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Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes
305
Biochimica et Biophysica Acta, 5 1 0 ( 1 9 7 8 ) 3 0 5 - - 3 1 5 © Elsevier/North-Holland
Biomedical Press
BBA 78056
CALCIUM-SENSITIVE UNIVALENT CATION CHANNEL FORMED BY LYSOTRIPHOSPHOINOSITIDE IN BILAYER LIPID MEMBRANES
FUMIO HAYASHI a,. MASAHIRO SOKABE b,** MASAYUKI HAYASHI c and UICHIRO KISHIMOTO a
T A K A G I a, K O H E I
a Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, b Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, and c Department of Biochemistry, Faculty of Medicine, Gumma University, Gumma 371 (Japan) (Received October 31st, 1977)
Summary A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to ur?ivalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb÷> Cs+> Na+> K+> Li+. The conductance of the membrane was increased up to a value of about 10 -2 o h m - ' / cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn 2+ > Cd2+ > Ca 2+ > Sr 2+ > Mg2+.
Introduction Inositol phospholipids have been known to be present in a wide variety of mammalian tissues and their various physiological activities have been reviewed recently [1]. Triphosphoinositide has particularly been regarded as an important constituent of the nervous system, because of the high turnover rate of their phosphate moiety [2--4]. The predominant localization of brain triphosphoinositide in the myeline sheath has been well established [5--7]. However, the rapid post-mortem depletion of triphosphoinositide has also been reported * P r e s e n t addresses: D e p a r t m e n t of Biology, College of General Education, Kobe University, Nada, Kobe 657, Japan. ** Department of Behaviorology, Faculty of H u m a n Sciences, Osaka University, Yamada, Suita, Osaka 565, Japan.
306 [8--10] and recently Eichberg and Hauser have claimed the localization of this substance at the cell m e m b r a n e of nervous systems including synapse or axon [11,12]. Triphosphoinositide has unique physicochemical characteristics. Since triphosphoinositide has five net negative charges at physiological pH, it can be dissolved into water very smoothly. On the ot her hand, it shows an extraordinarily high affinity for calcium ion, resulting in a highly h y d r o p h o b i c c o mp lex with Ca 2÷ ]13,14]. Thus, triphosphoinositide shows a drastic change in hydrophilicity, when the univalent-divalent cation exchange reaction occurs. A hypothesis was proposed to explain the molecular mechanism of nerve excitation by Tasaki and his coworkers [15]. In their hypothesis, it was proposed that an essential process of nerve excitation is a kind of phase transition of a certain c o m p o n e n t of the excitable membrane; the phase transition is caused by desorption of calcium ion from the m em brane surface. No material has, however, been proposed as the key substance which undergoes such a phase transition in the excitable membrane. We intended to incorporate the triphosphoinositide into an artificial membrane as a c o m p o n e n t which could undergo a h y d r o p h i l i c - h y d r o p h o b i c micelle transition with univalent-divalent cation exchange. However, triphosphoinositide had no effect on the c o n d u c t a n c e of artificial bilayer m em brane of oxidized cholesterol. In contrast, a very little a m o u n t of lysotriphosphoinositide which was an oxidative breakdown p r o d u c t of triphosphoinositide was f o u n d to give a remarkably large c o n d u c t a n c e to the artificial membrane. The lysotriphosphoinositide could penetrate into the m em brane and formed a channel system which was selectively permeable to univalent cations. Moreover, the univalent cation c o n d u c t a n c e of the m e m brane was greatly influenced by the c o n c e n t r a t i o n of calcium ion in the aqueous phase. We will r ep o r t some features of this univalent cation channel constructed from the polar head groups of lysotriphosphoinositides. Materials and Methods
Extraction of triphosphoinositide Triphosphoinositide was obtained by Folch fractionation of bovine brain [16]. The crude lipid was further purified by c h r o m a t o g r a p h y on DEAEcellulose as described by Hendrickson et al. [17[. This sample gave a single spot in f o r m a l d e h y d e - t r e a t e d paper c h r o m a t o g r a p h y and could be stored safely in wet c h l o r o f o r m at --20°C in the dark. The formaldehyde-treated papers were prepared by the m e t h o d of Kai et al. [18].
Preparation of lysotriphosphoinositide An aqueous solution of triphosphoinositide (3 mg/ml) in a quartz cell was irradiated with ultraviolet rays (2537 /~ in wavelength from a sterilizing lamp; a b o u t 3300 erg/cm 2 per s) for a week at r o o m temperature. The triphosphoinositide molecules a ut ooxi di z e d and the material obtained gave a single spot in formaldehyde-treated paper c h r o m a t o g r a p h y just below the spot of triphosphoinositide. With the aim of characterizing this sample the molar ratio of ester fatty
307 acid to p h o s p h o r us was determined. The f a t t y acid ester was determined by the m e t h o d o f S n yde r et al. [19] and the phosphorus was determined by the m e t h o d of Bartlett [20]. It was f o u n d t ha t the oxidized triphosphoinositide contained one f a t t y acid ester per three phosphate residues. This result implied t h a t triphosphoinositide molecules were completely oxidized and broken down into lysotriphosphoinositide molecules. It was also f o u n d that this oxidized sample contained o t h e r c o m p o n e n t s than lysotriphosphoinositide such as free f a t t y acids or malonaldehyde. Free fa t t y acid was observed on silicic acid thin layer c h r o m a t o g r a p h y and also observed by colorimetric analysis [21]. Malonaldehyde which indicates the degree o f lipid p e r oxi da t i on was determined with a TBA test [22]. But none o f them seems to c ont r i but e to following results (See Discussion).
Membrane formation and modification Oxidized cholesterol was obtained by the m e t h o d of Jain [23]. That is, a 4 p er cen t solution of cholesterol (99% pure, Sigma Chemical Co.) in 25 ml of 1 : 1 mixture of n-decane and n-tetradecane was refluxed at boiling t e m p e r a t u r e for 20 h and stirred vigorously with a Teflon magnetic stirrer bar. The resulting solution was then cooled for overnight and a supernatant was obtained. The supernatant was useful for forming a bilayer m em brane stable for ab o u t a mo n th. The alkanes were of the best grade available commercially. The ch amb er shown in Fig. 1 was used for forming membranes. The chamber had vertical septum (1.5 mm thickness) in which the membrane-forming orifice (1.3 mm diameter) was located. The volume of each c o m p a r t m e n t was about 7 ml. The chamber was made of Daiflon (a resin of t r i f l u o r o m o n o c h l o r o ethylene, purchased f r om Daikin Engineering Co. Ltd.). A feeder hole (0.7 m m diameter) was drilled through the chamber septum to the ~nembrane hole. This passage was c o n n e c t e d externally to a microsyringe by means of a thin Teflon tube. The glass window was attached to the chamber with Araldite (Ciba-Geigy) and left for over two weeks in order to eliminate the irritating vapor from the Araldite.
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308 Each chamber was filled with 5 ml of 10 mM Tris/C1 (pH 7.5) and a thick biconcave lens of oxidized cholesterol solution was formed at the hole by introducing an adequate volume of the solution. The oxidized cholesterol was then sucked up carefully by the syringe so t hat a m e m b r a n e of the desired area might be formed. Lysotriphosphoinositide and CaCl2 were added to b o th c o m p a r t m e n t s through a m i c r opi pet t e after the m em brane had become completely black. The bitayer membranes were incubated in this solution for ab o u t five minutes with gentle stirring. A small quantity of concent rat ed solution of electrolytes was added to either one or bot h aqueous phases with a m i c ro p ip ette in order to change the ionic composition. All electrolytes emp l o yed were the best grade available commercially, and which were dissolved in the double-distilled water.
