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Effect of angiotensin II on artificial lipid membranes
448
Biochimica et Biophysica Acta, 464 ( 1 9 7 7 ) 4 4 8 - - 4 5 2 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA Report BBA 7 1 2 8 1
E F F E C T OF ANG I O T E N SI N II ON A R T I F I C I A L LIPID MEMBRANES
PETER SCHLIEPER*
Instituto de Biolog[a Celular, Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires (Argentina) (Received O c t o b e r 19th, 1 9 7 6 )
Summary (5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between AG = 5 . 1 0 -11 ~-1 and 3 . 5 . 1 0 -1° ~ - 1 . These fluctuations depend on the voltage and the hydrostatic pressure. The m e mb r an e resistance is lowered by AR = 6.1. 1 0 7 ~ • cm 2 . With (5-isoleucine, 8-1eucine)-angiotensin II the jumps are of a single amplitude (A G = 2 . 1 0 -1° ~2-1 ). In both cases water and ions are transported across the membrane.
The so-called ionophoric antibiotics are characterized by the p r o p e r t y o f transporting ions, water and neutral molecules across membranes; among them, the channel or pore-forming ones are t hought to produce stable transm e mb r an e structures that could mediate ion codduct ance in " l i q u i d " as well as " s o lid " bilayers [ 1]. One of the most conspicuous manifestations of pore f o rmatio n is the appearance of discrete conduct ance steps in an artificial black lipid membrane, which suggests the opening of individual channels. Several o f the pore-forming antibiotics are short cyclic polypeptides, as in the classical example of alamethicin, or linear polypeptides, as in the case of the gramicidins (for reviews, see refs. 2, 3). For example, in the presence of very small amounts of gramicidin A, the current across a voltage-clamped black lipid m e mb r an e fluctuates between discrete and constant values with conduct ance jumps of ab o u t 4 . 1 0 - l l ~ - I [ 4] . These findings have led to the study of the effect o f natural polypeptides on artificial membranes. Fettiplace et al. [ 5] observed that lysine-vasopressin increased the conduct ance of artificial bilayers, but t h e y did not describe the f o r m a t i o n of typical pores. In the present work we have studied the interaction of the octapeptide h o r m o n e angiotensin II on black lipid membranes and have observed the *Fellow of the Deutsche Forschungsgemeinschaft.
449
production of typical conductance fluctuations, characteristic of a poreforming ionophore. These changes are accompanied by the translocation of ions and water across the membrane. Black lipid membranes were made according to Mueller et al. [6] on a 0.9 mm hole traversing a Teflon septum that separates two aquous phases. Ag-AgC1 electrodes were used to apply a definite voltage across the membrane. Measurements of conductance were done by means of a Keithley 610C microammeter. A storage oscilloscope (Tektronix 5103 N), connected to a preamplifier (Philbrick 1029) and a Keithley inscriptor device were used for recording the voltage and current changes. The membranes were made in an horizontal position, using a 2% solution of egg lecithin (Sigma) in decane. The bathing solution on both sides of the membrane contained 100 mM NaC1 or KC1 with 1 mM Tris/PO4 buffer, pH 7.3. The drugs in 10--25-pl aliquots were applied on one side near the membrane. (5-Isoleucine)-angiotensin II and the homologue (5-isoleucine, 8-1eucine)angiotensin II, kindly provided by Prof. A.C.M. Paiva (Escola Paulista de Medicina, 04023 Sao Paulo, Brazil) were synthesized by the solid phase method. The amino acid sequence of (5-isoleucine)-angiotensin II is: Asp-Arg-Val-TyrIle-His-Pro-Phe; in (5-isoleucine, 8-1eucine)-angiotensin II, the phenylalanine in the 8th position is replaced by leucine. The addition of (5-isoleucine)-angiotensin II produced discrete changes in membrane conductance, while keeping the membrane clamped at a constant potential. In Fig. l , a short time after the addition of 25 pl of a 0.1% solution of the polypeptide, typical current jumps appear with several levels of conductance Most of these have a value of AG = 5- 10 -11 ~2-1 , but there are others of higher amplitude, which may reach 3.5- 10 -l° ~Z-1. In this particular case there were
]
2 0 mV
lOmV
lO-'°A/cm
2
1 min F i g . 1. T r a c e o f c u r r e n t v e r s u s t i m e . V o l t a g e s o f 1 0 a n d 2 0 m V a r e a p p l i e d a c r o s s a m e m b r a n e f o r m e d w i t h a 2 % l e c i t h i n - d e c a n e s o l u t i o n . A n g i o t e n s i n II ( 5 - i s o l e u c i n e ) w a s a d d e d n e a r t h e m e m b r a n e . T h e s m a l l e s t c o n d u c t a n c e c h a n g e s c o r r e s p o n d to AG = 5 o 1 0 -1152 -1 .
