Release of Proteins from the Inner Surface of Squid Axon Membrane Labeled with Tritiated N-Ethylmaleimide I S A O I N O U E , H A R I S H C. P A N T , I C H I J I T A S A K I , and H A R O L D G A I N E R From the Laboratory of Neurobiology, National Institute of Mental Health, and The Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014, and from the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT Proteins in the inner surface of the squid axon membrane were labeled by intracellular perfusion of [aH]N-ethylmaleimide (NEM), which forms covalent bonds with free sulfhydryl groups. The excitability of the axon was unaffected by the [aH]NEM perfusion. After washout of the unbound label, the perfusate was monitored for the release of labeled proteins. Labeled proteins were released from the inner membrane surface by potassium depolarization of the axon only in the presence of external calcium ions. Replacement of the fluoride ion in the perfusion medium by various anions also caused labeled protein release. The order of effectiveness was SCN- >> Br- > C1- > F-. The extent of labeled protein release by the various anions was correlated with their effects on axonal excitability. The significance of these results is discussed. INTRODUCTION
Squid giant axons are k n o w n to m a i n t a i n their electrical excitability a f t e r extensive r e m o v a l o f the a x o p l a s m with proteases ( T a k e n a k a et al., 1968). P r o l o n g e d t r e a t m e n t o f the a x o n m e m b r a n e with intracellularly a d m i n i s t e r e d p r o n a s e initially p r o d u c e s a decrease in the m a x i m u m inward c u r r e n t u n d e r voltage c l a m p (Sato et al., 1973), b e f o r e the decrease in a m p l i t u d e a n d p r o l o n g a t i o n o f the d u r a t i o n o f the action potential is o b s e r v e d ( T a k e n a k a a n d Yamashigi, 1966; A r m s t r o n g et al., 1973). Ultimately, the proteolytic action causes inexcitability o f the a x o n (Tasaki, 1968). Such observations suggest that proteins associated with the internal surface o f the a x o n m e m b r a n e are involved in the regulation o f excitability. In an earlier investigation (Gainer et al., 1974), the proteins in the internal m e m b r a n e surface o f the squid a x o n w e r e labeled with I2~1 by using an enzymatic radio-iodination p r o c e d u r e in c o m b i n a t i o n with the intracellular p e r f u s i o n technique. T h i s study d e m o n s t r a t e d that a p r o t e i n with a m o l e c u l a r weight equal to a b o u t 12,000 daltons was associated with the internal m e m b r a n e surface, a n d the ability o f this protein to be iodinated was selectively r e d u c e d by p o t a s s i u m depolarization o f the a x o n . Recent studies by T a k e n a k a et al. (1976) have conTHE JOURNAL OF GENERAL PHYSIOLOGY' VOLUME 68, 1 9 7 6 " p a g e s 3 8 5 - 3 9 5
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f i r m e d these f i n d i n g s , a n d s u g g e s t t h a t t h e l a b e l e d 12,000-dalton p r o t e i n is r e l e a s e d i n t o the p e r f u s a t e a f t e r p o t a s s i u m d e p o l a r i z a t i o n o r electrical activity o f t h e a x o n . I n the p r e s e n t s t u d y , we have l a b e l e d the p r o t e i n s in t h e m e m b r a n e by i n t r a c e l l u l a r p e r f u s i o n o f [ 3 H ] N - e t h y l m a l e i m i d e ([3H]NEM) a n d h a v e m o n i t o r e d t h e p e r f u s a t e f o r release o f l a b e l e d p r o t e i n s a f t e r v a r i o u s e x p e r i m e n t a l t r e a t m e n t s o f the a x o n . We r e p o r t h e r e t h a t l a b e l e d p r o t e i n s a r e r e l e a s e d f r o m the i n t e r n a l s u r f a c e o f the m e m b r a n e by p o t a s s i u m d e p o l a r i z a t i o n (in t h e prese n c e o f e x t e r n a l c a l c i u m ions only), a n d by the i n t e r n a l p e r f u s i o n o f v a r i o u s anions. MATERIALS
AND
METHODS
Electrophysiotogical Methods Giant axons of squid Loligo pealei available at the Marine Biological Laboratory, Woods Hole, Mass., were used throughout the present experiments. The diameter of the axons used was between 280 and 380/.~m. T h e major portion of small nerve fibers and other adherent tissues s u r r o u n d i n g the giant fiber were removed u n d e r a dissecting microscope. The axon was then transferred to a Lucite chamber filled with artificial seawater (ASW) in which internal perfusion with two glass cannulae was performed (Lerman et al., 1969). T h e outside diameter of the inlet cannula was 120/xm (see Fig, 1, right). T h e standard internal perfusion solution contained 400 meq/liter K-ion, 350 meq/liter F-ion, phosphate (potassium salt) buffer adjusted to a pH between 7.2 and 7.4, and glycerol (4% by volume). In the present study the axoplasm within a long portion of the axon, 28-30 mm in length, was removed extensively by means of enzymatic digestion. Axons used were initially perfused intracellularly with the KF solution containing 0.01 mg/ml pronase (Calbiochem, San Diego, Calif.) until the maximum inward c u r r e n t u n d e r voltage clamp was reduced to about 80% of the level before the start of internal perfusion (approximately 12-20 min). After replacement of the enzyme-containing solution with an enzymefree one, the perfusion zone was shortened by inserting both of the cannulae 5 mm deeper into the axon so that the axoplasm remaining in the unperfused zone was separated from the perfusion zone by the nonflowing KF solution. T h e length of the perfusion zone was thus between 18 and 20 mm. The flow rate of the internal perfusion fluid was maintained at a level between 20 and 35/zl/min. T o study the effects of internal anions, the KF in the standard perfusion solution described above was replaced by an equimolar a m o u n t of KCI, KBr, or KSCN. The composition of the ASW used was 423 mM NaCI, 9 mM KC1, 23 mM MgCI2, 25 MgSO~, 10 mM CaCI2, and 5 mM Tris (the pH of the external solution was adjusted to between 7.8 and 8.0). T h e composition of the external potassium depolarization solution was identical to the above ASW except that the NaC1 was reduced to 250 mM and the KCI raised to 200 mM. When Ca-ions were removed from the K-rich ASW, ethylene glycol bis (/3-aminoethyl ether) tetraacetic acid (EGTA) was added to a concentration of 4 raM. In all cases the osmotic pressures of the various external solutions were equated by the addition of small amounts of glycerol, and the pH was between 7.8 and 8.0. The potential difference across the m e m b r a n e was measured at the center of the perfusion zone with a glass pipette Ag-AgCI electrode filled with a 3 M KC1 solution (70 /zm in diameter). T h e pipette was filled with KCl-agar gel near the tip to reduce outflow of the KCI solution. A calomel electrode immersed in the external medium was used as the reference point for potential measurements. T h e external fluid medium was g r o u n d e d through a large coil of platinized platinum wire. Stimulating shocks were delivered through a pair of platinum electrodes to the unperfused zone of the axon when
INOUE, PANT, TASAKI, AND GAINER Sqt~d Axolrt Membran¢ Proteins
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p r o p a g a t i o n o f impulses across the p e r f u s i o n z o n e was m o n i t o r e d . When application o f either c u r r e n t pulses or voltage pulses to the m e m b r a n e was required, a platinized platinum wire (50/~m in diameter) was inserted into the axon t h r o u g h the outlet cannula.
Biochemical Methods Tritiated N-ethylmaleimide ([SH]NEM, sp act 230 mCi/mmol) was purchased from New England Nuclear, Boston, Mass. T h e [SH]NEM (250/~Ci) was dissolved in 20 ml of the 400 mM KF solution. T h e NEM concentration in this solution was approximately 50/zM. T h e [3H]NEM-containing KF solution was i n t r o d u c e d into the axon from which the axoplasm had been removed by the method described above, and the axon was perfused with this cpm ~K-DEPO.~-~
400ram KF
l
I ,N ._j --
200
°
~_ OUT //
AXON LABELED WITH [3HINEM
....
