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The modeling of biological membrane properties by means of filters impregnated with lipid-like substances
Journal of Membrane Science, 30 (198’7) 39-46 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
39
THE MODELING OF BIOLOGICAL MEMBRANE PROPERTIES BY MEANS OF FILTERS IMPREGNATED WITH LIPID-LIKE SUBSTANCES
N.M. KOCHERGINSKY,” Yu. Sh. MOSHKOVSKY
IS. OSAK, L.E. BROMBERG,
Institute of Chemical Physics, U.S.S.R. Academy
V.A. KARYAGIN,
of Sciences, Moscow (U.S.S.R.)
and G.S. LESKIN M.F. Vladimirsky Moscow Regional Research Clinical Institute, Moscow (U.S.S.R.) (Received
July 22,1985;
accepted in revised form July 29,1986)
Summary Nitrocellulose ultrafilters (ca. 0.01 cm thick), impregnated with fatty acids, their esters and other lipid-like substances, are shown to be suitable for modeling some properties of biological membranes. Electric conductance, capacitance, and water permeability of these filters were determined. Recalculations of these parameters for a thickness of 50 A gave values typical for biological membranes. As in the case of squid giant axon and contrary to what is observed for unmodified bilayer lipid membranes, the electric capacitance of the filters was found to depend on the frequency of the applied voltage. These facts, together with the ionic selectivity of the filters, are due to the presence of a nitrocellulose matrix with fixed ion-exchange groups impregnated with lipid-like liquids, and water molecules interacting with these groups.
Introduction Extensive studies of the physico-chemical processes occurring in biological membranes are frequently discouraged by the complex structure and multicomponent nature of these membranes. It is this fact that is responsible for the great interest in the properties of simple model systems, among which bilayer lipid membranes (BLMs) and liposomes are most commonly studied [l-- -31. However, even those model systems have some demerits such as fragility and small dimensions, which preclude many experiments. Moreover, unmodified BLMs made up of neutral lipids differ greatly from biological membranes in certain physico-chemical characteristics. In particular, they are practically devoid of ionic selectivity, and their electric resistance is at least four orders of magnitude greater than that of biological membranes 11731. Some authors have studied model membranes in the form of porous substrates, such as ultrafilters impregnated with organic solvents [4] and *To whom correspondence
should be addressed.
40
lipids dissolved therein [ 5-101. The thickness of such impregnated filters (IFS) is as large as lo-’ cm, which is ca. lo4 times that of biological membranes. The properties of such models depend on the kind of solvent used; therefore, in some cases this solvent is specifically and carefully removed by evaporation [lo]. Sometimes the membranes thus obtained have a conductance of 10-2-10-3 51-l [ 5,6]. After being recalculated for a thickness of 50--100 W, this value exceeds that of biological membranes by over four orders of magnitude. On the contrary, polycarbonate---lipid-decane systems have an extremely low conductance, viz., 10e9 LY’ [ 91. In our experiments we have used nitrocellulose filters impregnated with various lipid-like substances, such as oleic and capric acids, esters of fatty acids (isobutyl laurate, methyl oleate, etc.), as well as castor oil and other vegetable oils. Owing to the use of liquid substances and nitrocellulose filters we have not only managed to avoid the additional introduction of solvent but were also able to obtain model membranes with some specific physico-chemical characteristics approximating those of biological membranes [ 11,121. Experimental The experiments were carried out on ultrafilters manufactured by Synpore (Czechoslovakia), lo-* cm thick and with a mean pore size of 2.5 X 10e4 cm. The filters were impregnated by simple immersion into the lipid-like substances equilibrated with the aqueous solution used for filling the cell. After such treatment the weight of each filter increased by 8--10 mg/cm*, thus indicating that the ultrafilter pores were completely filled with the impregnating liquid. In experiments on the dependence of the model membrane properties on the state of the polymeric matrix, the ultrafilters, before being impregnated, were soaked in Hz0 or an aqueous solution of 0.1 M CaCl, . After that they were dried in a warm air stream. Thus obtained impregnated filters were fixed in a thermostatted cell in such a way that this cell was divided by the membrane into two equal parts, each of 25 ml volume. The effective area of the membrane was 2.5 cm*. Its capacitance and conductance in the frequency range 20-20,000 Hz were measured by means of planar platinum electrodes connected to a circuit incorporating a capacitance bridge, a zero indicator, and a signal generator. The electric transmembrane potential was measured by use of Ag/AgCl electrodes with agar bridges. Other Ag/AgCl electrodes were used for reference purposes when measuring the pH of aqueous solutions by means of flow-thermostatted electrodes OP-743 (Radelkis, Hungary). Tritium-labeled water flow was measured with a Beta pulse counter (U.S.S.R.). The solution was circulated and stirred with a peristaltic pump. The arrangement of the experimental device is shown in Fig. 1. The isobutyl laurate used for impregnation was predistilled in uucuo; oleic and capric acids as well as all remaining reagents were used without
41
Fig. 1. Schematic diagram of the experimental system: 1 - Beta pulse counter; 2 - chart recorder; 3 - pH meter; 4 - “zero” indicator; 5 - signal generator; 6 - capacitance bridge; 7 - peristaltic pump.
