Electrical Oscillatory Phenomena in Protein Membranes BY VICTORE. SHASHOUA McLean Hospital Biological Research Laboratory and Department of Biological Chemistry Harvard Medical School Belmont Mass. 02178 U.S.A. Received 5th August 1974 Membranes were prepared by interacting polycations with polyanions at an interface to give a structured system in which a cationic phase was separated from an anionic phase by a neutral poly- ampholyte zone. Such a membrane system with a cation +anion junction exhibits electrical oscillations in an electric field. Measurements of the (current voltage) characteristics of the membranes under current clamp conditions shows a "negative resistance " region coincident with the polarization conditions required for producing electrical oscillations.Both proteins and poly- nucleic acids can be used as the polyelectrolyte components of the membrane. Biological membranes exhibit many types of electrical oscillatory properties. These include the relatively slow phenomena characteristic of plant cells and the fast events observed in nerve and muscle cells. Tasaki and Takenaka,I in an analysis of the electrical properties of the squid giant axon demonstrated that substantially all the electrical excitability and conduction properties of axons can be attributed to the cell membrane alone i.e. these properties remain intact even when practically all cytoplasmic components have been perfused out of the axons. Thus the axonal membrane consisting of a 70A thick layer of proteins coinplexed with lipids can generate the electrical properties of neurons.A number of model systems have been proposed for simulating various aspects of the excitability properties of axonal membranes. One of the first models was described by Lillie.2 He showed that an iron wire covered with glass tubing can propagate an electrical impulse in a manner suggestive of the characteristics of myelinated nerve fibers. In 1954 Teorell and subsequently Franck showed that glass membranes can generate slow oscillatory electrical signals. More recently lipid membrane systems based on the bilipid layer concept of Davson and Danielli have been the subject of many investigations.6-10 Mueller and Rudin showed that when lipid bilayers were modified by certain macromolecules they then could generate many of the electrical characteristics of neuronal membranes.This type of model suggests that specific macromolecules can convert a lipid bilayer which has the high electrical resistance characteristics of a good insulator (lo8 ohm/cm2) into a membrane with a low resistance (lo2 ohm/crn2) and a capacity to generate electrical oscillations. In addition "semiconductor like " properties are obtained for some protein-lipid interactions. Clearly no such properties can be predicted from an analysis of the bulk properties of either the lipid or the protein constituents of the membrane. Both classes of these molecules are insulators in the dry state. Lipids behave like detergents and proteins become polyelectrolytes in aqueous solutions. In an effort to find out if there are any fundamental characteristics of proteins or more generally polyelectrolytes which may be useful for defining the electrical properties of biological membranes we explored the possibility that specific changes might occur when polyelectrolyte membranes are organized into layered structures.* 174 V. E. SHASHOUA In this way we attempted to simulate the interaction of proteins with charged lipid monolayers organized in a smectic phase. The possibility exists that new properties may be obtained following such an interfacial interaction. ELECTRICALLY ACTIVE POLYELECTROLYTE MEMBRANES In the initial experiments aqueous solutions of polyacids were layered onto solutions of polybases.12 The membranes produced at the interface were found to be capable of generating random electrical oscillations in an applied electric field (see fig.1 A). In subsequent work,13 some membrane systems produced sustained electrical oscillations (see fig. 1B C,D). Essentially these membranes could simulate a transduction process with the properties of an electrical oscillator circuit to convert a d.c. potential into an a.c. output with " spike-like " characteristics. The amplitude (1-1OOmV) and duration (1-10ms) of the oscillations were of the same order of magnitude as those of the neuronal spikes. These properties were found to be directly attributable to the " sandwich-like " structure of the membrane in which a cation exclusion barrier (polycationic phase) was separated from an anion exclusion barrier (polyanionic phase) by a neutral polyampholyte zone.This type of structure may be called a polycation c-)polyanion (c f-) a) junction membrane. The double arrow in the c t)a symbol is used to designate the presence of a neutral polyampholyte layer between the two poIyelectrolyte phases of the membrane. FIG.2.