e n t half-activation constant for the effect o f Na0 on Ca efflux can be calculated: KA values' were c o m p u t e d by interpolation o f that Nao concentration at which the net N a - d e p e n d e n t Ca efflux (defined as the Ca efflux level observed at 441 mM o f Na0 minus that seen at 0 Nao) is r e d u c e d to half. For the e x p e r i m e n t shown in Fig. 10, a value o f 37 + 7 mM o f Na0 was calculated for the A T P - f r e e condition while 43 • 5 mM o f Nao 1.0~
~8 0.8 0
,.r
0
LLI
E
0.6
Co,"
o
9 300 o0.1
~S g
pM
pM
o4
L
0.2
I
I
I
I
I
20
40
60
80
I00
FIGURE 8. The effect on Ca efflux of internal Na at constant Nao (440 m M) in the presence of ATP. The straight line was fitted by least squares method to the experimental data. The slope of the line is -0.0025 mM -I. Ca efflux levels were normalized in each experiment with reference to the Ca efflux level observed at zero Nat to which the value of 1.0 was assigned. (0) Axoplasmic ionized Ca set at 300 v.M; (O) ionized Ca buffered to 0.1 ~M. was calculated for that part o f the e x p e r i m e n t in which the axon was fueled with A T P . It is clear that if Nai is very low (5 mM) A T P has no effect on the activation p r o d u c e d by external Na. This conclusion is s u p p o r t e d by experiments d o n e at various levels o f Na~, the results o f which are s u m m a r i z e d in Table IX. It can be seen that with Nai nominally zero the half-activation constant for external Na o f axons dialyzed without A T P is indistinguishable f r o m that o f axons dialyzed with A T P . This is also true for axons dialyzed with 30 mM o f Na~. However, at h i g h e r concentrations o f internal Na the a p p a r e n t half-activation constant for The uncertainty in determining the Ka by this procedure is reflected as the upper and lower limits set for the KAreported here.
REQUENA
463
C a E f f l u x a n d N a in S q u z d A x o n
Na o becomes d e p e n d e n t upon ATP. Specifically, one axon dialyzed with 100 mM Na~ showed a KA o f 112 mM in the absence of ATP while in the presence o f ATP, the same axon showed aKA of 39 mM Nao. T h e relationship between the apparent half-activation constant for Nao and the concentration of internal Na is better seen in Fig. 11. Although it is evident that in the presence of A T P the KA for Nao is independent of Nai, in the absence o f A T P the apparent half-activation constant for Nao depends upon the concentration of internal Na. T h e pronounced sigmoidal shape of the curve relating Ka for Nao to Nai, observed in the absence of ATP, varies from 41 mM TABLE
VII
EFFECT OF Nat ON Ca EFFLUX IN THE PRESENCE OF ATP Axon reference
Ca,++
Internal sodium concentration I~M
Flux units
mM 0
0
R 210677
0.10
20 50 Ca efflux
36.0 40.0
38.5
100
32.5
24.3
fmol cm~.s "
R 220677 R 230677 R 130577
0.10 0.10 300
31.7 36.4 6.5
33.0 -
28.7 26.1 -
22.5 25.0 6.0
R 090677 R 140677 R 270477
300 300 300
11.0 103 6.8
9.5 -
9.5 8.9 6.6
7.3 8.4 5.7
TABLE
pmol ClTILS " " "'
VIII
EFFECT OF ATP ON Ca EFFLUX IN THE ABSENCE OF INTERNAL Na Ratio (Ca eff]ux)^Tp
Ca efflux Axon reference
Ca,+* taM
R 130577 R 090677 R 270677
300 300 300
No ATP
ATP
(Ca effluX)so^'rp
pmol .cm - 2.s - ~
6.4 11.2 6.6
6.5 11.0 6.8
1.02 0.98 1.03 Mean
1.01
Nao at low Nai (