D
COLLECTIONOF PERFUSATE
¢ PROTEIN PRECIPITATION WITH TCA
10C
O
T
COUNTING RADIOACTIVITY OF LABELED PROTEINS
10
20
30 rnin
FIGURE 1. Schematic illustration on the right shows the experimental arrangement used for measuring the a m o u n t o f [aH]NEM-labeled proteins a p p e a r i n g in the perfusate. IN and OUT r e p r e s e n t the inlet and outlet cannulae, respectively. T h e internal perfusion fluid used was a 400 mM KF solution (see text). Left, release o f the radioactive proteins into the internal fluid m e d i u m d u r i n g normal resting state and d u r i n g potassium depolarization. T h e depolarization was p r o d u c e d by switching the external seawater to a K-rich m e d i u m containing CaC12. T h e salt composition of the K-rich solution is given in the text. solution for 15 min. During this period, the axon was immersed in rapidly circulating (50 ml/min) artificial seawater. No detectable deterioration of the m e m b r a n e excitability was p r o d u c e d by the [aH]NEM treatment u n d e r the present experimental conditions. After switching to a [aH]NEM-free KF solution, continuous flow of both the internal and external fluid media was maintained for 15-20 min in o r d e r to eliminate free [aH]NEM from this system. T h e radioactivity o f the total proteins that could be extracted from one whole single fiber p r e p a r a t i o n was o f the o r d e r o f 104 cpm. (A small amount o f efflux o f [3H]NEM across the m e m b r a n e [20-30 cpm/s, cm 2] was observed d u r i n g internal application o f the [3H]NEM-containing KF solution.) Although NEM is commonly considered specific for sulfhydryl groups, it can also react to some extent with amino groups. At the p H of the internal perfusion fluid, the reaction rate o f NEM with thiols is about 1,000-fold greater than with amino c o m p o u n d s (Means and Feeny, 1971). T h e r e f o r e , u n d e r these conditions, it is likely that the principal reaction of the [aH]NEM in this study was with the free sulfhydryl groups in m e m b r a n e proteins. T h e labeled proteins released from the m e m b r a n e into the internal perfusing m e d i u m were measured by collecting fractions o f perfusate from the outlet cannula onto pads of W h a t m a n 3 mm filter p a p e r (Fig, 1). Contamination by free [aH]NEM in each collected
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sample was eliminated by precipitating proteins with trichloroacetic acid (TCA). The TCA precipitation procedure consisted of two washes of the pads in ice-cold 10% TCA (30 min each), followed by a hot (95°C) extraction in 5% TCA (30 min), another 30 min in cold 5% TCA, a 30-min wash of the pads in ethanol:ether (1:1, vol/vot), and then 30 min in 100% ether. In control experiments it was demonstrated that any protein-bound [3H]NEM and not free [aH]NEM remained in the pad after this procedure. The radioactivity of the proteins remaining on the filter paper was measured with a Beckman liquid scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). In one series of experiments perfusate was collected in small glass test tubes. Standard proteins (bovine serum albumin, 68,000 daltons; ovalbumin, 45,000 daltons; and cytochrome c, 12,000 daltons) were added as carrier, and the proteins in the tubes were precipitated with TCA. The pellet was washed with ether to remove the TCA, and then dissolved in 1% SDS, and reduced by the addition of/3-mercaptoethanol. The proteins in these samples were then separated by polyacrylamide gel disc electrophoresis in SDS (Neville, 1971). The standard proteins in the coprecipitate also served as internal molecular weight markers on each gel. The gels were sliced, liquid scintillation cocktail was added, and the radioactivity of each slice was measured with a Beckman liquid scintillation counter. All the experiments were conducted at room temperature (20° -+ l°C). RESULTS
Induction of Protein Release from Membrane by Potassium Depolarization A f t e r the labeling o f the squid axons with [3H]NEM a n d the washout o f the u n b o u n d label (see Materials a n d Methods), the axons were continuously perfused with the s t a n d a r d 400 m M KF solution in circulating ASW. U n d e r these conditions the axons maintain their ability to p r o p a g a t e n e r v e impulses across the p e r f u s i o n zone for m o r e t h a n 2 h. T h e resting potential o f the exitable m e m b r a n e was b e t w e e n - 5 5 a n d - 6 0 m V , and the m e m b r a n e resistance was usually between 0.7 and 1.5 k 1"/. cm 2. T h e external ASW was t h e n switched to a K-rich solution, which d e p o l a r i z e d the a x o n by a p p r o x i m a t e l y 50 m V . T h e m e m b r a n e resistance o f the depolarized a x o n was a b o u t 200 1/. cm ~. T h e effect o f such m e m b r a n e depolarization on the release o f proteins into the internal fluid m e d i u m was e x a m i n e d . T h e e x p e r i m e n t a l p r o c e d u r e is schematically illustrated on the right side o f Fig. 1. Perfusate was collected at 3-min intervals, a n d the radioactivity o f proteins in each collected sample was m e a sured. A typical e x a m p l e o f the results obtained is r e p r e s e n t e d on the left side o f the figure. T h e abscissa indicates the time in minutes a f t e r the start o f the collection o f p e r f u s a t e . T h e rate o f release o f proteins (cpm/3 min) was maintained at a low level as long as the a x o n was i m m e r s e d in ASW. A f t e r replacem e n t o f the e x t e r n a l seawater with the K-rich solution, t h e r e was a significant rise in the rate o f protein release followed by a g r a d u a l decline. R e p l a c e m e n t o f the K-rich external solution by seawater outside the a x o n b r o u g h t a b o u t a rapid fall in the rate o f protein release which finally r e t u r n e d to the original low level. In general, b o t h the m e m b r a n e potential a n d m e m b r a n e resistance gradually a p p r o a c h e d the original values o f the resting state a f t e r recirculation o f seawater. C o n d u c t i o n could usually be restored d u r i n g repolarization o f the m e m brane. In a separate e x p e r i m e n t , the radioactive proteins which a p p e a r e d in the
389
INOUE, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
perfusate were analyzed by SDS gel electrophoresis. In Fig. 2 the broken line shows the molecular weight distribution of proteins released into the internal fluid medium before depolarization, and the solid line shows the molecular weight distribution of proteins released during depolarization. T h e depolarization was produced by immersing the axon in the K-rich solution. One distinct peak with approximately 12,000 daltons was produced by the potassium depolarization. These results are consistent with those reported in previous papers 68
4ol
45
I •
I
12 mol wt/I,O00
1
K-DEPO.
-..o..- NORMAL -- 3 0
_1 03 _1 Ld LD ~. 2 0
E
I0
0
~O.o I0
20
SLICE NUMBER
FIGURE 2. Molecular weight distribution on SDS polyacrylamide gels of a fraction of [nH]NEM-labeled proteins which were present in the perfusate under normal resting state (NORMAL) and during potassium depolarization (K-DEPO.). The depolarization was produced by replacing the external seawater with the K-rich solution containing CaCI2. Note that a 12,000-dalton labeled protein was released by potassium depolarization. (Gainer et al., 1974; Takenaka et al., 1976) in which membrane labeling was done by radio-iodination.