450
no fluctuations at 10 mV, but t hey appeared at 20 mV. Such current fluctuations appear also to be related to the hydrostatic pressure applied to the membrane. If 0.5 ml excess solution (which corresponds to 2.6- 10 -s atm) is applied on one side, then the first pores appear 2 min after the addition of angiotension II. When there is no pressure applied, it may take 20 min for the pores to show up and only when higher potentials (80--100 mV) are applied. Membranes, undergoing interaction with angiotensin IL(5-isoleucine) for 30 min, lowered their resistance f r o m R ~ = 6 . 7 - 1 0 7 ~ 2 - c m 2 t o R 2 = 5 . 6 " 1 0 6 ~ ' c m 2 ( A R = 6.1-107~2.cm 2) Simultaneously there was the building up of a hydrostatic pressure in the upper chamber. In this case the m e m br a ne showed a concavity looking upwards. If the pressure is equilibrated by removal of liquid from the upper c o m p a r t m e n t , the m e mb r an e flattens, the pores disappear and the initial membrane resistance is restored. This p h e n o m e n o n is reversible and pores can be seen again after application of a hydrostatic pressure. With time, no m em brane potential could be detected, suggesting that the pores are not ion selective and that t hey p r o b ably allow the passage of Na ÷ and C1- as well as water. With KC1 in the bathing solution similar current jumps could be observed; however their frequency was lower and also the decrease in resistance of the membrane was smaller than with NaC1 (AR = 1 . 1 - 1 0 7 ~ .cm 2 ). The effect o f (5-isoleucine, 8-1eucine)-angiotensin II on the artificial lipid membrane was slightly different. Immediately after the application of the polypeptide, pores having a AG = 2 . 1 0 -l° ~-1 appeared. As shown in Fig. 2 these pores in general are of a single amplitude and very seldom different levels of conductances were observed; also here the hydrostatic pressure and the resistance of the membrane changes with time. In the case o f the pore-forming antibiotics it has been postulated that the channels result from the special organization of the molecules within the bilayer. Transmembrane structures are t h o u g h t to be form ed of repeating
Fig. 2. T r a c e of c u r r e n t versus t i m e p h o t o g r a p h e d f r o m t h e o s c i l l o s c o p e . 4 0 m V w e r e a p p l i e d across the m e m b r a n e m a d e w i t h a 2% l e c i t h i n - d e c a n e s o l u t i o n . A n g i o t e n s i n II ( 5 - i s o l e u c i n e , 8-1eucine) was a d d e d u p o n o n e s i d 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 c h a n g e s c o r r e s p o n d t o A G = 2" ] 0 - 1 ° ~ - - 1 .
451
units which may arrange in series or in parallel (i.e. as the staves of a barrel) and which may exhibit a dependence on the electrical field across the lipid bilayers. Bauman and Mueller [7] have proposed that the assembly of a channel is made by the voltage-dependent aggregation of m o n o m e r i c precursors. According to this hypothesis the gating mechanism for the channel involves the voltage-induced insertion of the precursor from the m em brane surface into the h y d r o c a r b o n region of the bilayer [8]. Juliano and Paiva [9] and Deslauriers et al. [10] have discussed the several molecular models that have been proposed for angiotensin II. From a carbon-13 spin-lattice relaxation study Paiva and co-workers conclude that (5-isoleucine)-angiotensin II is probably folded so that the N- and C-terminals are held in closer p r o x i m i t y than expected for a random conformation. The molecular model that emerges is that of a molecule folded on itself and having a radius of ab o u t 8 A. The (5-isoleucine, 8-1eucine)-angiotensin II which has only 1% of the physiological activity of the other, showed a 10% increase in spin relaxation time, indicating a loosening of the c o n f o r m a t i o n in the b a ck b o n e of the molecule. The present study shows that bot h polypeptides of angiotensin II are able to interact with lipid bilayers made of lecithin to create pores. We may speculate that the interaction of these polypeptides with the lipid bilayer leads to a rearrangement of several of these folded molecules, so that the more h y d r o p h o b i c parts of the chains are held toward the lipidic milieu, while the hydrophilic groups face towards the center of the channel. The relative wide diameter of the pore f or m ed may explain the flux of water and the non-ionic