additional purification. The state of the lipid-like substances in the impregnated filters was additionally characterized by fatty acid spin labels, ‘H-NMR spectroscopy and polarization microscopy. Results and discussion The physico-chemical parameters of the impregnated membrane, characterizing its barrier properties with respect to ions and water, are listed in Table 1. It was shown experimentally that such parameters as conductance, capacitance, water permeability and fatty acid permeability vary inversely proportional to the thickness of the membrane, i.e., they are controlled by the properties of the membrane itself rather than by the properties of the unstirred water layers. Therefore it would be fairly reasonable to compare these parameters, recalculated for thicknesses of 50-100 8, with the corresponding properties of biological membranes. These values, as well as the activation energy for water transport and electric potentials characterizing the K’/Na’ and K’/Cl- selectivities, are presented in Table 1. It is evident that orders of magnitude are all close to those of natural membranes. No quantitative comparison of the properties of the IFS, BLMs, and biological membranes has been made in previous works. A more detailed study of the values obtained is presented elsewhere [ 121. In a note to Table 1 one can find the specific systems to which the data refer. Interestingly, the capacitance--frequency relationship of the IF resembles that of the squid giant axon; capacitance decrease with increasing frequency in a frequency range where no such change is seen for BLMs altogether [21,22] (Fig. 2). In addition, as in the case of the axon [21] , the IF conductance increases with frequency (data not shown). As opposed to the
0.5-1.3 t11
x lo7
(0.4-0.8)
0.3-1.0 [I31
0.8-1.6
40
2 x lo-?
-
-
Oleic acid permeabilitya (cm/see)
20
X lo-*
1 00-400b
(0.5-2)
4-l Ill
1131
1.0-400
Water permeability (lo-” cm/set)
5.9-9.2c
5.9-9.2c
12-15 [201
6.0 [19] 9.6 [13]
Activation energy for water transport (kcal/mol)
25-40
25-40
[I31
O-10
60-90 [I41
Bi-ionic K’/Na’ potential (mV)
20--40
20-40
O-10 1131
1151
20--58
Potential in in lo-fold gradient of KC1 (mV)
aData on the permeability of biological membranes and BLMs with respect tooleic acid are not known to us. Permeability through BLMs in the case of less hydrophobic butyric and salicylic acids is 0.5 X lo-’ and 0.8 cm/see, respectively [ 181. Permeability of picric acid through biological membranes is 0.4 cm/set [16], that of 4,5,6,7-tetrachloro-2-tr~fluoromethylbenzimadazole is lo-30 cm/set [17], and that of carbonylcyanide-m-chlorophenylhydrazone is 11 cm/set [17]. bThe lower value was obtained when the filter was impregnated with lanoline, the higher one when the filter was impregnated with isobutyl lam-ate. ‘The activation energy was measured on filters impregnated with capric acid. A value of 9.2 kcal/mol was obtained at a solution pH of 2.0, and a value of 5.9 kcal/mol at pH 4.5.