Perfusion technique A perfusion technique was used to eliminate the lysotriphosphoinositide from the aqueous phases. The perfusing apparatus consisted of two buffering vessels, two siphons and a level controlling siphon. All of them were made of glass. The level-controlling siphon (m) was vertically movable and had a needle tip at one end. The surface tension kept the water from flowing out when the tip was detached from the surface of the solution in the second buffering vessel
(j). The p r o ced u r e of the perfusion was as follows. The tip of the level controlling siphon was lowered into the second buffering vessel in order to suck up the extensive solution as soon as a new solution ran into the first buffering vessel f r o m the inlet. Th e water level of the test side c o m p a r t m e n t was kept constant by adjusting the height of the controlling siphon (m).
Electrical measurement The current-voltage curve of this m e m brane was linear provided that the applied voltage was less than 20 inV. Therefore, zero current conduct ance of the m e m b r a n e was obtained by applying a 10 mV rectangular voltage of 0.5-s duration and by measuring the corresponding current using a low-drift FET input operational amplifier (Teledyne-Philbrick, 1023). Rectangular voltage was applied to the membrane by using a stimulator (Nihonkoden, MSE-3R) and isolater (Nihonkoden, MSE-JM) followed by a low o u t p u t impedance operational amplifier, 1009. A two channel recorder (National, VP-6521-A) connected to the FET input operational amplifier (Teledyne-Philbrick, 1009) through one channel allowed the recording of the voltage applied or the membrane potential. The ot her channel was connect ed to the o u t p u t of the amm e t e r operational amplifier to record the corresponding current. An Ag/AgC1 electrode was e m p l o y e d for electrical measurement. It made c o n t a c t with the saturated KC1 solution in the shank of the glass tube which was c o n n e c t e d to the aqueous phase of the cell by means of a saturated KC1/ agar bridge (See Fig 1.). The measurements were done at r oom t em p erat ure (20 ± 2°C). Results The basic observation is shown in Fig. 2. Upon addition of 10 -6 g/ml lysotriphosphoinositide and 1 mM CaC12 in final c o n c e n t r a t i o n to bot h sides of
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conductance c h a n g e of a b l a c k m e m b r a n e m o d i f i e d w i t h 10 -6 g / m l 1 m M CaCl 2. T h e f i r s t a r r o w i n d i c a t e s t h e t i m e a t w h i c h l y s o t r i p h o s p h o i n o s i t i d e a n d C a C l 2 w e r e a d d e d t o b o t h t h e a q u e o u s p h a s e s c o n t a i n i n g 10 m M Tris/C1 ( p H 7.5)• A l i q u o t s ( 1 0 0 #1) o f 2 M N a C 1 w e r e a d d e d t o b o t h a q u e o u s p h a s e s • F i n a l c o n c e n t r a t i o n s a r e i n d i c a t e d i n t h e f i g u r e . of the membrane
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o f NaCl w a s i n c r e a s e d i n t h e p r e s e n c e o f 1 m M CaC12. L o w e r c u r v e w a s o b t a i n e d of CaC12 w a s i n c r e a s e d i n t h e a b s e n c e o f g r o u p I A c a t i o n . T h e d a t a r e p r e s e n t
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a black membrane made of oxidized cholesterol, an increase in the membrane conductance was observed. The conductance gradually increased from about 10 -9 o h m - 1 / c m 2 to about 10 -6 o h m - 1 / c m 2 within 5 minutes. However, no further increase in conductance was found by the subsequent additions of CaC12 (lower curve in Fig. 3). The n e x t great step of increase in the membrane conductance was observed when a small aliquot of a concentrated solution of uni-univalent electrolyte was microinjected into both aqueous phases (indicated by arrows in Fig. 2). The increase in the conductance caused by the addition of uni-univalent electrolyte depended on the concentration of the electrolyte, and the conductance could be increased to as large as the order of 10 -5 o h m - 1 / c m 2. As shown in Fig. 3, the conductance was proportional to about the second power of the concentration of the uni-univalent electrolyte (upper curve in Fig. 3). No change in the membrane conductance was observed when the membrane was incubated in the solution containing triphosphoinositide and CaC12. That is, triphosphoinositide which was n o t subjected to autoxidation was unable to m o d i f y the oxidized cholesterol membrane. When the membrane was
310
n o t black, i.e. colored membrane or oil film, no conductance change was generated even with the lysotriphosphoinositide. In order to know the species of ion which could permeate this membrane, membrane potential was measured first against the concentration gradient of KC1 across the membrane. As shown in Fig. 4, the membrane potential could be fitted successfully with the Goldman equation, if the ratio of the permeability coefficient (Pcl-/PK +) was assumed to be 0.03. On the other hand, a concentration-gradient of CaCL2 generated no membrane potential. Concentration gradient of CaC12 (1 : 20 mM) did n o t influence the membrane potential which was generated by univalent cation. These results implied that this membrane was highly selectively permeable to the univalent cation, and is only very sparingly permeable to both calcium ion and anion. Fig 5 presents the "fingerprint" characteristic of the membrane modified with lysotriphosphoinositide. The logarithm of the experimentally observed permeability ratio [24] has been plotted as a function of the reciprocal of the naked [25] cation radius. Selectivity sequence among group IA cations was Rb ÷ > Cs ÷ > Na + > K + > Li +, and NH~ was markedly "supra-IA" [ 2 6 ] . No effect on the membrane conductance was observed by the elimination of lysotriphosphoinositide in the aqueous phase by perfusing the solution with a lysotriphosphoinositide-free saline solution. This fact suggested that the lysotriphosphoinositide molecule once incorporated in the membrane could never be dissolved into the aqueous phase. The univalent cation conductance was greatly influences by calcium or other divalent cations. Fig. 6 shows the change in membrane conductance when CaC12 was added to one side in the presence (Fig. 6a) and in the absence (Fig. 6b) of lysotriphosphoinositide in the aqueous solution. As shown in
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Fig. 4. M e m b r a n e p o t e n t i a l g e n e r a t e d b y t h e c o n c e n t r a t i o n g r a d i e n t o f KC1. T h e c o n c e n t r a t i o n o f KC1 at t h e r e f e r e n c e side w a s f i x e d at 8 m M . T h e s u b s c r i p t s " t " a n d "r" r e p r e s e n t t h e t w o a q u e o u s p h a s e s s e p a r a t e d b y t h e m e m b r a n e ( t e s t a n d r e f e r e n c e side). T h e solid line i n d i c a t e s t h e m e m b r a n e p o t e n t i a l calculated from the Goldman equation by assuming PC1-/PK + = 0.03. Fig. 5. S e l e c t i v i t y " f i n g e r p r i n t " for p e r m e a t i o n t h r o u g h t h e c h a n n e l m a d e o f l y s o t r i p h o s p h o i n o s i t i d e . T h e l o g a r i t h m o f t h e p e r m e a b i l i t y ratios relative t o K ÷ d e t e r m i n e d f r o m z e r o - c u r r e n t p o t e n t i a l m e a s u r e m e n t s o f a b i l a y e r m e m l ~ r a n e is p l o t t e d against t h e r e c i p r o c a l o f t h e n a k e d c a t i o n radius. T h e i o n i c s t r e n g t h o f t h e e a c h a q u e o u s p h a s e w a s e q u a l i z e d . R e f e r e n c e side: 4 0 0 m M KC1 + 4 0 m M MeC1 ( M e : g r o u p I A c a t i o n ) . T e s t side: 4 0 m M K C l + 4 0 0 m M MeC1.