-Experimental arrangement for study of electrical oscillatory properties of c ++a junction membranes :S Agar salt bridges with 0.15 N NaCl ;V and A are a voltmeter and ammeter ;output to oscilloscope is through a d.c. amplifier with high impedance ;insert shows diagram of the structure of a matrix supported c Ha membrane. In a typical experiment a c c-)a membrane separates two compartments containing 0.15 N NaCl (see fig. 2).The current is passed through the system via two agar salt bridges containing 0.15 N NaCl. These connect each compartment to a silver/ silver chloride electrode-immersed in 0.15 N NaC1. The potential across the membrane is detected by means of two Ag/AgCl electrodes connected to a d.c. differential amplifier with a very high input impedance (Metametrics Corp. Cambridge Mass.). This essentially draws no current from the system. The output of the amplifier is fed into an oscilloscope to display the pattern of the potential changes obtained. When a current is passed through the membrane to drive anions into the polycation phase and cations into the polyanion phase a sequence of electrical and 176 ELECTRICAL OSCILLATORY PHENOMENA IN PROTEIN MEMBRANES mechanical events takes place as a function of the applied voltage.At first the record- ing electrodes show that there is an instability region and that the output voltage recorded across the membrane becomes very sensitive to mechanical vibrations. Further increase in the applied voltage results in a loss of the mechanical instability followed by the generation of electrical transients with " spike-like " characteristics. Higher voltages produce a breakdown of the membrane. Thus there is a critical voltage range at which oscillations take place. MATRIX SUPPORTED c ++ a MEMBRANES The preparation of c c-) a membranes by the direct interaction of a polyacid with a polybase was found to be difficult to control. The membranes frequently had regions of imperfection and holes where short-circuiting occurred.In order to obtain a more experimentally feasible situation a matrix supported system was developed. This was achieved by using a matrix membrane to act as a neutral hydrophilic support polymer for the two polyelectrolyte phases. Fig. 2 shows a diagram of the cross- section of such a polymer matrix membrane cemented in place at the 2 mrn aperture of a cellulose nitrate tube separating the two electrode compartments. The membrane is generally about 1200A thick and contains a distribution of pores of 50-3000Ain diameter as shown by electron microscopy. This " sieve-like " structure acts as a support for the two polyelectrolyte components which are electrophoretically loaded into the films to produce a c w a membrane.The matrix membrane was prepared by evaporating thin layers of chloroforin solutions of the polyamide poly (sebacyl piperazine). (Pip-8) l4 onto a glass plate. The polymer and solvent were care-fully purified to eliminate contamination with dust particles water and traces of other organic matter. Pip-8 has a combination of properties which provide for a suitable matrix material. It is a neutral hydrophilic but water-insoluble polymer and has no peptide NH groups which could promote denaturation of any proteins that may be used as membrane additives. It generates no oscillatory activity in an electric field. The porous structure of Pip-8 is prepared by adding varying amounts of a water soluble impurity to g/ml) such as glycerol or polyethylene glycol to the 0.5 % Pip-8 solution.These are incompatible with Pip8 in the solid phase but remain in solution in CHC13. The porous Pip-8 structure is obtained because the added impurities separate into isolated regions as the film dries. These can then be extracted out with water. Fig. 3 shows a series of electron micrographs of the Pip-8 membranes showing the effect of additives to produce the porous types of films as well as the polyelectrolyte loaded systems. Experimentally the Pip-8 matrix is loaded simultaneously with a polyacid and polybase from opposite sides. The conditions of loading such as concentrations of the polyelectrolyte viscosity of the solutions and pH were adjusted so that the mobilities of the polyacid and polybase were the same and that they could interact within the pores to form films and thus the required barrier for a c c.)a structure.This type of experimental method was used to investigate a variety of synthetic polyelectrolytes proteins and polynucleic acids as membrane components (see table 1). EXPERIMENTAL PREPARATION OF MATRIX MEMBRANES The polymer for the matrix membrane was synthesized by the interfacial polycondensation method from sebacyl chloride and piperazine (Eastman Organic Chemicals Co.).l4 The polymer was rigorously purified by repeated extraction with 1 M sodium carbonate and water. The wet polymer was then dissolved in chloroform and precipitated by a mixture FIG. 1.