10*-lo3
IFS (recalculated for 50 A)
2 x lob--2
IFS, 0.01 cm thick
loG-lo9 [I31
x 1O-4
Capacitance (ctF/cm’)
BLMs from neutral lipids
lo’-los t1,131
Biological membranes
Resistance (a-cm’ )
Comparison of the properties of biological membranes, BLMs, and IFS
TABLE 1
43
unmodified BLMs made up of neutral lipids, the IFS exhibit high cation/ anion and cation/cation selectivities [23,24]. In our experiments the addition of KC1 to the solution on one side of the membrane and NaCl to its other side in 0.1 A4 concentration resulted in a bi-ionic potential resembling the rest potential of a nerve cell. The value of this potential was as high as 27 mV (positive on the NaCl side) (Fig. 3). Decreasing pH brought about protonation of the cation-exchange groups in the IF with the result that the membrane potential, $, dropped to zero. This indicates a disappearance of K’/Na’ selectivity. The dependence of $ on pH shows a sharp change at
1.2 . wg
l.l_
‘2 x
l.O-
_
?J O.Qf ;
OB-
0.1
1 FREQUENCY,
10 kHz
100
Fig. 2. Frequency dependences of IF, BLM, and squid giant axon capacitances: 1 - The filter is impregnated with isobutyl laurate equilibrated with distilled water and HCl at pH 2.0 and 20” C. Capacitances are recalculated for a thickness of 50 A. 2 - Squid giant axon with an introduced 100 mAf solution of tetraethylammonium, 20°C [21]. 3 - A bilayer prepared from egg lecithin in benzene [22] and placed in a saturated NaCl solution.
Fig. 3. pH dependence of a bi-ionic K’INa’ potential. The membrane is impregnated with isobutyl laurate.
44
pH 4.5. This value is somewhat smaller than the pK, of the carboxyl groups present in nitrocellulose membranes [ 231. Both the appearance of the K’/Na+ potential and its dependence on the pH of aqueous solutions can be attributed to the interaction of these fixed ion-exchange groups with immobilized water molecules [ 23,241. The seven measured physico-chemical parameters of the IF correlate fairly well with those of biological membranes (see Table l), which is due to the presence of the following three major components in the IF: (1) a polysaccharide matrix, (2) a lipid-like impregnating liquid, and (3) water. Experiments show that pretreatment of the filters with aqueous salt solutions, followed by drying and impregnation, replacement of the lipid-like species by liquid mixtures of lipids (e.g., castor oil or lanoline), as well as thermoinduced gel-liquid crystal phase transition, are responsible for variations in the parameters measured. In particular, IFS pretreated with CaCl, solution become one order of magnitude more permeable to tritium-labeled water. An increased impregnating liquid viscosity, as determined by spin probe technique, in the series isobutyl laurate < oleic acid < lanoline is responsible for the higher electric resistance and lower model membrane permeability to HZ0 [ 271. The liquid--solid phase transition of capric acid in the cooling of filters impregnated with this acid results in a loss of K’/Na+ selectivity and a sharp (jump-wise) growth of membrane conductance. Analysis of the frequency dependences of the electric parameters (Fig. 2) shows our model to behave like a polar substance with dielectric losses. To a large measure, these properties are controlled by the presence of ionexchange impurities such as carboxyl groups [23,24] in the polymeric matrix and aqueous associates (“channels”) interacting with them. The presence of membrane-bound water was demonstrated directly by us using tritium-labeled water. In the case of lecithin BLMs there are no ion-exchange groups and water inside the membrane, resulting in a very high electric resistance, the absence of capacity dispersion in the frequency range studied, etc. In other words, IFS are more complex models than liposomes and BLMs; it is just this complexity that allows the match of a variety of macroscopic properties of this model and biological membranes. Interestingly, the state and dynamic characteristics of the impregnating liquid in the ultrafilter pores differ from those in a free liquid. In particular, the changes in the fatty acid spin label ESR spectra suggest the immobilization of the liquid in the course of ultrafilter impregnation. This immobilization increases as the pore radius decreases from 2.5 to 0.2 pm. The ‘H-NMR spectra of ultrafilters impregnated with oleic acid show the presence of a broad line in the region typical of CH1 and CH3 groups. Subsequent introduction of Ccl4 into the IF-containing tube, resulting in the extraction of the impregnating liquid, leads to the appearance of well-resolved lines which are usually observed in a true oleic acid solution. Previously, the possibility of formation of lamellar liquid-crystal structures in phospholipide-filled ultrafilter pores has been demonstrated directly by electron microscopy [ 61.