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Fig. 6a, there were two opposite aspects in the effect of calcium ion in the membrane conductance. The one is a rapid decrease just after the microinjection of calcium ion, and the other is a subsequent gradual increase. However, if the lysotriphosphoinositide was eliminated from the bulk solution by per1.0
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Fig. 7, T h e d e p e n d e n c e o f t h e rate o f c o n d u c t a n c e i n c r e a s e t o t h e c o n c e n t r a t i o n o f c a l c i u m i o n a d d e d to o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d 1 0 -6 g / m l l y s o t r i p h o s p h o i n o s i t i d e , 2 7 5 m M NaC1, 1 raM CaCl 2 a n d 1 0 m M Tris/Cl ( p H 7 . 5 ) initially. T h e d a t a r e p r e s e n t m e a n v a l u e s (-+S.D.) o f at l e a s t t h r e e m e m branes. Fig. 8. P l o t o f relative c o n d u c t a n c e vs. c o n c e n t r a t i o n o f CaCl 2 a d d e d t o o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d initially 1 0 -6 g / m l l y s o t r i p h o s p h o i n o s i t i d e , 1 raM CaC12, 2 7 5 m M NaC1 a n d 1 0 m M Tris/Cl ( p H 7 . 5 ) . A f t e r t h e t e s t side a q u e o u s p h a s e w a s p e r f u s e d w i t h l y s o t r i p h o s p h o i n o s i t i d e - f r e e s o l u t i o n c o n t a i n i n g s a m e salt c o n t e n t s , 10-~1 a l i q u o t s o f 5 0 0 m M CaC12 w e r e a d d e d t o t h e t e s t side t h r o u g h m i c r o p i p e t t e . T h e relative c o n d u c t a n c e w a s o b t a i n e d b y d i v i d i n g t h e v a l u e o f m e m b r a n e c o n d u c t a n c e w i t h t h a t o f t h e m e m b r a n e in t h e initial c o n d i t i o n (1 m M CaC12 ). T h e d a t a r e p r e s e n t m e a n values (-+S.D.) o f 6 m e m branes.
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Fig. 9. P l o t o f r e l a t i v e c o n d u c t a n c e vs. c o n c e n t r a t i o n o f d i v a l e n t c a t i o n a d d e d to o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d i n i t i a l l y 1 m M C a C l 2 , 2 7 5 m M NaC1 a n d 10 m M Tris/C1 ( p H 7.5). T h e 10 pl a l i q u o t s o f 5 0 0 m M s o l u t i o n o f d i v a l e n t c a t i o n w e r e a d d e d t o t h e t e s t side a f t e r t h e p e r f u s i o n .
fusion, the later c o n d u c t a n c e increase disappeared. The presence o f lysotriphosphoinositide in the aqueous phase, therefore, is the cause for the latter effect o f calcium ion on the m e m br a ne conductance. The rate of rise in c o n d u c t a n c e changed with the concent rat i on of calcium ion as shown in Fig. 6a. The rate was p l o t t e d against the c o n c e n t r a t i o n of calcium ion in Fig. 7. The curve had a peak for the CaC12 concent rat i on at about 2--3 mM. Fig. 8 illustrates the relative c o n d u c t a n c e as a function of the concent rat i on o f calcium ion which was added to one side of the m em brane after the elimination o f lysotriphosphoinositide by perfusion. The relative c o n d u c t a n c e in this figure means a value of the m e m br a ne c o n d u c t a n c e relative to that observed in the presence o f 1 mM CaC12 in both c o m p a r t m e n t s . Though divalent cations o t h e r than calcium could also depress the membrane c o n d u c t a n c e , their effectiveness differed as shown in Fig. 9. A small aliquot o f 500 mM solution of divalent cation was added to one side of the m em brane and the a m o u n t of c o n d u c t a n c e decrease was measured. Both aqueous phases always contained 275 mM NaC1, 1 mM CaC12 and 10 mM Tris/C1 (pH 7.5). The order of the efficiencies was Zn 2÷ > Cd 2÷ > Ca z÷ > SP ÷ > Mg 2÷. Discussion It is k n o wn that a phospholipid having double bonds in its h y d r o c a r b o n chain is susceptible to the pe r oxi dat i on induced by ionizing radiation, ascorbate or metal ions [ 22, 27, 32] , and that the subsequent chain of metabolic conversion o f phospholipid acyls thus started forms lysophospholipid and ot her p e r o x i d a t i o n p r o duct s [27--32]. Most of the ester fat t y acid of triphosphoinositide is arachidic acid (C20:4) at the fl-position [33]. We found t hat the oxidized triphosphoinositide contained three phosphorus atoms per one fatty
313 acid ester. Therefore, it is highly likely that triphosphoinositide was broken down into lysotriphosphoinositide being subjected to peroxidation induced by ultraviolet irradiation. On the other hand, the oxidized triphosphoinositide used in this experiment contained free fatty acids, malonaldehyde and possibly short chain acyl fragments as the products of lipid peroxidation. However, we found that purified lysotriphosphoinositide obtained from DEAE-cellulose column caused similar phenomena in bilayer membranes of oxidized cholesterol (unpublished). These c o m p o n e n t s other than lysotriphosphoinositide might not have a role in this ion-conducing system. Such a lipid peroxidation has been observed in some biological membranes. For example, a light-dependent lipid peroxidation was observed in rod outer segment of retina [34] and in the solution of chlorophyll [35]. Also, such a lipid peroxidation was observed in mitochondria [36--38] and microsomes [28,30,32], when they were incubated with ascorbate of NADPH or Fe ÷. The lipid peroxidation in these membranes may bring a b o u t a formation of lysophospholipid including lysotriphosphoinositide. It is well known that lysophospholipid such as lysolecithin behaves as a detergent and has lytic activity to the membrane. Relatively low concentrations cause complete disorganization and disruption of lipid bilayers [39--41]. On the contrary, lysotriphosphoinositide did not disrupt bilayer membranes even if its concentration was high. Lysotriphosphoinositide caused several order higher conductance than lysophosphatidylcholine and the conductance was susceptible to the concentration of calcium ion. As mentioned in the Results, lysotriphosphoinositide could not induce ionic conductance in the thick (colored) membrane which had a bulk oil phase between two oil-water interfaces. This result implied that the lysotriphosphoinositide could n o t carry ions through the oil phase as a "carrier". Therefore, it is suggested that they formed a ion conducting channel mechanism in the membrane. In this experiment, calcium ion caused two opposite effects on the membrane conductance in the presence of a relatively high concentration of univalent cation (Fig. 6a). As previously mentioned, the conductance increased at a constant rate by addition of calcium ion just after the initial decrease. This latter increase could n o t be observed in the absence of lysotriphosphoinositide in the aqueous phase (Fig. 6b). Therefore, the increase in conductance can be attributed to the additional incorporation of lysotriphosphoinositide from the bulk solution into the membrane. Abramson et al. [42] showed that the h y d r o p h o b i c i t y of acidic phospholipid in the presence of calcium ion was reduced by coexistence of a relatively high concentration of univalent cation. Therefore, the h y d r o p h o b i c i t y of lysotriphosphoinositide in 1 mM CaC12 solution should be depressed to considerably low value by addition of enough a m o u n t of Na ÷. Thus, the mechanism of the later increase in the conductance be explained as followings. The h y d r o p h o b i c i t y of lysotriphosphpinositide increases with increase in the concentration of calcium ion (1 mM to several mM). Thereby, incorporation of lysotriphosphoinositide into the membrane will be accelerated. The absorbed lysotriphosphoinositide molecules immediately form univalent cation channels, resulting in the increase of the membrane conductance.