Ca ++-Dependence of Protein Release T h e data in Fig. 3 illustrate the dependence of protein release upon Ca ++ in the internal medium. Axons were internally perfused with the KF solution. T h e external solutions used are given in the figure (see Materials and Methods for composition of solutions). As illustrated in Fig. 3 A and B, no significant alteration of the rate of release of radioactive proteins in the internal perfusion fluid could be produced if the membrane was depolarized with the K-EGTA solution. However, as shown in Fig. 3 B, there was sharp rise in the rate of protein release when CaCI2 was added to the external K-rich medium by replacing the K-EGTA solution with the K-Ca solution. In both o f the experiments in Fig. 3, conduction
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o f the action potential could be r e s t o r e d by recirculation o f seawater outside the axons. T h e m e m b r a n e potentials a n d resistance o f the depolarized a x o n in the K - E G T A a n d K-Ca ++ solutions did not significantly differ. Variation o f the Mg ~+ concentration o f the K - E G T A solution between 5 and 50 m M did not affect the rate o f protein release. T h e s e findings suggest strongly that the release o f proteins d u r i n g p o t a s s i u m depolarization arises f r o m the interaction between external Ca-ions a n d the m a c r o m o l e c u l e s at the i n n e r surface o f the m e m b r a n e , p r e s u m a b l y d u e to the e n h a n c e d rate o f Ca 2+ e n t r y into the m e m b r a n e d u r i n g potassium depolarization (Baker, 1972).
A
B cpm
SEA
SEA K-EGTA -WATER ~: "1"
K EGTA K-Ca
WATER~
SEA WATER--
T
SEA
~ WATER--
50
100
50
10
2;0
30
0
10
20
30
rain
FIGURE 3. Effect of external Ca++-ion on release of [JH]NEM-labeled proteins during potassium depolarization. Two kinds of K-rich solutions containing 200 mM KC1 were used to depolarize the membrane. K-EGTA solution contained 4 mM EGTA but no Ca++-ions. K-Ca solution contained 10 mM CaCI2, but no EGTA.
Effects of Internal Anions on Protein Release and Excitability of the Axon Previous studies have d e m o n s t r a t e d that the excitability o f the p e r f u s e d squid a x o n is strongly affected by the anionic species used in the p e r f u s i o n fluid (Tasaki et al., 1965). T h e efficacy o f internal anions in m a i n t a i n i n g n o r m a l excitability increased in the o r d e r o f S C N - < I - < B r - < C1- < SO4 =, < H P O 4 =, < F - . This anion sequence c o r r e s p o n d s to the lyotropic (or H o f m e i s t e r ) series a n d is directly related to the salting-out effect o f anions on proteins, w h e r e S C N is least effective a n d F - most effective (see Tasaki et al., 1965, for a discussion o f these p h e n o m e n a in relation to the squid axon). T h e following e x p e r i m e n t s indicate that such anion effects on m e m b r a n e excitability are correlated with the ability o f the anions to release m e m b r a n e proteins into the internal fluid m e d i u m . T h e [JH]NEM-labeled squid axons i m m e r s e d in ASW were first p e r f u s e d intracellularly with a 400 mM KF solution, a n d then with a 400 m M solution o f KCI, KBr, or KSCN for a period o f 6 min. A f t e r these t r e a t m e n t s , the KF solution was r e i n t r o d u c e d into the axons. Excitability was s u p p r e s s e d within a short time a f t e r r e p l a c e m e n t o f the internal KF solution with o t h e r K-anion solutions. (Deterioration o f excitability was a c c o m p a n i e d by a fall in m e m b r a n e resistance.) T h e a v e r a g e survival time after onset of internal p e r f u s i o n with the KC1 solution, as d e t e r m i n e d by eight
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INOUE, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
individual m e a s u r e m e n t s , was 3.4 -+ 2.4 (SD) min; the average survival time with the KBr solution internally was 2.1 +- 1.7 (SD) min (nine axons). Loss o f excitability took place (within 30 s). It should be n o t e d that the average survival time obtained in the present study was almost 10% o f that r e p o r t e d by Tasaki et al., (1965). This e n h a n c e d effect was solely d e p e n d e n t on the extent o f removal o f the axoplasm f r o m the axon, a n d not on the [3H]NEM treatment. R e i n t r o d u c t i o n o f the KF solution to the axon interior t e n d e d to restore m e m b r a n e excitability previously suppressed by internal t r e a t m e n t with the o t h e r K-anion solutions. T h e d e g r e e o f recovery was strongly d e p e n d e n t on the anion species in the internal perfusion fluid. T o c o m p a r e the anion effect on the ability o f the axon to restore its excitability, attempts were m a d e to m e a s u r e the m e m b r a n e c u r r e n t u n d e r voltage clamp before and after t r e a t m e n t o f the axon with K-salt solutions. T h e voltage c u r r e n t relationships illustrated in Fig. 4 show A
mA.,,~2
-5o
B
1
KCI
o
50
-I
C 1
ioo
mv
-5o
ol . . . . -I
-2 -3
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~-
-5o
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-
-3
-4
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-5"
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-6 -7'
FICURE 4. Voltage-current relationships of the axon membrane before and after perfusion with 400 mM solutions of KC1 (A), KBr (B), and KSCN (C). Each anion produced an irreversible alteration of the electrical properties of the membrane. The period of perfusion with the solution listed above was 6 min. Curve marked a was obtained before switching the internal solution (400 mM KF) to each K-salt solution. Curve marked b was taken when a maximum recovery of excitability was attained by reintroduction of the KF solution into the axon. such anion effects. C u r v e a in each d i a g r a m represents the control obtained before a p p l y i n g the K-salt solution to the axon interior. Curve b was obtained f r o m the same axon when the m a x i m u m recovery o f excitability was attained by r e i n t r o d u c t i o n o f the KF solution. Fig. 4 A was taken f r o m an axon treated internally with the KCI solution. T h e m a g n i t u d e o f the action potential associated with excitation could be restored almost completely but recovery o f the m a x i m u m inward c u r r e n t was only 50%. With this d e g r e e o f recovery, c o n d u c tion o f action potential could be p r o d u c e d by electric stimulations. As shown in Fig. 4 B, the d e g r e e o f recovery was p o o r e r when an axon had been treated intracellularly with the KBr solution instead o f with the KCI solution. T h e r e was
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no c o m p l e t e recovery of the m a g n i t u d e o f the e m f j u m p associated with excitation. T h e m a x i m u m inward c u r r e n t was r e s t o r e d by only 20%. In general, axons lost their ability to p r o p a g a t e action potentials across the p e r f u s i o n zone u n d e r these conditions. As shown in Fig. 4 C , the axon was no longer capable o f p r o d u c i n g inward c u r r e n t in r e s p o n s e to d e p o l a r i z i n g voltage pulses. No all-orn o n e action potential could be elicited f r o m the a x o n in this case. M e m b r a n e resistance could be r e s t o r e d only to a level o f 20% o f that b e f o r e the K S C N treatment. A large a m o u n t o f protein was released into the internal fluid m e d i u m d u r i n g internal p e r f u s i o n with these K-anion solutions. T h e a m o u n t o f protein released was strongly d e p e n d e n t on the anion species in the internal p e r f u s i o n fluid; SCN ions released a b o u t 20%, Br ions released a b o u t 6%, and C1 ions released a b o u t 3% o f the total radioactivity in the squid fiber. Typical e x a m p l e s o f the results obtained are shown in Fig. 5. Fig. 5 A was obtained f r o m an axon internally
00 s(
C
-
OUTSIDE : ARTIFICIAL SEA WATER
-
KF
KF--
1,40~ A
Boo
cpm
-
-
KF-~"KBr"F--KF-300
200
200
200
I00
I00
100
0
Ts
30 0
15
30
is
30mi n
FIGURE 5. Effect of internal perfusion of media containing different anions on the release of [3H]NEM-labeled proteins into the internal fluid medium. The composition of the internal solutions used which contained 400 meq/liter of K-ions is given in the text. p e r f u s e d with the KC1 solution. T h e rate o f release o f proteins into the internal fluid m e d i u m (cpm/3 min) was e n h a n c e d a b o u t t h r e e times by r e p l a c e m e n t o f the KF solution with the KCI solution. R e i n t r o d u c t i o n o f the KF solution into the a x o n b r o u g h t a b o u t a fall in the rate o f protein release which a p p r o a c h e d the original low level within 6 min. Fig. 5B shows that intracellular application o f the KBr solution released proteins at a rate a p p r o x i m a t e l y six times as high as that u n d e r internal p e r f u sion with the KF solution. Fig. 5 C shows that i n t r o d u c t i o n o f the KSCN solution to the a x o n interior p r o d u c e d a d r a m a t i c rise in the rate o f protein release