45
It is believed that IFS impregnated with liquid and liquid-crystal lipid-like species, owing to their large dimensions, high stability, controllable composition, possibility of using spectral and other methods, as well as favorable physico-chemical properties may be used as model membranes in studies of ion transport employing low-molecular ionophores such as fatty acids [25], and current-generating proteins [ 71. Another important application is studies of mechanisms describing the action of membrane-active chemical compounds such as chloropromazine and antidepressants [26]. An essential characteristic is that the filters can be impregnated with lipids that are unable to form BLMs altogether. Water transport through such models is discussed in our work [ 271, showing the possibility of modeling a variety of water channel properties. It should be remembered, however, that the proposed model system has only limited applications in studies of the specific functions of biological membranes. This can be attributed to their composition and microscopic structure. Further microstructural studies and comparison of the impregnated filters with other model and biological membranes are to provide a deeper knowledge of the role of liquid-crystal and, particularly, bilayer lipid regions in the functional properties of biological membranes. References
5 6
7
8
9 10 11
H.T. Tien, Bilayer Lipid Membranes (BLM): Theory and Practice, Marcel Dekker, New York, NY, 1974, p. 655. G. Gregoriadis and A.C. Alison (Eds.), Liposomes in Biological Systems, John Wiley and Sons, Chichester, 1980, p. 384. V.G. Ivkov and G.N. Berestovsky, Dynamic Structure of the Lipid Bilayer, Nauka, Moscow, 1981, p. 293. A. Ilani, Ion discrimination by “Millipore” filters saturated with organic solvents. I. Cation selectivity, mobility, and relative permeability of membranes saturated with bromobenzene, Biochim. Biophys. Acta, 94 (1965) 405. H.A. Nash and J.M. Tobias, Phospholipid membrane model: Importance of phosphatidylserine and its cation exchanger nature, Proc. Natl. Acad. Sci. U.S.A., 51 (1964) 476. S. Tokutomi, K. Ohki and S. Ohnishi, Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes, Biochim. Biophys. Acta, 192 (1980) 596. V.P. Skulachev, Membrane potential and reconstitution, in: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 55, Part F, Academic Press, New York, NY, 1979, p. 586. P.K. Schien, J. Lanyi and L. Packer, Lipid-impregnated filter membranes: A stable membrane system for essay of electrical properties across reconstituted liposomes and natural membrane vesicles, in: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 55, Part F, Academic Press, New York, NY, 1979, p. 604. M. Thompson, R.B. Lennox and R.A. McClelland, Structure and electrochemical properties of microfiltration filter-lipid membrane systems. Anal. Chem., 54 (1982) 76. H. Sandermann, Preparation of stable and solvent-free model membranes, Biochem. Biophys. Res. Commun., 87 (1979) 789. N.M. Kocherginsky, I.S. Osak and Yu. Sh. Moshkovsky, The modeling of electrochemical properties of biomembranes on impregnated ultrafilters, in: Abstracts 1st All-Union Conference on Electrochemistry, Moscow, Vol. II, 1982, p. 162.