selectivity. As in the case of other pore-forming polypeptides the gating mechanism for angiotensin II would be electrically driven. As shown in Fig. 1 the pores are mainly opened under an applied positive potential. A not he r factor that appears to be involved in the gating mechanism is the hydrostatic pressure that produces a stretching of the membrane; such a pressure may be externally imposed upon the membrane or it may be built by the preferential flux of water and ions toward the c o m p a r t m e n t containing the polypeptide. While the two homologues of angiotensin II used in the present study are able to pr oduce channels, the (5-isoleucine, 8-1eucine)-angiotensin II seems to be m or e active and this could be related to the more relaxed c o n f o r m a t i o n of this molecule [ 10] . The observations r epor t e d here suggest that the octapeptide angiotensin II may have a direct interaction with the lipid bilayers of the membrahe. However, the relevance of this interaction in the m ode of action of the h o r m o n e in the biological system, in which a specific receptor is involved, remains open to further investigations. I am indebted to Prof. A.C.M. Paiva for providing the Angiotensin and to Prof. E. De Robertis for helpful discussions and support of this work. References 1 K r a s n e , S., E i s e m a n , G. and S z a b o , G. ( 1 9 7 1 ) Scicnce 174, 4 1 2 - - 4 1 5 2 M c L a u g h l i n , S. a n d E i s e n b e r g , M. ( 1 9 7 5 ) A n n . Rev. B i o p h y s . Bioeng. 4 , 3 3 5 - - 3 6 6
452
3 Shamoo, A.E. (Ed.) (1975) Carriers and Channels in Biological Systems, Ann. New York Acad. Sci. 264, 1--485 4 I-Iladky, S.B. and Haydon, D.A. (1970) Nature 2 2 5 , 4 5 1 - - 4 5 3 5 Fettiplace, R., Haydon, D.A. and Knowles, C.D. (1971) J. Physiol. 221, 18--19 6 Mueller, P., Rudin, D.O,, Tien, H.T. and Wescott, W.C. (1963) J. Phys, Chem., 67, 534--535 7 Baumann, G. and Mueller, P. (1974) J. Supramol. Struet. 2, 538--547 8 Mueller, P. (1975) Ann. New York Acad. Sci. 264, 247--264 9 Juliano, L. and Paiva, A.C.M. (1974) Biochemistry 13, 2445--2450 10 Deslauriers, R., Paiva, A.C.M., Sehaumburg, K. and Smith, I.C.P. (1975) Biochemistry 14, 878--886
Biochimica et Biophysica Acta, 464 ( 1 9 7 7 ) 4 4 8 - - 4 5 2 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA Report BBA 7 1 2 8 1
E F F E C T OF ANG I O T E N SI N II ON A R T I F I C I A L LIPID MEMBRANES
PETER SCHLIEPER*
Instituto de Biolog[a Celular, Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires (Argentina) (Received O c t o b e r 19th, 1 9 7 6 )
Summary (5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between AG = 5 . 1 0 -11 ~-1 and 3 . 5 . 1 0 -1° ~ - 1 . These fluctuations depend on the voltage and the hydrostatic pressure. The m e mb r an e resistance is lowered by AR = 6.1. 1 0 7 ~ • cm 2 . With (5-isoleucine, 8-1eucine)-angiotensin II the jumps are of a single amplitude (A G = 2 . 1 0 -1° ~2-1 ). In both cases water and ions are transported across the membrane.
The so-called ionophoric antibiotics are characterized by the p r o p e r t y o f transporting ions, water and neutral molecules across membranes; among them, the channel or pore-forming ones are t hought to produce stable transm e mb r an e structures that could mediate ion codduct ance in " l i q u i d " as well as " s o lid " bilayers [ 1]. One of the most conspicuous manifestations of pore f o rmatio n is the appearance of discrete conduct ance steps in an artificial black lipid membrane, which suggests the opening of individual channels. Several o f the pore-forming antibiotics are short cyclic polypeptides, as in the classical example of alamethicin, or linear polypeptides, as in the case of the gramicidins (for reviews, see refs. 2, 3). For example, in the presence of very small amounts of gramicidin A, the current across a voltage-clamped black lipid m e mb r an e fluctuates between discrete and constant values with conduct ance jumps of ab o u t 4 . 1 0 - l l ~ - I [ 4] . These findings have led to the study of the effect o f natural polypeptides on artificial membranes. Fettiplace et al. [ 5] observed that lysine-vasopressin increased the conduct ance of artificial bilayers, but t h e y did not describe the f o r m a t i o n of typical pores. In the present work we have studied the interaction of the octapeptide h o r m o n e angiotensin II on black lipid membranes and have observed the *Fellow of the Deutsche Forschungsgemeinschaft.