INOUZ, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
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followed by a quick fall. In this case, the rate was reduced to a level much lower than the original level by replacement of the internal KSCN solution with the KF solution. Thus, it seems that the major portion of releasable proteins appeared in the perfusate within a short time after onset of internal perfusion with the KSCN solution. Results similar to those shown in Fig. 5 could be obtained from the [SH]NEMlabeled squid axons immersed in Ca2+-free ASW containing EGTA. The release of proteins by internally applied anions does not require the presence of Ca-ion in the external medium. DISCUSSION
Release of proteins from the inner surface of the squid axon plasma membrane was produced either by exposing the external surface of the axon to K+-rich solutions or by application of various anions to the internal surface of the axon membrane. The dependence of protein release by K + depolarization upon external calcium ions suggests that this phenomenon is related to an increased calcium concentration at the inner membrane under these conditions. The empirical application of salt perturbation in protein systems has been widely, employed by biochemists. In particular, CaCI~ and KSCN have been used for protein subunit dissociation (von Hippel and Schleich, 1969). The order of effectiveness of these ions in promoting stable protein conformations (i.e. helical and native conformations, loss of solubility, referred to as "salting-out") and causing decreased structural stability associated with greater solubility (i.e. random coil structure or denaturation, referred to as "salting-in") follows the classical Hofmeister series (Bungenberg d e J o n g , 1949; yon Hippel and Schleich, 1969). This ranking corresponds to the order of effectiveness of anions found to release proteins from inner membrane surface (Fig. 5). Singer and Nicolson (1972) have classified membrane proteins into "peripheral" (extrinsic) and "integral" (intrinsic) proteins largely on the basis of their solubility characteristics. Integral membrane proteins are hydrophobic and amphipathic, and involved in the structural integrity of membranes; whereas peripheral membrane proteins are considered to be loosely bound to the membrane and not stabilized by hydrophobic interactions. It is of interest to note that it is the latter which are uniquely associated with the cytoplasmic surface of the membrane (Steck, 1974), and that it may be this population of proteins which are being released in our experiments. As pointed out earlier, it has been demonstrated that ions which tend to solubilize proteins in the squid axon also tend to suppress membrane excitability when applied internally. For example, introduction of Ca-ions into the interior of an axon causes a fall in the membrane resistance and eventual loss of excitability (Tasaki, 1968). The adverse effect of the internal anions on excitability was demonstrated by Tasaki et al. (1965) as well as in the present study (Fig. 4). In contrast, the external surface of the axon membrane is quite insensitive to alterations of the anion composition in the external fluid medium, and calcium in the external medium plays an essential role in stabilizing the membrane macromolecules which are responsible for conformational changes associated with nerve excitation.
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A l t h o u g h considerable attention has recently been directed at the subsurface structure b e n e a t h the cytoplasmic m e m b r a n e ( H y d e n , 1974; Metuzals, 1969; Metuzals a n d Izzard, 1969; Metuzals a n d Mushynski, 1974; Le Beux and Willemot, 1975), it is still too early to identify those s u b m e m b r a n e structures related to the released proteins in this study. Recently, the fine structure o f the pronasetreated squid axon m e m b r a n e was e x a m i n e d by scanning electron microscopy (Metuzals a n d Tasaki, 1976). T h e results obtained clearly indicated the existence o f a longitudinally oriented structure m a d e o f fibrous material a d h e r i n g to the i n n e r surface o f the m e m b r a n e . Extensive pronase t r e a t m e n t o f the axon interior r e d u c e d the a m o u n t o f fibrous material but did not completely destroy the longitudinally oriented structure. It is therefore plausible that the major portion o f the proteins which a p p e a r e d in the perfusate was released from these fibrous s u b m e m b r a n e structures. I f that is the case, this s u b m e m b r a n e structure may be involved in the regulation o f excitability and hence should be r e g a r d e d as a part o f the excitable m e m b r a n e . Received for publication 9 February 1976. REFERENCES
C. M., F. BEZANILLA, and E. ROJAS. 1973. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J. Gen. Physiol. 62:375-391. BAKER, P. F. 1972. Transport and metabolism of calcium ions in nerve. Prog. Biophys. Mol. Biol. 6:177-223. BUNGENBERG DE JONG, H. G. 1949. Crystallization-coacervation-flocculation, and complex colloidal systems. In Colloid Science. H. R. Kruyt, editor. Elsevier Publishing Co., 2:232-258; 335-432. GAINER, H . , E. CARBONE, [. SINGER, K. SISCO, and I. TASAKI. 1974. Depolarizationinduced change in the enzymatic radio-iodination of a protein on the internal surface of the squid giant axon membrane. Comp. Biochem. Physiol. 47 A:477-484. HYDEN, H. 1974. A calcium-dependent mechanism for synapse and nerve cell membrane modulation. Proc. Natl. Acad. Sci. U. S. A. 71:2965-2968. LE BEUX, Y. J., and J. WILLEMOT.1975. An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. Cell Tissue Res. 160:37-68. LEmaAN, L., A. WATANABE,and I. TASAKI. 1969. Intracellular perfusion of squid giant axons: recent findings and interpretations. Neurosci. Res. 2"71-105. MEANS, G. E., and R. E. FEENY. 1971. Chemical modification of proteins. Holden-Day Inc., San Francisco, Calif. 1-245. METUZALS,J. 1969. Configuration of a filamentous network in the axoplasm of the squid (Loligo pealii L.) giant nerve fiber. J. Cell Biol. 43:480. METUZALS, J., and C. S. IZZARD. 1969. Spatial patterns of threadlike elements in the axoplasm of the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interface microscopy and by electron microscopy. J. Cell Biol. 43:456. METUZALS, J., and W. E. MUSHYNSKI. 1974. Electron microscope and experimental investigations of the neurofilamentous network in Deiters neurons. Relationship with the cell surface and nuclear pores. J. Cell Biol. 61:701-722. ARMSTRONG,
INouz, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
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METUZALS, J., and 1. TASAKI. 1976. Scanning electron microscopy o f the filamentous network on the cytoplasmic face o f the axolemma. J. Cell Biol. 70(2, Pt. 2):117a. (Abstr.). NEVILLE, D. M. 1971. Molecular weight determination o f protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system.J. Biol. Chem. 246:63286334. SATO, H., I. TASAKI, E. CARBONE,and M. HALLETT. 1973. Changes in axon birefringence associated with excitation: implications for the structure o f the axon m e m b r a n e . J. Mechanochem. Cell Motil. 2:209-217. SINGER, S. J., and G. L. NICOLSON. 1972. T h e fluid mosaic model of the structure of cell membranes. Science ( Wash. D.C.). 175:720-731. STECK, T . L. 1974. T h e organization o f proteins in the h u m a n red blood cell m e m b r a n e . A review.J. Cell Biol. 62:1-19. TAKENAKA,T., R. HIRAKOW,and S. Y. YAMAGISHI.1968. Ultrastructural examination o f the squid giant axon perfused intraceilularly with protease. J. Ultrastruct. Res. 25:408416. TAKENAKA, T . , and S. YAMASmGI. 1966. Intracellular perfusion o f squid giant axons with protease solution. Proc. Jpn. Acad. 42:521-526. TAKENAKA, T., T. YOSHIOKA, H. HORIE, and F. WATANABE. 1976. Changes in 1251-labeled m e m b r a n e proteins d u r i n g excitation of the squid giant axon. Comp. Biochem. Physiol. In press. TASAK~, I. 1968. Nerve excitation: a macromolecular approach. Charles C Thomas, Publisher, Springfield, Ill. 1-201. TASAKI, I., I. SINGER, and T. TAKENAKA. 1965. Effects o f internal and external ionic environment on excitability of squid giant axon. J. Gen. Physiol. 48:1095-1123. VON HIPPEL, P. H., and T. SCHLEICH. 1969. T h e effects o f neutral salts on the structure and conformational stability o f macromolecules in solution. In Structure and Stability of Biological Macromolecules. S. N. T i m a s h e f f and G. D. Fasman, editors. Marcel Dekker, Inc., New York.