46 12 13 14 15 16
17 18 19 20 21 22
23 24 25
26
27
N.M. Kocherginsky, I.S. Osak and Yu. Sh. Moshkovsky, The impregnated ultrafilter as a model of biological membranes, Biophysics, 29 (1984) 624. F.A. Henn and T.E. Thompson, Synthetic lipid bilayer membranes, Ann. Rev. Biothem., 38 (1969) 241. B. Katz, Nerve, Muscle and Synapse, McGraw-Hill, New York, NY, 1966, p. 193. P.S. Nobel, Plant Cell Physiology. A Physico-Chemical Approach, W.H. Freeman, San Francisco, CA, 1970, p. 267. W.G. Hanstein and R. Kiehl, Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles, Biochem. Biophys. Res. Commun., 100 (1981) 1118. G.A. Stuart, J. McLaughlin and P. Dilger, Transport of protons across membranes by weak acids, Physiol. Rev., 60 (1980) 825. A. Walter, D. Hastings and S. Gutknecht, Weak acid permeability through lipid bilayer membranes. Role of chemical reactions in the unstirred layer, J. Gen. Physiol., 79 (1982) 917. C.V. Paganelli and A.K. Solomon, The rate of exchange of tritiated water across the human red cells membrane, J. Gen. Physiol., 41 (1957) 259. B.E. Cohen, The permeability of liposomes to nonelectrolytes, J. Membrane Biol., 20 (1975) 205. S. Takashima and H.P. Schwan, Passive electrical properties of squid axon membrane, J. Membrane Biol., 17 (1974) 51. T. Hanai, D.A. Haydon and J.-L. Taylor, An investigation by electrical methods of lecithin-in-hydrocarbon films in aqueous solutions, Proc. Roy. Sot. London, A281 (1964) 377. E. Shohami and A. Ilani, Model hydrophobic ion exchange membrane, Biophys. J., 13 (1973) 1242. A.A. Lev, Modeling of the Ionic Selectivity of Cell Membranes, Nauka, Moscow, 1976, p. 45. N.M. Kocherginsky and IS. Osak, Free fatty acids as membrane ionophores in the Symporegulation of oxidative phosphorylation, in: Abstracts IIIrd Soviet-Swiss sium on Biological Membranes, Structure and Functions, Tashkent, 1983, p, 192. N.M. Kocherginsky, V.A. Karyagin and IS. Osak, The chloropromazine and some antidepressant effects on electrochemical properties of model membranes, in: Abstracts 1st All-Union Biophysical Congress, Moscow, 1982, Vol. III, p. 73. N.M. Kocherginsky, L.E. Bromberg, I.S. Osak, Yu. Sh. Moshkovsky, Yu.M. Popkov and Ya.B. Skuratnik, The modeling of biomembrane water transport on impregnated ultrafilters, Biol. Membranes, U.S.S.R. (English translation), 1 (1984) 954.
39
THE MODELING OF BIOLOGICAL MEMBRANE PROPERTIES BY MEANS OF FILTERS IMPREGNATED WITH LIPID-LIKE SUBSTANCES
N.M. KOCHERGINSKY,” Yu. Sh. MOSHKOVSKY
IS. OSAK, L.E. BROMBERG,
Institute of Chemical Physics, U.S.S.R. Academy
V.A. KARYAGIN,
of Sciences, Moscow (U.S.S.R.)
and G.S. LESKIN M.F. Vladimirsky Moscow Regional Research Clinical Institute, Moscow (U.S.S.R.) (Received
July 22,1985;
accepted in revised form July 29,1986)
Summary Nitrocellulose ultrafilters (ca. 0.01 cm thick), impregnated with fatty acids, their esters and other lipid-like substances, are shown to be suitable for modeling some properties of biological membranes. Electric conductance, capacitance, and water permeability of these filters were determined. Recalculations of these parameters for a thickness of 50 A gave values typical for biological membranes. As in the case of squid giant axon and contrary to what is observed for unmodified bilayer lipid membranes, the electric capacitance of the filters was found to depend on the frequency of the applied voltage. These facts, together with the ionic selectivity of the filters, are due to the presence of a nitrocellulose matrix with fixed ion-exchange groups impregnated with lipid-like liquids, and water molecules interacting with these groups.