314
The rate of conductance increase by addition of CaC12 had a peak value at about 2--3 mM CaC12 (Fig. 7). If the conductance increase is due to the incorporation of lysotriphosphoinositide from the aqueous phase as mentioned before, the rate of its incorporation will increase with its hydrophobicity. From turbidity measurement, we have also observed a maximum hydrophobicity of lysotriphosphoinositide in an aqueous medium at a defined concentration of CaC12 (unpublished). Such a decrease of hydrophobicity may be caused by the charge reversal of its micelle by binding with excess calcium ion. Dawson et al. [43] showed that the excess calcium ion reversed the sign of the surface potential of an acidic phospholipid monotayer on an air/water interface. If a similar phenomenon occurs at the surface of lysotriphosphoinositide micelle in the water, their hydrophobicity will become m a x i m u m when the charge of the micelle is neutralized by calcium ions. On the other hand, the conductance decrease by addition of calcium ion could not be interpreted simply in terms of electrostatic concepts, since the effectiveness for decreasing the membrane conductance differed among divalent cations (Fig. 9). Such an effectiveness seems to depend on the affinity of divalent cation to the head group of lysotriphosphoinositide. Blaustein and Goldman [44] studied the shift of activation of sodium channel of nerve with changes of external divalent cation concentration by means of voltage clamp. With regard to the effectiveness for the shift, they ranked divalent cations in the following sequence: La > > Ni > > Zn > > Co > > C d 2÷> Ba 2÷> Ca 2÷> Mg2÷. Hille et al. [45] also showed similar sequence or the affinities of divalent cations to negative surface charge near sodium channels of nerve. These sequences are same as ours. Our results show that calcium ion having high affinity for lysotriphosphoinositide has extremely low permeability in the membrane, whereas univalent cation having relatively low affinity for the lysotriphosphoinositide has very high permeability. These results suggest that calcium ion causes some modification of the channel made of lysotriphosphinositide and blocks the channel passway.
Acknowledgement The authors are sincerely grateful to Professor R. Suzuki and Dr. T. T o k i m o t o for their continuing interest and encouragement. The careful reading of the manyscript by Ms. J. R u y a k is gratefully acknowledged. Thanks are due to Ms. Y. Hamada for typing this manuscript.
References 1 2 3 4 5 6 7 8 9
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E i c h b e r g , J. a n d H a u s e r , G. ( 1 9 6 7 ) B i o c h i m . B i o p h y s . A c t a 1 4 4 , 4 1 5 - - 4 2 2 H a u s e r , G. a n d E i c h b e r g , J. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 2 6 , 2 0 1 - - 2 0 9 E i c h b e r g , J. a n d H a u s e r , G. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 2 6 , 2 1 0 - - 2 2 3 H e n d r i c k s o n , H.S. a n d F u l l i n g t o n , J . G . ( 1 9 6 5 ) B i o c h e m i s t r y 4, 1 5 9 9 - - 1 6 0 5 D a w s o n , R . M . C . ( 1 9 6 5 ) B i o c h e m . J. 9 7 , 1 3 4 - - 1 3 8 T a s a k i , I. ( 1 9 6 8 ) in N e r v e E x c i t a t i o n : a M a c r o m o l e c u l a r A p p r o a c h , C. T h o m a s , S p r i n g f i e l d F o l c h - P i , J. ( 1 9 4 9 ) J. Biol. C h e m . 1 7 7 , 4 9 7 - - 5 0 4 H e n d r i c k s o n , H.S. a n d B a l l o u , C.E. ( 1 9 6 4 ) J. Biol. C h e m . 2 3 9 , 1 3 6 9 - - 1 3 7 3 Kai, M. a n d H a w t h o r n e , J.M. ( 1 9 6 6 ) B i o c h e m . J. 9 8 , 6 2 - - 6 7 S n y d e r , F. a n d S t e p h e n s , N. ( 1 9 5 9 ) B i o c h i m . B i o p h y s . A c t a 3 4 , 2 4 4 - - 2 4 5 B a r t l e t t , G . R . ( 1 9 5 9 ) J. Biol. C h e m . 2 3 4 , 4 6 6 - - 4 6 8 K u s h i r o , H. ( 1 9 7 0 ) J a p . J. Clin. P a t h o l . 18, 8 3 3 - - 8 3 7 D a M e , L . K . , Hill, E . G . a n d H o l m a n , R . T . ( 1 9 6 2 ) A r c h . B i o c h e m . B i o p h y s . 9 8 , 2 5 3 - - 2 6 1 J a i n , M . K . , Mehl, L.E. a n d C o r d e s , E . H . ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 51, 1 9 2 - - 1 9 7 S z a b o , G., E i s e n m a n , G. a n d Ciani, S. ( 1 9 6 9 ) J. M e m b r a n e Biol., 1, 3 4 6 - - 3 8 2 P a u l i n g , L. ( 1 9 6 0 ) in T h e N a t u r e o f t h e C h e m i c a l B o n d , C o r n e l l U n i v e r s i t y Press, I t h a c a , N e w Y o r k E i s e n m a n , G. a n d K r a s n e , S.J. ( 1 9 7 3 ) in M T P I n t e r n a t i o n a l R e v i e w o f S c i e n c e , B i o c h e m i s t r y Series, Vol. 2 ( F o x , C . F . , e d . ) , B u t t e r w o r t h s , L o n d o n M a y , H . E . a n d M c C a y , P.B. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 2 2 8 8 - - 2 2 9 5 J a c o b , G.S. a n d L u x , S.E. ( 1 9 6 8 ) B l o o d 3 2 , 5 4 9 - - 5 6 8 Wills, E . D . ( 1 9 6 9 ) B i o c h e m . J. 1 1 3 , 3 1 5 - - 3 2 4 L e i b o w i t z , M.E. a n d J o h n s o n , M.C. ( 1 9 7 1 ) J. L i p i d Res. 12, 6 6 2 - - 6 7 0 Wills, E . D . ( 1 9 7 1 ) B i o c h e m . J. 1 2 3 , 9 8 3 - - 9 9 1 S m o l e n , J . E . a n d S h o h e t , S.B. ( 1 9 7 4 ) J. L i p i d Res. 1 5 , 2 7 3 - - 2 8 0 H o l u b , B.J., K u k s i s , A. a n d T h o m p s o n . W. ( 1 9 7 0 ) J. L i p i d Res. 11, 5 5 8 - - 5 6 4 K a g a n , V . E . , S h v e d o v a , A . A . , N o v i k o v , K.M. a n d K o z l o v , Y u . P . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 3 0 , 76--79 Hall, G . E . a n d R o b e r t s , D . G . ( 1 9 6 6 ) J. C h e m . S o c . 1 1 0 9 - - 1 1 1 2 H u n t e r , F . E . , S c o t t , A., H o f f s t e n , P.E., G u e r r a , R., W e i n s t e i n , J., S c h n e i d e r , A., S c h u t z , B., F i n k , J., F o r d , L. a n d S m i t h , E. ( 1 9 6 4 ) J. Biol. C h e m . 2 3 9 , 6 0 4 - - 6 1 3 U t s u m i , K., Y a m a m o t o , G. a n d I n a b a , K. ( 1 9 6 5 ) B i o c h i m . B i o p h y s . A c t a 1 0 5 , 3 6 8 - - 3 7 1 V a n Z u t p h e n , H. a n d V a n D e e n e n , L . L . M . ( 1 9 6 7 ) C h e m . P h y s . L i p i d s 1, 3 8 9 - - 3 9 1 Reman, F.C. and Van Deenen, L.L.M. (1967) Biochim. Biophys. Acta 137, 592--594 M a n d e r s l o o t , J . G . , R e m a n , F . C . , V a n D e e n e n , L . L . M . a n d De Gier, J. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 382, 22--26 A b r a m s o n , M.B., C o l a c i c c o , G., C u r c i , R . a n d R a p p o r t , M.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 1 6 9 2 - - 1 6 9 8 D a w s o n , R . M . C . a n d H a u s e r , G. ( 1 9 7 0 ) in C a l c i u m a n d C e l l u l a r F u n c t i o n ( C u t h b e r d , A.W., e d . ) , Macmillan and Company B l a u s t e i n , M.P. a n d G o l d m a n , E . D . ( 1 9 6 8 ) J. G e n . P h y s i o l . 5 1 , 3 8 8 - - - 4 0 0 Hille, B., W o o d h u l l , A . M . a n d S h a p i r o , B.I. ( 1 9 7 5 ) Phil. T r a n s . R. S o c . L o n d . B. 2 7 0 , 3 0 1 - - 3 1 8