Squid giant axons are k n o w n to m a i n t a i n their electrical excitability a f t e r extensive r e m o v a l o f the a x o p l a s m with proteases ( T a k e n a k a et al., 1968). P r o l o n g e d t r e a t m e n t o f the a x o n m e m b r a n e with intracellularly a d m i n i s t e r e d p r o n a s e initially p r o d u c e s a decrease in the m a x i m u m inward c u r r e n t u n d e r voltage c l a m p (Sato et al., 1973), b e f o r e the decrease in a m p l i t u d e a n d p r o l o n g a t i o n o f the d u r a t i o n o f the action potential is o b s e r v e d ( T a k e n a k a a n d Yamashigi, 1966; A r m s t r o n g et al., 1973). Ultimately, the proteolytic action causes inexcitability o f the a x o n (Tasaki, 1968). Such observations suggest that proteins associated with the internal surface o f the a x o n m e m b r a n e are involved in the regulation o f excitability. In an earlier investigation (Gainer et al., 1974), the proteins in the internal m e m b r a n e surface o f the squid a x o n w e r e labeled with I2~1 by using an enzymatic radio-iodination p r o c e d u r e in c o m b i n a t i o n with the intracellular p e r f u s i o n technique. T h i s study d e m o n s t r a t e d that a p r o t e i n with a m o l e c u l a r weight equal to a b o u t 12,000 daltons was associated with the internal m e m b r a n e surface, a n d the ability o f this protein to be iodinated was selectively r e d u c e d by p o t a s s i u m depolarization o f the a x o n . Recent studies by T a k e n a k a et al. (1976) have conTHE JOURNAL OF GENERAL PHYSIOLOGY' VOLUME 68, 1 9 7 6 " p a g e s 3 8 5 - 3 9 5
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f i r m e d these f i n d i n g s , a n d s u g g e s t t h a t t h e l a b e l e d 12,000-dalton p r o t e i n is r e l e a s e d i n t o the p e r f u s a t e a f t e r p o t a s s i u m d e p o l a r i z a t i o n o r electrical activity o f t h e a x o n . I n the p r e s e n t s t u d y , we have l a b e l e d the p r o t e i n s in t h e m e m b r a n e by i n t r a c e l l u l a r p e r f u s i o n o f [ 3 H ] N - e t h y l m a l e i m i d e ([3H]NEM) a n d h a v e m o n i t o r e d t h e p e r f u s a t e f o r release o f l a b e l e d p r o t e i n s a f t e r v a r i o u s e x p e r i m e n t a l t r e a t m e n t s o f the a x o n . We r e p o r t h e r e t h a t l a b e l e d p r o t e i n s a r e r e l e a s e d f r o m the i n t e r n a l s u r f a c e o f the m e m b r a n e by p o t a s s i u m d e p o l a r i z a t i o n (in t h e prese n c e o f e x t e r n a l c a l c i u m ions only), a n d by the i n t e r n a l p e r f u s i o n o f v a r i o u s anions. MATERIALS
AND
METHODS
Electrophysiotogical Methods Giant axons of squid Loligo pealei available at the Marine Biological Laboratory, Woods Hole, Mass., were used throughout the present experiments. The diameter of the axons used was between 280 and 380/.~m. T h e major portion of small nerve fibers and other adherent tissues s u r r o u n d i n g the giant fiber were removed u n d e r a dissecting microscope. The axon was then transferred to a Lucite chamber filled with artificial seawater (ASW) in which internal perfusion with two glass cannulae was performed (Lerman et al., 1969). T h e outside diameter of the inlet cannula was 120/xm (see Fig, 1, right). T h e standard internal perfusion solution contained 400 meq/liter K-ion, 350 meq/liter F-ion, phosphate (potassium salt) buffer adjusted to a pH between 7.2 and 7.4, and glycerol (4% by volume). In the present study the axoplasm within a long portion of the axon, 28-30 mm in length, was removed extensively by means of enzymatic digestion. Axons used were initially perfused intracellularly with the KF solution containing 0.01 mg/ml pronase (Calbiochem, San Diego, Calif.) until the maximum inward c u r r e n t u n d e r voltage clamp was reduced to about 80% of the level before the start of internal perfusion (approximately 12-20 min). After replacement of the enzyme-containing solution with an enzymefree one, the perfusion zone was shortened by inserting both of the cannulae 5 mm deeper into the axon so that the axoplasm remaining in the unperfused zone was separated from the perfusion zone by the nonflowing KF solution. T h e length of the perfusion zone was thus between 18 and 20 mm. The flow rate of the internal perfusion fluid was maintained at a level between 20 and 35/zl/min. T o study the effects of internal anions, the KF in the standard perfusion solution described above was replaced by an equimolar a m o u n t of KCI, KBr, or KSCN. The composition of the ASW used was 423 mM NaCI, 9 mM KC1, 23 mM MgCI2, 25 MgSO~, 10 mM CaCI2, and 5 mM Tris (the pH of the external solution was adjusted to between 7.8 and 8.0). T h e composition of the external potassium depolarization solution was identical to the above ASW except that the NaC1 was reduced to 250 mM and the KCI raised to 200 mM. When Ca-ions were removed from the K-rich ASW, ethylene glycol bis (/3-aminoethyl ether) tetraacetic acid (EGTA) was added to a concentration of 4 raM. In all cases the osmotic pressures of the various external solutions were equated by the addition of small amounts of glycerol, and the pH was between 7.8 and 8.0. The potential difference across the m e m b r a n e was measured at the center of the perfusion zone with a glass pipette Ag-AgCI electrode filled with a 3 M KC1 solution (70 /zm in diameter). T h e pipette was filled with KCl-agar gel near the tip to reduce outflow of the KCI solution. A calomel electrode immersed in the external medium was used as the reference point for potential measurements. T h e external fluid medium was g r o u n d e d through a large coil of platinized platinum wire. Stimulating shocks were delivered through a pair of platinum electrodes to the unperfused zone of the axon when
INOUE, PANT, TASAKI, AND GAINER Sqt~d Axolrt Membran¢ Proteins
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p r o p a g a t i o n o f impulses across the p e r f u s i o n z o n e was m o n i t o r e d . When application o f either c u r r e n t pulses or voltage pulses to the m e m b r a n e was required, a platinized platinum wire (50/~m in diameter) was inserted into the axon t h r o u g h the outlet cannula.