Introduction Extensive studies of the physico-chemical processes occurring in biological membranes are frequently discouraged by the complex structure and multicomponent nature of these membranes. It is this fact that is responsible for the great interest in the properties of simple model systems, among which bilayer lipid membranes (BLMs) and liposomes are most commonly studied [l-- -31. However, even those model systems have some demerits such as fragility and small dimensions, which preclude many experiments. Moreover, unmodified BLMs made up of neutral lipids differ greatly from biological membranes in certain physico-chemical characteristics. In particular, they are practically devoid of ionic selectivity, and their electric resistance is at least four orders of magnitude greater than that of biological membranes 11731. Some authors have studied model membranes in the form of porous substrates, such as ultrafilters impregnated with organic solvents [4] and *To whom correspondence
should be addressed.
40
lipids dissolved therein [ 5-101. The thickness of such impregnated filters (IFS) is as large as lo-’ cm, which is ca. lo4 times that of biological membranes. The properties of such models depend on the kind of solvent used; therefore, in some cases this solvent is specifically and carefully removed by evaporation [lo]. Sometimes the membranes thus obtained have a conductance of 10-2-10-3 51-l [ 5,6]. After being recalculated for a thickness of 50--100 W, this value exceeds that of biological membranes by over four orders of magnitude. On the contrary, polycarbonate---lipid-decane systems have an extremely low conductance, viz., 10e9 LY’ [ 91. In our experiments we have used nitrocellulose filters impregnated with various lipid-like substances, such as oleic and capric acids, esters of fatty acids (isobutyl laurate, methyl oleate, etc.), as well as castor oil and other vegetable oils. Owing to the use of liquid substances and nitrocellulose filters we have not only managed to avoid the additional introduction of solvent but were also able to obtain model membranes with some specific physico-chemical characteristics approximating those of biological membranes [ 11,121. Experimental The experiments were carried out on ultrafilters manufactured by Synpore (Czechoslovakia), lo-* cm thick and with a mean pore size of 2.5 X 10e4 cm. The filters were impregnated by simple immersion into the lipid-like substances equilibrated with the aqueous solution used for filling the cell. After such treatment the weight of each filter increased by 8--10 mg/cm*, thus indicating that the ultrafilter pores were completely filled with the impregnating liquid. In experiments on the dependence of the model membrane properties on the state of the polymeric matrix, the ultrafilters, before being impregnated, were soaked in Hz0 or an aqueous solution of 0.1 M CaCl, . After that they were dried in a warm air stream. Thus obtained impregnated filters were fixed in a thermostatted cell in such a way that this cell was divided by the membrane into two equal parts, each of 25 ml volume. The effective area of the membrane was 2.5 cm*. Its capacitance and conductance in the frequency range 20-20,000 Hz were measured by means of planar platinum electrodes connected to a circuit incorporating a capacitance bridge, a zero indicator, and a signal generator. The electric transmembrane potential was measured by use of Ag/AgCl electrodes with agar bridges. Other Ag/AgCl electrodes were used for reference purposes when measuring the pH of aqueous solutions by means of flow-thermostatted electrodes OP-743 (Radelkis, Hungary). Tritium-labeled water flow was measured with a Beta pulse counter (U.S.S.R.). The solution was circulated and stirred with a peristaltic pump. The arrangement of the experimental device is shown in Fig. 1. The isobutyl laurate used for impregnation was predistilled in uucuo; oleic and capric acids as well as all remaining reagents were used without
41
Fig. 1. Schematic diagram of the experimental system: 1 - Beta pulse counter; 2 - chart recorder; 3 - pH meter; 4 - “zero” indicator; 5 - signal generator; 6 - capacitance bridge; 7 - peristaltic pump.