Biochimica et Biophysica Acta, 5 1 0 ( 1 9 7 8 ) 3 0 5 - - 3 1 5 © Elsevier/North-Holland
Biomedical Press
BBA 78056
CALCIUM-SENSITIVE UNIVALENT CATION CHANNEL FORMED BY LYSOTRIPHOSPHOINOSITIDE IN BILAYER LIPID MEMBRANES
FUMIO HAYASHI a,. MASAHIRO SOKABE b,** MASAYUKI HAYASHI c and UICHIRO KISHIMOTO a
T A K A G I a, K O H E I
a Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, b Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, and c Department of Biochemistry, Faculty of Medicine, Gumma University, Gumma 371 (Japan) (Received October 31st, 1977)
Summary A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to ur?ivalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb÷> Cs+> Na+> K+> Li+. The conductance of the membrane was increased up to a value of about 10 -2 o h m - ' / cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn 2+ > Cd2+ > Ca 2+ > Sr 2+ > Mg2+.
Introduction Inositol phospholipids have been known to be present in a wide variety of mammalian tissues and their various physiological activities have been reviewed recently [1]. Triphosphoinositide has particularly been regarded as an important constituent of the nervous system, because of the high turnover rate of their phosphate moiety [2--4]. The predominant localization of brain triphosphoinositide in the myeline sheath has been well established [5--7]. However, the rapid post-mortem depletion of triphosphoinositide has also been reported * P r e s e n t addresses: D e p a r t m e n t of Biology, College of General Education, Kobe University, Nada, Kobe 657, Japan. ** Department of Behaviorology, Faculty of H u m a n Sciences, Osaka University, Yamada, Suita, Osaka 565, Japan.
306 [8--10] and recently Eichberg and Hauser have claimed the localization of this substance at the cell m e m b r a n e of nervous systems including synapse or axon [11,12]. Triphosphoinositide has unique physicochemical characteristics. Since triphosphoinositide has five net negative charges at physiological pH, it can be dissolved into water very smoothly. On the ot her hand, it shows an extraordinarily high affinity for calcium ion, resulting in a highly h y d r o p h o b i c c o mp lex with Ca 2÷ ]13,14]. Thus, triphosphoinositide shows a drastic change in hydrophilicity, when the univalent-divalent cation exchange reaction occurs. A hypothesis was proposed to explain the molecular mechanism of nerve excitation by Tasaki and his coworkers [15]. In their hypothesis, it was proposed that an essential process of nerve excitation is a kind of phase transition of a certain c o m p o n e n t of the excitable membrane; the phase transition is caused by desorption of calcium ion from the m em brane surface. No material has, however, been proposed as the key substance which undergoes such a phase transition in the excitable membrane. We intended to incorporate the triphosphoinositide into an artificial membrane as a c o m p o n e n t which could undergo a h y d r o p h i l i c - h y d r o p h o b i c micelle transition with univalent-divalent cation exchange. However, triphosphoinositide had no effect on the c o n d u c t a n c e of artificial bilayer m em brane of oxidized cholesterol. In contrast, a very little a m o u n t of lysotriphosphoinositide which was an oxidative breakdown p r o d u c t of triphosphoinositide was f o u n d to give a remarkably large c o n d u c t a n c e to the artificial membrane. The lysotriphosphoinositide could penetrate into the m em brane and formed a channel system which was selectively permeable to univalent cations. Moreover, the univalent cation c o n d u c t a n c e of the m e m brane was greatly influenced by the c o n c e n t r a t i o n of calcium ion in the aqueous phase. We will r ep o r t some features of this univalent cation channel constructed from the polar head groups of lysotriphosphoinositides. Materials and Methods
Extraction of triphosphoinositide Triphosphoinositide was obtained by Folch fractionation of bovine brain [16]. The crude lipid was further purified by c h r o m a t o g r a p h y on DEAEcellulose as described by Hendrickson et al. [17[. This sample gave a single spot in f o r m a l d e h y d e - t r e a t e d paper c h r o m a t o g r a p h y and could be stored safely in wet c h l o r o f o r m at --20°C in the dark. The formaldehyde-treated papers were prepared by the m e t h o d of Kai et al. [18].
Preparation of lysotriphosphoinositide An aqueous solution of triphosphoinositide (3 mg/ml) in a quartz cell was irradiated with ultraviolet rays (2537 /~ in wavelength from a sterilizing lamp; a b o u t 3300 erg/cm 2 per s) for a week at r o o m temperature. The triphosphoinositide molecules a ut ooxi di z e d and the material obtained gave a single spot in formaldehyde-treated paper c h r o m a t o g r a p h y just below the spot of triphosphoinositide. With the aim of characterizing this sample the molar ratio of ester fatty
307 acid to p h o s p h o r us was determined. The f a t t y acid ester was determined by the m e t h o d o f S n yde r et al. [19] and the phosphorus was determined by the m e t h o d of Bartlett [20]. It was f o u n d t ha t the oxidized triphosphoinositide contained one f a t t y acid ester per three phosphate residues. This result implied t h a t triphosphoinositide molecules were completely oxidized and broken down into lysotriphosphoinositide molecules. It was also f o u n d that this oxidized sample contained o t h e r c o m p o n e n t s than lysotriphosphoinositide such as free f a t t y acids or malonaldehyde. Free fa t t y acid was observed on silicic acid thin layer c h r o m a t o g r a p h y and also observed by colorimetric analysis [21]. Malonaldehyde which indicates the degree o f lipid p e r oxi da t i on was determined with a TBA test [22]. But none o f them seems to c ont r i but e to following results (See Discussion).