Biochemical Methods Tritiated N-ethylmaleimide ([SH]NEM, sp act 230 mCi/mmol) was purchased from New England Nuclear, Boston, Mass. T h e [SH]NEM (250/~Ci) was dissolved in 20 ml of the 400 mM KF solution. T h e NEM concentration in this solution was approximately 50/zM. T h e [3H]NEM-containing KF solution was i n t r o d u c e d into the axon from which the axoplasm had been removed by the method described above, and the axon was perfused with this cpm ~K-DEPO.~-~
400ram KF
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FIGURE 1. Schematic illustration on the right shows the experimental arrangement used for measuring the a m o u n t o f [aH]NEM-labeled proteins a p p e a r i n g in the perfusate. IN and OUT r e p r e s e n t the inlet and outlet cannulae, respectively. T h e internal perfusion fluid used was a 400 mM KF solution (see text). Left, release o f the radioactive proteins into the internal fluid m e d i u m d u r i n g normal resting state and d u r i n g potassium depolarization. T h e depolarization was p r o d u c e d by switching the external seawater to a K-rich m e d i u m containing CaC12. T h e salt composition of the K-rich solution is given in the text. solution for 15 min. During this period, the axon was immersed in rapidly circulating (50 ml/min) artificial seawater. No detectable deterioration of the m e m b r a n e excitability was p r o d u c e d by the [aH]NEM treatment u n d e r the present experimental conditions. After switching to a [aH]NEM-free KF solution, continuous flow of both the internal and external fluid media was maintained for 15-20 min in o r d e r to eliminate free [aH]NEM from this system. T h e radioactivity o f the total proteins that could be extracted from one whole single fiber p r e p a r a t i o n was o f the o r d e r o f 104 cpm. (A small amount o f efflux o f [3H]NEM across the m e m b r a n e [20-30 cpm/s, cm 2] was observed d u r i n g internal application o f the [3H]NEM-containing KF solution.) Although NEM is commonly considered specific for sulfhydryl groups, it can also react to some extent with amino groups. At the p H of the internal perfusion fluid, the reaction rate o f NEM with thiols is about 1,000-fold greater than with amino c o m p o u n d s (Means and Feeny, 1971). T h e r e f o r e , u n d e r these conditions, it is likely that the principal reaction of the [aH]NEM in this study was with the free sulfhydryl groups in m e m b r a n e proteins. T h e labeled proteins released from the m e m b r a n e into the internal perfusing m e d i u m were measured by collecting fractions o f perfusate from the outlet cannula onto pads of W h a t m a n 3 mm filter p a p e r (Fig, 1). Contamination by free [aH]NEM in each collected
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sample was eliminated by precipitating proteins with trichloroacetic acid (TCA). The TCA precipitation procedure consisted of two washes of the pads in ice-cold 10% TCA (30 min each), followed by a hot (95°C) extraction in 5% TCA (30 min), another 30 min in cold 5% TCA, a 30-min wash of the pads in ethanol:ether (1:1, vol/vot), and then 30 min in 100% ether. In control experiments it was demonstrated that any protein-bound [3H]NEM and not free [aH]NEM remained in the pad after this procedure. The radioactivity of the proteins remaining on the filter paper was measured with a Beckman liquid scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). In one series of experiments perfusate was collected in small glass test tubes. Standard proteins (bovine serum albumin, 68,000 daltons; ovalbumin, 45,000 daltons; and cytochrome c, 12,000 daltons) were added as carrier, and the proteins in the tubes were precipitated with TCA. The pellet was washed with ether to remove the TCA, and then dissolved in 1% SDS, and reduced by the addition of/3-mercaptoethanol. The proteins in these samples were then separated by polyacrylamide gel disc electrophoresis in SDS (Neville, 1971). The standard proteins in the coprecipitate also served as internal molecular weight markers on each gel. The gels were sliced, liquid scintillation cocktail was added, and the radioactivity of each slice was measured with a Beckman liquid scintillation counter. All the experiments were conducted at room temperature (20° -+ l°C). RESULTS
Induction of Protein Release from Membrane by Potassium Depolarization A f t e r the labeling o f the squid axons with [3H]NEM a n d the washout o f the u n b o u n d label (see Materials a n d Methods), the axons were continuously perfused with the s t a n d a r d 400 m M KF solution in circulating ASW. U n d e r these conditions the axons maintain their ability to p r o p a g a t e n e r v e impulses across the p e r f u s i o n zone for m o r e t h a n 2 h. T h e resting potential o f the exitable m e m b r a n e was b e t w e e n - 5 5 a n d - 6 0 m V , and the m e m b r a n e resistance was usually between 0.7 and 1.5 k 1"/. cm 2. T h e external ASW was t h e n switched to a K-rich solution, which d e p o l a r i z e d the a x o n by a p p r o x i m a t e l y 50 m V . T h e m e m b r a n e resistance o f the depolarized a x o n was a b o u t 200 1/. cm ~. T h e effect o f such m e m b r a n e depolarization on the release o f proteins into the internal fluid m e d i u m was e x a m i n e d . T h e e x p e r i m e n t a l p r o c e d u r e is schematically illustrated on the right side o f Fig. 1. Perfusate was collected at 3-min intervals, a n d the radioactivity o f proteins in each collected sample was m e a sured. A typical e x a m p l e o f the results obtained is r e p r e s e n t e d on the left side o f the figure. T h e abscissa indicates the time in minutes a f t e r the start o f the collection o f p e r f u s a t e . T h e rate o f release o f proteins (cpm/3 min) was maintained at a low level as long as the a x o n was i m m e r s e d in ASW. A f t e r replacem e n t o f the e x t e r n a l seawater with the K-rich solution, t h e r e was a significant rise in the rate o f protein release followed by a g r a d u a l decline. R e p l a c e m e n t o f the K-rich external solution by seawater outside the a x o n b r o u g h t a b o u t a rapid fall in the rate o f protein release which finally r e t u r n e d to the original low level. In general, b o t h the m e m b r a n e potential a n d m e m b r a n e resistance gradually a p p r o a c h e d the original values o f the resting state a f t e r recirculation o f seawater. C o n d u c t i o n could usually be restored d u r i n g repolarization o f the m e m brane. In a separate e x p e r i m e n t , the radioactive proteins which a p p e a r e d in the
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INOUE, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
perfusate were analyzed by SDS gel electrophoresis. In Fig. 2 the broken line shows the molecular weight distribution of proteins released into the internal fluid medium before depolarization, and the solid line shows the molecular weight distribution of proteins released during depolarization. T h e depolarization was produced by immersing the axon in the K-rich solution. One distinct peak with approximately 12,000 daltons was produced by the potassium depolarization. These results are consistent with those reported in previous papers 68
4ol
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FIGURE 2. Molecular weight distribution on SDS polyacrylamide gels of a fraction of [nH]NEM-labeled proteins which were present in the perfusate under normal resting state (NORMAL) and during potassium depolarization (K-DEPO.). The depolarization was produced by replacing the external seawater with the K-rich solution containing CaCI2. Note that a 12,000-dalton labeled protein was released by potassium depolarization. (Gainer et al., 1974; Takenaka et al., 1976) in which membrane labeling was done by radio-iodination.
Ca ++-Dependence of Protein Release T h e data in Fig. 3 illustrate the dependence of protein release upon Ca ++ in the internal medium. Axons were internally perfused with the KF solution. T h e external solutions used are given in the figure (see Materials and Methods for composition of solutions). As illustrated in Fig. 3 A and B, no significant alteration of the rate of release of radioactive proteins in the internal perfusion fluid could be produced if the membrane was depolarized with the K-EGTA solution. However, as shown in Fig. 3 B, there was sharp rise in the rate of protein release when CaCI2 was added to the external K-rich medium by replacing the K-EGTA solution with the K-Ca solution. In both o f the experiments in Fig. 3, conduction
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o f the action potential could be r e s t o r e d by recirculation o f seawater outside the axons. T h e m e m b r a n e potentials a n d resistance o f the depolarized a x o n in the K - E G T A a n d K-Ca ++ solutions did not significantly differ. Variation o f the Mg ~+ concentration o f the K - E G T A solution between 5 and 50 m M did not affect the rate o f protein release. T h e s e findings suggest strongly that the release o f proteins d u r i n g p o t a s s i u m depolarization arises f r o m the interaction between external Ca-ions a n d the m a c r o m o l e c u l e s at the i n n e r surface o f the m e m b r a n e , p r e s u m a b l y d u e to the e n h a n c e d rate o f Ca 2+ e n t r y into the m e m b r a n e d u r i n g potassium depolarization (Baker, 1972).