additional purification. The state of the lipid-like substances in the impregnated filters was additionally characterized by fatty acid spin labels, ‘H-NMR spectroscopy and polarization microscopy. Results and discussion The physico-chemical parameters of the impregnated membrane, characterizing its barrier properties with respect to ions and water, are listed in Table 1. It was shown experimentally that such parameters as conductance, capacitance, water permeability and fatty acid permeability vary inversely proportional to the thickness of the membrane, i.e., they are controlled by the properties of the membrane itself rather than by the properties of the unstirred water layers. Therefore it would be fairly reasonable to compare these parameters, recalculated for thicknesses of 50-100 8, with the corresponding properties of biological membranes. These values, as well as the activation energy for water transport and electric potentials characterizing the K’/Na’ and K’/Cl- selectivities, are presented in Table 1. It is evident that orders of magnitude are all close to those of natural membranes. No quantitative comparison of the properties of the IFS, BLMs, and biological membranes has been made in previous works. A more detailed study of the values obtained is presented elsewhere [ 121. In a note to Table 1 one can find the specific systems to which the data refer. Interestingly, the capacitance--frequency relationship of the IF resembles that of the squid giant axon; capacitance decrease with increasing frequency in a frequency range where no such change is seen for BLMs altogether [21,22] (Fig. 2). In addition, as in the case of the axon [21] , the IF conductance increases with frequency (data not shown). As opposed to the
0.5-1.3 t11
x lo7
(0.4-0.8)
0.3-1.0 [I31
0.8-1.6
40
2 x lo-?
-
-
Oleic acid permeabilitya (cm/see)
20
X lo-*
1 00-400b
(0.5-2)
4-l Ill
1131
1.0-400
Water permeability (lo-” cm/set)
5.9-9.2c
5.9-9.2c
12-15 [201
6.0 [19] 9.6 [13]
Activation energy for water transport (kcal/mol)
25-40
25-40
[I31
O-10
60-90 [I41
Bi-ionic K’/Na’ potential (mV)
20--40
20-40
O-10 1131
1151
20--58
Potential in in lo-fold gradient of KC1 (mV)
aData on the permeability of biological membranes and BLMs with respect tooleic acid are not known to us. Permeability through BLMs in the case of less hydrophobic butyric and salicylic acids is 0.5 X lo-’ and 0.8 cm/see, respectively [ 181. Permeability of picric acid through biological membranes is 0.4 cm/set [16], that of 4,5,6,7-tetrachloro-2-tr~fluoromethylbenzimadazole is lo-30 cm/set [17], and that of carbonylcyanide-m-chlorophenylhydrazone is 11 cm/set [17]. bThe lower value was obtained when the filter was impregnated with lanoline, the higher one when the filter was impregnated with isobutyl lam-ate. ‘The activation energy was measured on filters impregnated with capric acid. A value of 9.2 kcal/mol was obtained at a solution pH of 2.0, and a value of 5.9 kcal/mol at pH 4.5.
10*-lo3
IFS (recalculated for 50 A)
2 x lob--2
IFS, 0.01 cm thick
loG-lo9 [I31
x 1O-4
Capacitance (ctF/cm’)
BLMs from neutral lipids
lo’-los t1,131
Biological membranes
Resistance (a-cm’ )
Comparison of the properties of biological membranes, BLMs, and IFS
TABLE 1
43
unmodified BLMs made up of neutral lipids, the IFS exhibit high cation/ anion and cation/cation selectivities [23,24]. In our experiments the addition of KC1 to the solution on one side of the membrane and NaCl to its other side in 0.1 A4 concentration resulted in a bi-ionic potential resembling the rest potential of a nerve cell. The value of this potential was as high as 27 mV (positive on the NaCl side) (Fig. 3). Decreasing pH brought about protonation of the cation-exchange groups in the IF with the result that the membrane potential, $, dropped to zero. This indicates a disappearance of K’/Na’ selectivity. The dependence of $ on pH shows a sharp change at
1.2 . wg
l.l_
‘2 x
l.O-
_
?J O.Qf ;
OB-
0.1
1 FREQUENCY,
10 kHz
100
Fig. 2. Frequency dependences of IF, BLM, and squid giant axon capacitances: 1 - The filter is impregnated with isobutyl laurate equilibrated with distilled water and HCl at pH 2.0 and 20” C. Capacitances are recalculated for a thickness of 50 A. 2 - Squid giant axon with an introduced 100 mAf solution of tetraethylammonium, 20°C [21]. 3 - A bilayer prepared from egg lecithin in benzene [22] and placed in a saturated NaCl solution.