Membrane formation and modification Oxidized cholesterol was obtained by the m e t h o d of Jain [23]. That is, a 4 p er cen t solution of cholesterol (99% pure, Sigma Chemical Co.) in 25 ml of 1 : 1 mixture of n-decane and n-tetradecane was refluxed at boiling t e m p e r a t u r e for 20 h and stirred vigorously with a Teflon magnetic stirrer bar. The resulting solution was then cooled for overnight and a supernatant was obtained. The supernatant was useful for forming a bilayer m em brane stable for ab o u t a mo n th. The alkanes were of the best grade available commercially. The ch amb er shown in Fig. 1 was used for forming membranes. The chamber had vertical septum (1.5 mm thickness) in which the membrane-forming orifice (1.3 mm diameter) was located. The volume of each c o m p a r t m e n t was about 7 ml. The chamber was made of Daiflon (a resin of t r i f l u o r o m o n o c h l o r o ethylene, purchased f r om Daikin Engineering Co. Ltd.). A feeder hole (0.7 m m diameter) was drilled through the chamber septum to the ~nembrane hole. This passage was c o n n e c t e d externally to a microsyringe by means of a thin Teflon tube. The glass window was attached to the chamber with Araldite (Ciba-Geigy) and left for over two weeks in order to eliminate the irritating vapor from the Araldite.
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6
g F i g . 1. S c h e m a t i c d i a g r a m o f a p p a r a t u s f o r t h e f o r m a t i o n o f t h e m e m b r a n e a n d f o r t h e p e r f u s i o n o f a q u e o u s s o l u t i o n , a, m e m b r a n e h o l e (1.3 m m dia.); b, f e e d e r h o l e (0.7 m m dia.); c, s e p t u m (1.5 m m t h i c k n e s s ) ; d, T e f l o n t u b e ; e, K C l [ a g a r b r i d g e a n d A g / A g C l e l e c t r o d e ; f, m a g n e t i c s t i r r e r b a r ; g, glass w i n d o w ; h, i n l e t o f p e r f u s i n g s o l u t i o n ( T e f l o n t u b e w i t h a s t o p c o c k ) ; i a n d j, b u f f e r i n g vessel; k a n d 1, siphon; m, level-controlling siphon.
308 Each chamber was filled with 5 ml of 10 mM Tris/C1 (pH 7.5) and a thick biconcave lens of oxidized cholesterol solution was formed at the hole by introducing an adequate volume of the solution. The oxidized cholesterol was then sucked up carefully by the syringe so t hat a m e m b r a n e of the desired area might be formed. Lysotriphosphoinositide and CaCl2 were added to b o th c o m p a r t m e n t s through a m i c r opi pet t e after the m em brane had become completely black. The bitayer membranes were incubated in this solution for ab o u t five minutes with gentle stirring. A small quantity of concent rat ed solution of electrolytes was added to either one or bot h aqueous phases with a m i c ro p ip ette in order to change the ionic composition. All electrolytes emp l o yed were the best grade available commercially, and which were dissolved in the double-distilled water.
Perfusion technique A perfusion technique was used to eliminate the lysotriphosphoinositide from the aqueous phases. The perfusing apparatus consisted of two buffering vessels, two siphons and a level controlling siphon. All of them were made of glass. The level-controlling siphon (m) was vertically movable and had a needle tip at one end. The surface tension kept the water from flowing out when the tip was detached from the surface of the solution in the second buffering vessel
(j). The p r o ced u r e of the perfusion was as follows. The tip of the level controlling siphon was lowered into the second buffering vessel in order to suck up the extensive solution as soon as a new solution ran into the first buffering vessel f r o m the inlet. Th e water level of the test side c o m p a r t m e n t was kept constant by adjusting the height of the controlling siphon (m).
Electrical measurement The current-voltage curve of this m e m brane was linear provided that the applied voltage was less than 20 inV. Therefore, zero current conduct ance of the m e m b r a n e was obtained by applying a 10 mV rectangular voltage of 0.5-s duration and by measuring the corresponding current using a low-drift FET input operational amplifier (Teledyne-Philbrick, 1023). Rectangular voltage was applied to the membrane by using a stimulator (Nihonkoden, MSE-3R) and isolater (Nihonkoden, MSE-JM) followed by a low o u t p u t impedance operational amplifier, 1009. A two channel recorder (National, VP-6521-A) connected to the FET input operational amplifier (Teledyne-Philbrick, 1009) through one channel allowed the recording of the voltage applied or the membrane potential. The ot her channel was connect ed to the o u t p u t of the amm e t e r operational amplifier to record the corresponding current. An Ag/AgC1 electrode was e m p l o y e d for electrical measurement. It made c o n t a c t with the saturated KC1 solution in the shank of the glass tube which was c o n n e c t e d to the aqueous phase of the cell by means of a saturated KC1/ agar bridge (See Fig 1.). The measurements were done at r oom t em p erat ure (20 ± 2°C). Results The basic observation is shown in Fig. 2. Upon addition of 10 -6 g/ml lysotriphosphoinositide and 1 mM CaC12 in final c o n c e n t r a t i o n to bot h sides of
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1 [yso TPI CaC[ z
u
I
182
113
2 7 5 m M Nr, CI
- - ---0--0-0- --(3- - - - 0 -
I
I
~ I
0 Time
Fig• 2. T i m e c o u r s e
(min)
tog10 CoCt 2 (M)
conductance c h a n g e of a b l a c k m e m b r a n e m o d i f i e d w i t h 10 -6 g / m l 1 m M CaCl 2. T h e f i r s t a r r o w i n d i c a t e s t h e t i m e a t w h i c h l y s o t r i p h o s p h o i n o s i t i d e a n d C a C l 2 w e r e a d d e d t o b o t h t h e a q u e o u s p h a s e s c o n t a i n i n g 10 m M Tris/C1 ( p H 7.5)• A l i q u o t s ( 1 0 0 #1) o f 2 M N a C 1 w e r e a d d e d t o b o t h a q u e o u s p h a s e s • F i n a l c o n c e n t r a t i o n s a r e i n d i c a t e d i n t h e f i g u r e . of the membrane
lysotriphosphoinositide
Fig. 3. Plot of m e m b r a n e when
the concentration concentration
and
conductance
vs. c o n c e n t r a t i o n
of electrolyte
solution.
Upper curve was obtained
o f NaCl w a s i n c r e a s e d i n t h e p r e s e n c e o f 1 m M CaC12. L o w e r c u r v e w a s o b t a i n e d of CaC12 w a s i n c r e a s e d i n t h e a b s e n c e o f g r o u p I A c a t i o n . T h e d a t a r e p r e s e n t
when
the
mean
v a l u e s (-+S.D.) o f a t l e a s t f o u r m e m b r a n e s .