A
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FIGURE 3. Effect of external Ca++-ion on release of [JH]NEM-labeled proteins during potassium depolarization. Two kinds of K-rich solutions containing 200 mM KC1 were used to depolarize the membrane. K-EGTA solution contained 4 mM EGTA but no Ca++-ions. K-Ca solution contained 10 mM CaCI2, but no EGTA.
Effects of Internal Anions on Protein Release and Excitability of the Axon Previous studies have d e m o n s t r a t e d that the excitability o f the p e r f u s e d squid a x o n is strongly affected by the anionic species used in the p e r f u s i o n fluid (Tasaki et al., 1965). T h e efficacy o f internal anions in m a i n t a i n i n g n o r m a l excitability increased in the o r d e r o f S C N - < I - < B r - < C1- < SO4 =, < H P O 4 =, < F - . This anion sequence c o r r e s p o n d s to the lyotropic (or H o f m e i s t e r ) series a n d is directly related to the salting-out effect o f anions on proteins, w h e r e S C N is least effective a n d F - most effective (see Tasaki et al., 1965, for a discussion o f these p h e n o m e n a in relation to the squid axon). T h e following e x p e r i m e n t s indicate that such anion effects on m e m b r a n e excitability are correlated with the ability o f the anions to release m e m b r a n e proteins into the internal fluid m e d i u m . T h e [JH]NEM-labeled squid axons i m m e r s e d in ASW were first p e r f u s e d intracellularly with a 400 mM KF solution, a n d then with a 400 m M solution o f KCI, KBr, or KSCN for a period o f 6 min. A f t e r these t r e a t m e n t s , the KF solution was r e i n t r o d u c e d into the axons. Excitability was s u p p r e s s e d within a short time a f t e r r e p l a c e m e n t o f the internal KF solution with o t h e r K-anion solutions. (Deterioration o f excitability was a c c o m p a n i e d by a fall in m e m b r a n e resistance.) T h e a v e r a g e survival time after onset of internal p e r f u s i o n with the KC1 solution, as d e t e r m i n e d by eight
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INOUE, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
individual m e a s u r e m e n t s , was 3.4 -+ 2.4 (SD) min; the average survival time with the KBr solution internally was 2.1 +- 1.7 (SD) min (nine axons). Loss o f excitability took place (within 30 s). It should be n o t e d that the average survival time obtained in the present study was almost 10% o f that r e p o r t e d by Tasaki et al., (1965). This e n h a n c e d effect was solely d e p e n d e n t on the extent o f removal o f the axoplasm f r o m the axon, a n d not on the [3H]NEM treatment. R e i n t r o d u c t i o n o f the KF solution to the axon interior t e n d e d to restore m e m b r a n e excitability previously suppressed by internal t r e a t m e n t with the o t h e r K-anion solutions. T h e d e g r e e o f recovery was strongly d e p e n d e n t on the anion species in the internal perfusion fluid. T o c o m p a r e the anion effect on the ability o f the axon to restore its excitability, attempts were m a d e to m e a s u r e the m e m b r a n e c u r r e n t u n d e r voltage clamp before and after t r e a t m e n t o f the axon with K-salt solutions. T h e voltage c u r r e n t relationships illustrated in Fig. 4 show A
mA.,,~2
-5o
B
1
KCI
o
50
-I
C 1
ioo
mv
-5o
ol . . . . -I
-2 -3
KSCN
KBr
5,o... w~/.,
~-
-5o
50~0~.. 10 0 .Z
-1 -2
-
-3
-4
-4
-5"
-5-
-6
-6 -7'
FICURE 4. Voltage-current relationships of the axon membrane before and after perfusion with 400 mM solutions of KC1 (A), KBr (B), and KSCN (C). Each anion produced an irreversible alteration of the electrical properties of the membrane. The period of perfusion with the solution listed above was 6 min. Curve marked a was obtained before switching the internal solution (400 mM KF) to each K-salt solution. Curve marked b was taken when a maximum recovery of excitability was attained by reintroduction of the KF solution into the axon. such anion effects. C u r v e a in each d i a g r a m represents the control obtained before a p p l y i n g the K-salt solution to the axon interior. Curve b was obtained f r o m the same axon when the m a x i m u m recovery o f excitability was attained by r e i n t r o d u c t i o n o f the KF solution. Fig. 4 A was taken f r o m an axon treated internally with the KCI solution. T h e m a g n i t u d e o f the action potential associated with excitation could be restored almost completely but recovery o f the m a x i m u m inward c u r r e n t was only 50%. With this d e g r e e o f recovery, c o n d u c tion o f action potential could be p r o d u c e d by electric stimulations. As shown in Fig. 4 B, the d e g r e e o f recovery was p o o r e r when an axon had been treated intracellularly with the KBr solution instead o f with the KCI solution. T h e r e was
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OF G E N E R A L P H Y S I O L O G Y • V O L U M E 6 8 • 1 9 7 6
no c o m p l e t e recovery of the m a g n i t u d e o f the e m f j u m p associated with excitation. T h e m a x i m u m inward c u r r e n t was r e s t o r e d by only 20%. In general, axons lost their ability to p r o p a g a t e action potentials across the p e r f u s i o n zone u n d e r these conditions. As shown in Fig. 4 C , the axon was no longer capable o f p r o d u c i n g inward c u r r e n t in r e s p o n s e to d e p o l a r i z i n g voltage pulses. No all-orn o n e action potential could be elicited f r o m the a x o n in this case. M e m b r a n e resistance could be r e s t o r e d only to a level o f 20% o f that b e f o r e the K S C N treatment. A large a m o u n t o f protein was released into the internal fluid m e d i u m d u r i n g internal p e r f u s i o n with these K-anion solutions. T h e a m o u n t o f protein released was strongly d e p e n d e n t on the anion species in the internal p e r f u s i o n fluid; SCN ions released a b o u t 20%, Br ions released a b o u t 6%, and C1 ions released a b o u t 3% o f the total radioactivity in the squid fiber. Typical e x a m p l e s o f the results obtained are shown in Fig. 5. Fig. 5 A was obtained f r o m an axon internally
00 s(
C
-
OUTSIDE : ARTIFICIAL SEA WATER
-
KF
KF--
1,40~ A
Boo
cpm
-
-
KF-~"KBr"F--KF-300
200
200
200
I00
I00
100
0
Ts
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15
30
is
30mi n
FIGURE 5. Effect of internal perfusion of media containing different anions on the release of [3H]NEM-labeled proteins into the internal fluid medium. The composition of the internal solutions used which contained 400 meq/liter of K-ions is given in the text. p e r f u s e d with the KC1 solution. T h e rate o f release o f proteins into the internal fluid m e d i u m (cpm/3 min) was e n h a n c e d a b o u t t h r e e times by r e p l a c e m e n t o f the KF solution with the KCI solution. R e i n t r o d u c t i o n o f the KF solution into the a x o n b r o u g h t a b o u t a fall in the rate o f protein release which a p p r o a c h e d the original low level within 6 min. Fig. 5B shows that intracellular application o f the KBr solution released proteins at a rate a p p r o x i m a t e l y six times as high as that u n d e r internal p e r f u sion with the KF solution. Fig. 5 C shows that i n t r o d u c t i o n o f the KSCN solution to the a x o n interior p r o d u c e d a d r a m a t i c rise in the rate o f protein release
INOUZ, PANT, TASAKI, AND GAINER Squid Axon Membrane Proteins
393
followed by a quick fall. In this case, the rate was reduced to a level much lower than the original level by replacement of the internal KSCN solution with the KF solution. Thus, it seems that the major portion of releasable proteins appeared in the perfusate within a short time after onset of internal perfusion with the KSCN solution. Results similar to those shown in Fig. 5 could be obtained from the [SH]NEMlabeled squid axons immersed in Ca2+-free ASW containing EGTA. The release of proteins by internally applied anions does not require the presence of Ca-ion in the external medium. DISCUSSION