449
production of typical conductance fluctuations, characteristic of a poreforming ionophore. These changes are accompanied by the translocation of ions and water across the membrane. Black lipid membranes were made according to Mueller et al. [6] on a 0.9 mm hole traversing a Teflon septum that separates two aquous phases. Ag-AgC1 electrodes were used to apply a definite voltage across the membrane. Measurements of conductance were done by means of a Keithley 610C microammeter. A storage oscilloscope (Tektronix 5103 N), connected to a preamplifier (Philbrick 1029) and a Keithley inscriptor device were used for recording the voltage and current changes. The membranes were made in an horizontal position, using a 2% solution of egg lecithin (Sigma) in decane. The bathing solution on both sides of the membrane contained 100 mM NaC1 or KC1 with 1 mM Tris/PO4 buffer, pH 7.3. The drugs in 10--25-pl aliquots were applied on one side near the membrane. (5-Isoleucine)-angiotensin II and the homologue (5-isoleucine, 8-1eucine)angiotensin II, kindly provided by Prof. A.C.M. Paiva (Escola Paulista de Medicina, 04023 Sao Paulo, Brazil) were synthesized by the solid phase method. The amino acid sequence of (5-isoleucine)-angiotensin II is: Asp-Arg-Val-TyrIle-His-Pro-Phe; in (5-isoleucine, 8-1eucine)-angiotensin II, the phenylalanine in the 8th position is replaced by leucine. The addition of (5-isoleucine)-angiotensin II produced discrete changes in membrane conductance, while keeping the membrane clamped at a constant potential. In Fig. l , a short time after the addition of 25 pl of a 0.1% solution of the polypeptide, typical current jumps appear with several levels of conductance Most of these have a value of AG = 5- 10 -11 ~2-1 , but there are others of higher amplitude, which may reach 3.5- 10 -l° ~Z-1. In this particular case there were
]
2 0 mV
lOmV
lO-'°A/cm
2
1 min F i g . 1. T r a c e o f c u r r e n t v e r s u s t i m e . V o l t a g e s o f 1 0 a n d 2 0 m V a r e a p p l i e d a c r o s s a m e m b r a n e f o r m e d w i t h a 2 % l e c i t h i n - d e c a n e s o l u t i o n . A n g i o t e n s i n II ( 5 - i s o l e u c i n e ) w a s a d d e d n e a r t h e m e m b r a n e . T h e s m a l l e s t c o n d u c t a n c e c h a n g e s c o r r e s p o n d to AG = 5 o 1 0 -1152 -1 .
450
no fluctuations at 10 mV, but t hey appeared at 20 mV. Such current fluctuations appear also to be related to the hydrostatic pressure applied to the membrane. If 0.5 ml excess solution (which corresponds to 2.6- 10 -s atm) is applied on one side, then the first pores appear 2 min after the addition of angiotension II. When there is no pressure applied, it may take 20 min for the pores to show up and only when higher potentials (80--100 mV) are applied. Membranes, undergoing interaction with angiotensin IL(5-isoleucine) for 30 min, lowered their resistance f r o m R ~ = 6 . 7 - 1 0 7 ~ 2 - c m 2 t o R 2 = 5 . 6 " 1 0 6 ~ ' c m 2 ( A R = 6.1-107~2.cm 2) Simultaneously there was the building up of a hydrostatic pressure in the upper chamber. In this case the m e m br a ne showed a concavity looking upwards. If the pressure is equilibrated by removal of liquid from the upper c o m p a r t m e n t , the m e mb r an e flattens, the pores disappear and the initial membrane resistance is restored. This p h e n o m e n o n is reversible and pores can be seen again after application of a hydrostatic pressure. With time, no m em brane potential could be detected, suggesting that the pores are not ion selective and that t hey p r o b ably allow the passage of Na ÷ and C1- as well as water. With KC1 in the bathing solution similar current jumps could be observed; however their frequency was lower and also the decrease in resistance of the membrane was smaller than with NaC1 (AR = 1 . 1 - 1 0 7 ~ .cm 2 ). The effect o f (5-isoleucine, 8-1eucine)-angiotensin II on the artificial lipid membrane was slightly different. Immediately after the application of the polypeptide, pores having a AG = 2 . 1 0 -l° ~-1 appeared. As shown in Fig. 2 these pores in general are of a single amplitude and very seldom different levels of conductances were observed; also here the hydrostatic pressure and the resistance of the membrane changes with time. In the case o f the pore-forming antibiotics it has been postulated that the channels result from the special organization of the molecules within the bilayer. Transmembrane structures are t h o u g h t to be form ed of repeating
Fig. 2. T r a c e of c u r r e n t versus t i m e p h o t o g r a p h e d f r o m t h e o s c i l l o s c o p e . 4 0 m V w e r e a p p l i e d across the m e m b r a n e m a d e w i t h a 2% l e c i t h i n - d e c a n e s o l u t i o n . A n g i o t e n s i n II ( 5 - i s o l e u c i n e , 8-1eucine) was a d d e d u p o n o n e s i d 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 c h a n g e s c o r r e s p o n d t o A G = 2" ] 0 - 1 ° ~ - - 1 .