Fig. 3. pH dependence of a bi-ionic K’INa’ potential. The membrane is impregnated with isobutyl laurate.
44
pH 4.5. This value is somewhat smaller than the pK, of the carboxyl groups present in nitrocellulose membranes [ 231. Both the appearance of the K’/Na+ potential and its dependence on the pH of aqueous solutions can be attributed to the interaction of these fixed ion-exchange groups with immobilized water molecules [ 23,241. The seven measured physico-chemical parameters of the IF correlate fairly well with those of biological membranes (see Table l), which is due to the presence of the following three major components in the IF: (1) a polysaccharide matrix, (2) a lipid-like impregnating liquid, and (3) water. Experiments show that pretreatment of the filters with aqueous salt solutions, followed by drying and impregnation, replacement of the lipid-like species by liquid mixtures of lipids (e.g., castor oil or lanoline), as well as thermoinduced gel-liquid crystal phase transition, are responsible for variations in the parameters measured. In particular, IFS pretreated with CaCl, solution become one order of magnitude more permeable to tritium-labeled water. An increased impregnating liquid viscosity, as determined by spin probe technique, in the series isobutyl laurate < oleic acid < lanoline is responsible for the higher electric resistance and lower model membrane permeability to HZ0 [ 271. The liquid--solid phase transition of capric acid in the cooling of filters impregnated with this acid results in a loss of K’/Na+ selectivity and a sharp (jump-wise) growth of membrane conductance. Analysis of the frequency dependences of the electric parameters (Fig. 2) shows our model to behave like a polar substance with dielectric losses. To a large measure, these properties are controlled by the presence of ionexchange impurities such as carboxyl groups [23,24] in the polymeric matrix and aqueous associates (“channels”) interacting with them. The presence of membrane-bound water was demonstrated directly by us using tritium-labeled water. In the case of lecithin BLMs there are no ion-exchange groups and water inside the membrane, resulting in a very high electric resistance, the absence of capacity dispersion in the frequency range studied, etc. In other words, IFS are more complex models than liposomes and BLMs; it is just this complexity that allows the match of a variety of macroscopic properties of this model and biological membranes. Interestingly, the state and dynamic characteristics of the impregnating liquid in the ultrafilter pores differ from those in a free liquid. In particular, the changes in the fatty acid spin label ESR spectra suggest the immobilization of the liquid in the course of ultrafilter impregnation. This immobilization increases as the pore radius decreases from 2.5 to 0.2 pm. The ‘H-NMR spectra of ultrafilters impregnated with oleic acid show the presence of a broad line in the region typical of CH1 and CH3 groups. Subsequent introduction of Ccl4 into the IF-containing tube, resulting in the extraction of the impregnating liquid, leads to the appearance of well-resolved lines which are usually observed in a true oleic acid solution. Previously, the possibility of formation of lamellar liquid-crystal structures in phospholipide-filled ultrafilter pores has been demonstrated directly by electron microscopy [ 61.
45
It is believed that IFS impregnated with liquid and liquid-crystal lipid-like species, owing to their large dimensions, high stability, controllable composition, possibility of using spectral and other methods, as well as favorable physico-chemical properties may be used as model membranes in studies of ion transport employing low-molecular ionophores such as fatty acids [25], and current-generating proteins [ 71. Another important application is studies of mechanisms describing the action of membrane-active chemical compounds such as chloropromazine and antidepressants [26]. An essential characteristic is that the filters can be impregnated with lipids that are unable to form BLMs altogether. Water transport through such models is discussed in our work [ 271, showing the possibility of modeling a variety of water channel properties. It should be remembered, however, that the proposed model system has only limited applications in studies of the specific functions of biological membranes. This can be attributed to their composition and microscopic structure. Further microstructural studies and comparison of the impregnated filters with other model and biological membranes are to provide a deeper knowledge of the role of liquid-crystal and, particularly, bilayer lipid regions in the functional properties of biological membranes. References
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