Release of proteins from the inner surface of the squid axon plasma membrane was produced either by exposing the external surface of the axon to K+-rich solutions or by application of various anions to the internal surface of the axon membrane. The dependence of protein release by K + depolarization upon external calcium ions suggests that this phenomenon is related to an increased calcium concentration at the inner membrane under these conditions. The empirical application of salt perturbation in protein systems has been widely, employed by biochemists. In particular, CaCI~ and KSCN have been used for protein subunit dissociation (von Hippel and Schleich, 1969). The order of effectiveness of these ions in promoting stable protein conformations (i.e. helical and native conformations, loss of solubility, referred to as "salting-out") and causing decreased structural stability associated with greater solubility (i.e. random coil structure or denaturation, referred to as "salting-in") follows the classical Hofmeister series (Bungenberg d e J o n g , 1949; yon Hippel and Schleich, 1969). This ranking corresponds to the order of effectiveness of anions found to release proteins from inner membrane surface (Fig. 5). Singer and Nicolson (1972) have classified membrane proteins into "peripheral" (extrinsic) and "integral" (intrinsic) proteins largely on the basis of their solubility characteristics. Integral membrane proteins are hydrophobic and amphipathic, and involved in the structural integrity of membranes; whereas peripheral membrane proteins are considered to be loosely bound to the membrane and not stabilized by hydrophobic interactions. It is of interest to note that it is the latter which are uniquely associated with the cytoplasmic surface of the membrane (Steck, 1974), and that it may be this population of proteins which are being released in our experiments. As pointed out earlier, it has been demonstrated that ions which tend to solubilize proteins in the squid axon also tend to suppress membrane excitability when applied internally. For example, introduction of Ca-ions into the interior of an axon causes a fall in the membrane resistance and eventual loss of excitability (Tasaki, 1968). The adverse effect of the internal anions on excitability was demonstrated by Tasaki et al. (1965) as well as in the present study (Fig. 4). In contrast, the external surface of the axon membrane is quite insensitive to alterations of the anion composition in the external fluid medium, and calcium in the external medium plays an essential role in stabilizing the membrane macromolecules which are responsible for conformational changes associated with nerve excitation.
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A l t h o u g h considerable attention has recently been directed at the subsurface structure b e n e a t h the cytoplasmic m e m b r a n e ( H y d e n , 1974; Metuzals, 1969; Metuzals a n d Izzard, 1969; Metuzals a n d Mushynski, 1974; Le Beux and Willemot, 1975), it is still too early to identify those s u b m e m b r a n e structures related to the released proteins in this study. Recently, the fine structure o f the pronasetreated squid axon m e m b r a n e was e x a m i n e d by scanning electron microscopy (Metuzals a n d Tasaki, 1976). T h e results obtained clearly indicated the existence o f a longitudinally oriented structure m a d e o f fibrous material a d h e r i n g to the i n n e r surface o f the m e m b r a n e . Extensive pronase t r e a t m e n t o f the axon interior r e d u c e d the a m o u n t o f fibrous material but did not completely destroy the longitudinally oriented structure. It is therefore plausible that the major portion o f the proteins which a p p e a r e d in the perfusate was released from these fibrous s u b m e m b r a n e structures. I f that is the case, this s u b m e m b r a n e structure may be involved in the regulation o f excitability and hence should be r e g a r d e d as a part o f the excitable m e m b r a n e . Received for publication 9 February 1976. REFERENCES
C. M., F. BEZANILLA, and E. ROJAS. 1973. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J. Gen. Physiol. 62:375-391. BAKER, P. F. 1972. Transport and metabolism of calcium ions in nerve. Prog. Biophys. Mol. Biol. 6:177-223. BUNGENBERG DE JONG, H. G. 1949. Crystallization-coacervation-flocculation, and complex colloidal systems. In Colloid Science. H. R. Kruyt, editor. Elsevier Publishing Co., 2:232-258; 335-432. GAINER, H . , E. CARBONE, [. SINGER, K. SISCO, and I. TASAKI. 1974. Depolarizationinduced change in the enzymatic radio-iodination of a protein on the internal surface of the squid giant axon membrane. Comp. Biochem. Physiol. 47 A:477-484. HYDEN, H. 1974. A calcium-dependent mechanism for synapse and nerve cell membrane modulation. Proc. Natl. Acad. Sci. U. S. A. 71:2965-2968. LE BEUX, Y. J., and J. WILLEMOT.1975. An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. Cell Tissue Res. 160:37-68. LEmaAN, L., A. WATANABE,and I. TASAKI. 1969. Intracellular perfusion of squid giant axons: recent findings and interpretations. Neurosci. Res. 2"71-105. MEANS, G. E., and R. E. FEENY. 1971. Chemical modification of proteins. Holden-Day Inc., San Francisco, Calif. 1-245. METUZALS,J. 1969. Configuration of a filamentous network in the axoplasm of the squid (Loligo pealii L.) giant nerve fiber. J. Cell Biol. 43:480. METUZALS, J., and C. S. IZZARD. 1969. Spatial patterns of threadlike elements in the axoplasm of the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interface microscopy and by electron microscopy. J. Cell Biol. 43:456. METUZALS, J., and W. E. MUSHYNSKI. 1974. Electron microscope and experimental investigations of the neurofilamentous network in Deiters neurons. Relationship with the cell surface and nuclear pores. J. Cell Biol. 61:701-722. ARMSTRONG,
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METUZALS, J., and 1. TASAKI. 1976. Scanning electron microscopy o f the filamentous network on the cytoplasmic face o f the axolemma. J. Cell Biol. 70(2, Pt. 2):117a. (Abstr.). NEVILLE, D. M. 1971. Molecular weight determination o f protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system.J. Biol. Chem. 246:63286334. SATO, H., I. TASAKI, E. CARBONE,and M. HALLETT. 1973. Changes in axon birefringence associated with excitation: implications for the structure o f the axon m e m b r a n e . J. Mechanochem. Cell Motil. 2:209-217. SINGER, S. J., and G. L. NICOLSON. 1972. T h e fluid mosaic model of the structure of cell membranes. Science ( Wash. D.C.). 175:720-731. STECK, T . L. 1974. T h e organization o f proteins in the h u m a n red blood cell m e m b r a n e . A review.J. Cell Biol. 62:1-19. TAKENAKA,T., R. HIRAKOW,and S. Y. YAMAGISHI.1968. Ultrastructural examination o f the squid giant axon perfused intraceilularly with protease. J. Ultrastruct. Res. 25:408416. TAKENAKA, T . , and S. YAMASmGI. 1966. Intracellular perfusion o f squid giant axons with protease solution. Proc. Jpn. Acad. 42:521-526. TAKENAKA, T., T. YOSHIOKA, H. HORIE, and F. WATANABE. 1976. Changes in 1251-labeled m e m b r a n e proteins d u r i n g excitation of the squid giant axon. Comp. Biochem. Physiol. In press. TASAK~, I. 1968. Nerve excitation: a macromolecular approach. Charles C Thomas, Publisher, Springfield, Ill. 1-201. TASAKI, I., I. SINGER, and T. TAKENAKA. 1965. Effects o f internal and external ionic environment on excitability of squid giant axon. J. Gen. Physiol. 48:1095-1123. VON HIPPEL, P. H., and T. SCHLEICH. 1969. T h e effects o f neutral salts on the structure and conformational stability o f macromolecules in solution. In Structure and Stability of Biological Macromolecules. S. N. T i m a s h e f f and G. D. Fasman, editors. Marcel Dekker, Inc., New York.