451
units which may arrange in series or in parallel (i.e. as the staves of a barrel) and which may exhibit a dependence on the electrical field across the lipid bilayers. Bauman and Mueller [7] have proposed that the assembly of a channel is made by the voltage-dependent aggregation of m o n o m e r i c precursors. According to this hypothesis the gating mechanism for the channel involves the voltage-induced insertion of the precursor from the m em brane surface into the h y d r o c a r b o n region of the bilayer [8]. Juliano and Paiva [9] and Deslauriers et al. [10] have discussed the several molecular models that have been proposed for angiotensin II. From a carbon-13 spin-lattice relaxation study Paiva and co-workers conclude that (5-isoleucine)-angiotensin II is probably folded so that the N- and C-terminals are held in closer p r o x i m i t y than expected for a random conformation. The molecular model that emerges is that of a molecule folded on itself and having a radius of ab o u t 8 A. The (5-isoleucine, 8-1eucine)-angiotensin II which has only 1% of the physiological activity of the other, showed a 10% increase in spin relaxation time, indicating a loosening of the c o n f o r m a t i o n in the b a ck b o n e of the molecule. The present study shows that bot h polypeptides of angiotensin II are able to interact with lipid bilayers made of lecithin to create pores. We may speculate that the interaction of these polypeptides with the lipid bilayer leads to a rearrangement of several of these folded molecules, so that the more h y d r o p h o b i c parts of the chains are held toward the lipidic milieu, while the hydrophilic groups face towards the center of the channel. The relative wide diameter of the pore f or m ed may explain the flux of water and the non-ionic selectivity. As in the case of other pore-forming polypeptides the gating mechanism for angiotensin II would be electrically driven. As shown in Fig. 1 the pores are mainly opened under an applied positive potential. A not he r factor that appears to be involved in the gating mechanism is the hydrostatic pressure that produces a stretching of the membrane; such a pressure may be externally imposed upon the membrane or it may be built by the preferential flux of water and ions toward the c o m p a r t m e n t containing the polypeptide. While the two homologues of angiotensin II used in the present study are able to pr oduce channels, the (5-isoleucine, 8-1eucine)-angiotensin II seems to be m or e active and this could be related to the more relaxed c o n f o r m a t i o n of this molecule [ 10] . The observations r epor t e d here suggest that the octapeptide angiotensin II may have a direct interaction with the lipid bilayers of the membrahe. However, the relevance of this interaction in the m ode of action of the h o r m o n e in the biological system, in which a specific receptor is involved, remains open to further investigations. I am indebted to Prof. A.C.M. Paiva for providing the Angiotensin and to Prof. E. De Robertis for helpful discussions and support of this work. References 1 K r a s n e , S., E i s e m a n , G. and S z a b o , G. ( 1 9 7 1 ) Scicnce 174, 4 1 2 - - 4 1 5 2 M c L a u g h l i n , S. a n d E i s e n b e r g , M. ( 1 9 7 5 ) A n n . Rev. B i o p h y s . Bioeng. 4 , 3 3 5 - - 3 6 6
452
3 Shamoo, A.E. (Ed.) (1975) Carriers and Channels in Biological Systems, Ann. New York Acad. Sci. 264, 1--485 4 I-Iladky, S.B. and Haydon, D.A. (1970) Nature 2 2 5 , 4 5 1 - - 4 5 3 5 Fettiplace, R., Haydon, D.A. and Knowles, C.D. (1971) J. Physiol. 221, 18--19 6 Mueller, P., Rudin, D.O,, Tien, H.T. and Wescott, W.C. (1963) J. Phys, Chem., 67, 534--535 7 Baumann, G. and Mueller, P. (1974) J. Supramol. Struet. 2, 538--547 8 Mueller, P. (1975) Ann. New York Acad. Sci. 264, 247--264 9 Juliano, L. and Paiva, A.C.M. (1974) Biochemistry 13, 2445--2450 10 Deslauriers, R., Paiva, A.C.M., Sehaumburg, K. and Smith, I.C.P. (1975) Biochemistry 14, 878--886