a black membrane made of oxidized cholesterol, an increase in the membrane conductance was observed. The conductance gradually increased from about 10 -9 o h m - 1 / c m 2 to about 10 -6 o h m - 1 / c m 2 within 5 minutes. However, no further increase in conductance was found by the subsequent additions of CaC12 (lower curve in Fig. 3). The n e x t great step of increase in the membrane conductance was observed when a small aliquot of a concentrated solution of uni-univalent electrolyte was microinjected into both aqueous phases (indicated by arrows in Fig. 2). The increase in the conductance caused by the addition of uni-univalent electrolyte depended on the concentration of the electrolyte, and the conductance could be increased to as large as the order of 10 -5 o h m - 1 / c m 2. As shown in Fig. 3, the conductance was proportional to about the second power of the concentration of the uni-univalent electrolyte (upper curve in Fig. 3). No change in the membrane conductance was observed when the membrane was incubated in the solution containing triphosphoinositide and CaC12. That is, triphosphoinositide which was n o t subjected to autoxidation was unable to m o d i f y the oxidized cholesterol membrane. When the membrane was
310
n o t black, i.e. colored membrane or oil film, no conductance change was generated even with the lysotriphosphoinositide. In order to know the species of ion which could permeate this membrane, membrane potential was measured first against the concentration gradient of KC1 across the membrane. As shown in Fig. 4, the membrane potential could be fitted successfully with the Goldman equation, if the ratio of the permeability coefficient (Pcl-/PK +) was assumed to be 0.03. On the other hand, a concentration-gradient of CaCL2 generated no membrane potential. Concentration gradient of CaC12 (1 : 20 mM) did n o t influence the membrane potential which was generated by univalent cation. These results implied that this membrane was highly selectively permeable to the univalent cation, and is only very sparingly permeable to both calcium ion and anion. Fig 5 presents the "fingerprint" characteristic of the membrane modified with lysotriphosphoinositide. The logarithm of the experimentally observed permeability ratio [24] has been plotted as a function of the reciprocal of the naked [25] cation radius. Selectivity sequence among group IA cations was Rb ÷ > Cs ÷ > Na + > K + > Li +, and NH~ was markedly "supra-IA" [ 2 6 ] . No effect on the membrane conductance was observed by the elimination of lysotriphosphoinositide in the aqueous phase by perfusing the solution with a lysotriphosphoinositide-free saline solution. This fact suggested that the lysotriphosphoinositide molecule once incorporated in the membrane could never be dissolved into the aqueous phase. The univalent cation conductance was greatly influences by calcium or other divalent cations. Fig. 6 shows the change in membrane conductance when CaC12 was added to one side in the presence (Fig. 6a) and in the absence (Fig. 6b) of lysotriphosphoinositide in the aqueous solution. As shown in
-IO0
I
I
I
~" --0.3 NH~
5
E
"~ - 0 . 2 C
&
o.1
"~
0.0
Rb+ ~
+
o_--50
k +0.1 I
; 10 [KcI]~/ [Kcq~
5~3 100
Cs+ Rb* K÷
+0.2 0.4
' ' NH,,~
Na+ --
1 LO'
-
i//n+ ( £ - ' )
Fig. 4. M e m b r a n e p o t e n t i a l g e n e r a t e d b y t h e c o n c e n t r a t i o n g r a d i e n t o f KC1. T h e c o n c e n t r a t i o n o f KC1 at t h e r e f e r e n c e side w a s f i x e d at 8 m M . T h e s u b s c r i p t s " t " a n d "r" r e p r e s e n t t h e t w o a q u e o u s p h a s e s s e p a r a t e d b y t h e m e m b r a n e ( t e s t a n d r e f e r e n c e side). T h e solid line i n d i c a t e s t h e m e m b r a n e p o t e n t i a l calculated from the Goldman equation by assuming PC1-/PK + = 0.03. Fig. 5. S e l e c t i v i t y " f i n g e r p r i n t " for p e r m e a t i o n t h r o u g h t h e c h a n n e l m a d e o f l y s o t r i p h o s p h o i n o s i t i d e . T h e l o g a r i t h m o f t h e p e r m e a b i l i t y ratios relative t o K ÷ d e t e r m i n e d f r o m z e r o - c u r r e n t p o t e n t i a l m e a s u r e m e n t s o f a b i l a y e r m e m l ~ r a n e is p l o t t e d against t h e r e c i p r o c a l o f t h e n a k e d c a t i o n radius. T h e i o n i c s t r e n g t h o f t h e e a c h a q u e o u s p h a s e w a s e q u a l i z e d . R e f e r e n c e side: 4 0 0 m M KC1 + 4 0 m M MeC1 ( M e : g r o u p I A c a t i o n ) . T e s t side: 4 0 m M K C l + 4 0 0 m M MeC1.
L~+ ]J .6
311 {a)
(b) mM CaC[ 2
gi E u 1.5
2 mM CaCI 2
3
E
8 to '7 o c
10
0,5
"5 ~ o.o o o
Time (min) Fig. 6. T i m e c o u r s e o f t h e m e m b r a n e c o n d u c t a n c e w h e n CaCI 2 w a s a d d e d to o n e side o f c o m p a r t m e n t (a) in t h e p r e s e n c e a n d ( b ) in t h e a b s e n c e o f l y s o t r i p h o s p h o i n o s i t i d e . B o t h a q u e o u s p h a s e s c o n t a i n e d initially 10 -5 g / m l l y s o t r i p h o s p h o i n o s i t i d e , 1 raM CaCI2, 2 7 5 m M NaC1 a n d 1 0 raM Tris/C1 ( p H 7.5). In t h e case (b), t h e a q u e o u s p h a s e w a s p e r f u s e d w i t h t h e l y s o t r i p h o s p h o i n o s i t i d e - f r e e s o l u t i o n c o n t a i n i n g s a m e salt c o n t e n t s , a n d t h e n 10 Dl a l i q u o t s o f 5 0 0 m M CaC12 w e r e a d d e d s u c c e s s i v e l y t o o n e side t h r o u g h a m i c r o p i p e t t e at t h e t i m e i n d i c a t e d .
Fig. 6a, there were two opposite aspects in the effect of calcium ion in the membrane conductance. The one is a rapid decrease just after the microinjection of calcium ion, and the other is a subsequent gradual increase. However, if the lysotriphosphoinositide was eliminated from the bulk solution by per1.0
u D o
o~ ~T 2 u c .c -~
o: 0.5
Vo
2c ~"
0.0 0
I
2
3 4 CaCI 2 ( m M )
5
6
,
2
5 CaCI 2 ( m M )
,
10
20
Fig. 7, T h e d e p e n d e n c e o f t h e rate o f c o n d u c t a n c e i n c r e a s e t o t h e c o n c e n t r a t i o n o f c a l c i u m i o n a d d e d to o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d 1 0 -6 g / m l l y s o t r i p h o s p h o i n o s i t i d e , 2 7 5 m M NaC1, 1 raM CaCl 2 a n d 1 0 m M Tris/Cl ( p H 7 . 5 ) initially. T h e d a t a r e p r e s e n t m e a n v a l u e s (-+S.D.) o f at l e a s t t h r e e m e m branes. Fig. 8. P l o t o f relative c o n d u c t a n c e vs. c o n c e n t r a t i o n o f CaCl 2 a d d e d t o o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d initially 1 0 -6 g / m l l y s o t r i p h o s p h o i n o s i t i d e , 1 raM CaC12, 2 7 5 m M NaC1 a n d 1 0 m M Tris/Cl ( p H 7 . 5 ) . A f t e r t h e t e s t side a q u e o u s p h a s e w a s p e r f u s e d w i t h l y s o t r i p h o s p h o i n o s i t i d e - f r e e s o l u t i o n c o n t a i n i n g s a m e salt c o n t e n t s , 10-~1 a l i q u o t s o f 5 0 0 m M CaC12 w e r e a d d e d t o t h e t e s t side t h r o u g h m i c r o p i p e t t e . T h e relative c o n d u c t a n c e w a s o b t a i n e d b y d i v i d i n g t h e v a l u e o f m e m b r a n e c o n d u c t a n c e w i t h t h a t o f t h e m e m b r a n e in t h e initial c o n d i t i o n (1 m M CaC12 ). T h e d a t a r e p r e s e n t m e a n values (-+S.D.) o f 6 m e m branes.
312 1.0
"5
g
0.0
,
,
2 5 ConcentrGtlon
10 (rnM)
2O
Fig. 9. P l o t o f r e l a t i v e c o n d u c t a n c e vs. c o n c e n t r a t i o n o f d i v a l e n t c a t i o n a d d e d to o n e side. B o t h a q u e o u s p h a s e s c o n t a i n e d i n i t i a l l y 1 m M C a C l 2 , 2 7 5 m M NaC1 a n d 10 m M Tris/C1 ( p H 7.5). T h e 10 pl a l i q u o t s o f 5 0 0 m M s o l u t i o n o f d i v a l e n t c a t i o n w e r e a d d e d t o t h e t e s t side a f t e r t h e p e r f u s i o n .
fusion, the later c o n d u c t a n c e increase disappeared. The presence o f lysotriphosphoinositide in the aqueous phase, therefore, is the cause for the latter effect o f calcium ion on the m e m br a ne conductance. The rate of rise in c o n d u c t a n c e changed with the concent rat i on of calcium ion as shown in Fig. 6a. The rate was p l o t t e d against the c o n c e n t r a t i o n of calcium ion in Fig. 7. The curve had a peak for the CaC12 concent rat i on at about 2--3 mM. Fig. 8 illustrates the relative c o n d u c t a n c e as a function of the concent rat i on o f calcium ion which was added to one side of the m em brane after the elimination o f lysotriphosphoinositide by perfusion. The relative c o n d u c t a n c e in this figure means a value of the m e m br a ne c o n d u c t a n c e relative to that observed in the presence o f 1 mM CaC12 in both c o m p a r t m e n t s . Though divalent cations o t h e r than calcium could also depress the membrane c o n d u c t a n c e , their effectiveness differed as shown in Fig. 9. A small aliquot o f 500 mM solution of divalent cation was added to one side of the m em brane and the a m o u n t of c o n d u c t a n c e decrease was measured. Both aqueous phases always contained 275 mM NaC1, 1 mM CaC12 and 10 mM Tris/C1 (pH 7.5). The order of the efficiencies was Zn 2÷ > Cd 2÷ > Ca z÷ > SP ÷ > Mg 2÷. Discussion It is k n o wn that a phospholipid having double bonds in its h y d r o c a r b o n chain is susceptible to the pe r oxi dat i on induced by ionizing radiation, ascorbate or metal ions [ 22, 27, 32] , and that the subsequent chain of metabolic conversion o f phospholipid acyls thus started forms lysophospholipid and ot her p e r o x i d a t i o n p r o duct s [27--32]. Most of the ester fat t y acid of triphosphoinositide is arachidic acid (C20:4) at the fl-position [33]. We found t hat the oxidized triphosphoinositide contained three phosphorus atoms per one fatty
313 acid ester. Therefore, it is highly likely that triphosphoinositide was broken down into lysotriphosphoinositide being subjected to peroxidation induced by ultraviolet irradiation. On the other hand, the oxidized triphosphoinositide used in this experiment contained free fatty acids, malonaldehyde and possibly short chain acyl fragments as the products of lipid peroxidation. However, we found that purified lysotriphosphoinositide obtained from DEAE-cellulose column caused similar phenomena in bilayer membranes of oxidized cholesterol (unpublished). These c o m p o n e n t s other than lysotriphosphoinositide might not have a role in this ion-conducing system. Such a lipid peroxidation has been observed in some biological membranes. For example, a light-dependent lipid peroxidation was observed in rod outer segment of retina [34] and in the solution of chlorophyll [35]. Also, such a lipid peroxidation was observed in mitochondria [36--38] and microsomes [28,30,32], when they were incubated with ascorbate of NADPH or Fe ÷. The lipid peroxidation in these membranes may bring a b o u t a formation of lysophospholipid including lysotriphosphoinositide. It is well known that lysophospholipid such as lysolecithin behaves as a detergent and has lytic activity to the membrane. Relatively low concentrations cause complete disorganization and disruption of lipid bilayers [39--41]. On the contrary, lysotriphosphoinositide did not disrupt bilayer membranes even if its concentration was high. Lysotriphosphoinositide caused several order higher conductance than lysophosphatidylcholine and the conductance was susceptible to the concentration of calcium ion. As mentioned in the Results, lysotriphosphoinositide could not induce ionic conductance in the thick (colored) membrane which had a bulk oil phase between two oil-water interfaces. This result implied that the lysotriphosphoinositide could n o t carry ions through the oil phase as a "carrier". Therefore, it is suggested that they formed a ion conducting channel mechanism in the membrane. In this experiment, calcium ion caused two opposite effects on the membrane conductance in the presence of a relatively high concentration of univalent cation (Fig. 6a). As previously mentioned, the conductance increased at a constant rate by addition of calcium ion just after the initial decrease. This latter increase could n o t be observed in the absence of lysotriphosphoinositide in the aqueous phase (Fig. 6b). Therefore, the increase in conductance can be attributed to the additional incorporation of lysotriphosphoinositide from the bulk solution into the membrane. Abramson et al. [42] showed that the h y d r o p h o b i c i t y of acidic phospholipid in the presence of calcium ion was reduced by coexistence of a relatively high concentration of univalent cation. Therefore, the h y d r o p h o b i c i t y of lysotriphosphoinositide in 1 mM CaC12 solution should be depressed to considerably low value by addition of enough a m o u n t of Na ÷. Thus, the mechanism of the later increase in the conductance be explained as followings. The h y d r o p h o b i c i t y of lysotriphosphpinositide increases with increase in the concentration of calcium ion (1 mM to several mM). Thereby, incorporation of lysotriphosphoinositide into the membrane will be accelerated. The absorbed lysotriphosphoinositide molecules immediately form univalent cation channels, resulting in the increase of the membrane conductance.
314
The rate of conductance increase by addition of CaC12 had a peak value at about 2--3 mM CaC12 (Fig. 7). If the conductance increase is due to the incorporation of lysotriphosphoinositide from the aqueous phase as mentioned before, the rate of its incorporation will increase with its hydrophobicity. From turbidity measurement, we have also observed a maximum hydrophobicity of lysotriphosphoinositide in an aqueous medium at a defined concentration of CaC12 (unpublished). Such a decrease of hydrophobicity may be caused by the charge reversal of its micelle by binding with excess calcium ion. Dawson et al. [43] showed that the excess calcium ion reversed the sign of the surface potential of an acidic phospholipid monotayer on an air/water interface. If a similar phenomenon occurs at the surface of lysotriphosphoinositide micelle in the water, their hydrophobicity will become m a x i m u m when the charge of the micelle is neutralized by calcium ions. On the other hand, the conductance decrease by addition of calcium ion could not be interpreted simply in terms of electrostatic concepts, since the effectiveness for decreasing the membrane conductance differed among divalent cations (Fig. 9). Such an effectiveness seems to depend on the affinity of divalent cation to the head group of lysotriphosphoinositide. Blaustein and Goldman [44] studied the shift of activation of sodium channel of nerve with changes of external divalent cation concentration by means of voltage clamp. With regard to the effectiveness for the shift, they ranked divalent cations in the following sequence: La > > Ni > > Zn > > Co > > C d 2÷> Ba 2÷> Ca 2÷> Mg2÷. Hille et al. [45] also showed similar sequence or the affinities of divalent cations to negative surface charge near sodium channels of nerve. These sequences are same as ours. Our results show that calcium ion having high affinity for lysotriphosphoinositide has extremely low permeability in the membrane, whereas univalent cation having relatively low affinity for the lysotriphosphoinositide has very high permeability. These results suggest that calcium ion causes some modification of the channel made of lysotriphosphinositide and blocks the channel passway.
Acknowledgement The authors are sincerely grateful to Professor R. Suzuki and Dr. T. T o k i m o t o for their continuing interest and encouragement. The careful reading of the manyscript by Ms. J. R u y a k is gratefully acknowledged. Thanks are due to Ms. Y. Hamada for typing this manuscript.
References 1 2 3 4 5 6 7 8 9
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