-Oscilloscope traces of spikes generated by polyelectrolyte membranes in 0.15 N NaCl (A) dextran sulphate -polylysine membranes in 0.15 N NaCI (A) dextran sulphate + poly-L-sarcosine; scan = 50 ms amplitude = 20 mV per major division.(B) RNAse ;scan 20 ms and amplitude 5 mV per major division. (C) methylacrylate/acrylic acid -Ca2+ membrane upper record is aTd.c. trace at 100 mV/cm. (D) RNA -Ca2+ membrane scan = 0.1 s amplitude 20 mV per major division. [Toface page 176 FIG.3.-Electron micrographs of Pip-8 membranes upper left shows the nonporous matrix nieni- brane ; upper right and lower left show a matrix membrane with pores ; lower right shows a matrix membrane loaded with yeast RNA; the deeply stained areas are regions of RNA in the membrane. A B FIG.4.-Oscillatory patterns from a polylysine *DNA membrane A-initial output €3 stabilized output ; scales are 2 niV/cm and 0.5 s/cm for each major division on oscilloscope screen.FIG.8.-Current-clamp data for a polylysine -polvglutamic acid junction membrane ; the upper photographs shows an oscilloscope trace of the (I V) characteristics of a cwa membrane ; lower photograph is for the unloaded matrix membrane. The x and y axes correspond to the current and voltage values. Each major division corresponds to 1 V and 0.1 mA respectively. The zero voltage and current value is at the centre of the photograph. V. E. SHASHOUA of ethanol and water. This was repeated three times to remove low molecular weight components. The white fibrous polymer was then dried at 40°C for several days. The matrix membranes were obtained by casting a 0.5 % Pip8 solution in chloroform over a glass plate coated with a thin layer of a water soluble polymer such as polyethylene glycol (American Cyanamid) or Dextran sulphate (Pharmacia Inc.).The thickness of the chloro- form solution was adjusted by a knife edge supported at a distance of 0.025 cm from the glass. After drying for 20 min at room temperature the position of the Pip-8 film on the glass surface was visible only by tilting the glass to produce interference fringes. The film was then cut with a razor blade into 3 crn squares and distilled water was added at the cut edges. The water dissolves the water soluble polymer under the Pip-8 and dislodges the segments which then float onto the water surface. Next a cellulose nitrate tube with a 2 mrn aperture was used to pick up the matrix membrane.The Pip-8 film was sealed onto the plastic tube with a cement of Pip-8 (10 % in chloroform) at the edges to form a window as shown in fig. 2. The electrophoretic loading with polyelectrolytes was accomplished by placing the polyacid in the outside compartment and the polybase in the inner compartment. Each c-a membrane requires its own experimental parameters. In one example of a polylysine t-)DNA preparation the polylysine hydrobrornide (polycation phase) at a pH of 3 was electrophoresized against a DNA (polyanion phase) at pH 3. Initially the resistance of the system of salt bridges and electrolytes was 1.5x lo6ohm. A current of 0.02 mA at 1.5 V was passed through the system.After 15 min the current dropped to less than 0.005 mA. This was considered to be an indication of complete loading of the matrix membrane. Fig. 4 shows the type of electrical oscillations obtained with this membrane when the inner and outer compartment electrolytes were 0.15 N NaCl and when the system was polarized at a potential of 1.2 V. Trace A is the initial type of sustained oscillations obtained when the critical voltage of 1.2V was first applied. Trace B (fig. 4) shows the pattern after about 1 min of firing. This membrane continued to generate electrical oscilla- tions for a total of 10 min and then abruptly stopped due to the formation of a short circuit. MEASUREMENT OF (CURRENT VOLTAGE) CHARACTERISTICS Two methods were used for measuring the current-voltage characteristics of the c +-) a membranes.Fig. 5 shows the experimental arrangement for the measurements. The first method used Ag/AgCI electrodes to apply a potential across the membrane M. The current FIG. 5.-Experimental arrangement for measurement of current-voltage characteristics of c t*a membranes-S salt bridges ; Vm,voltage across membrane. flow in this circuit was detected by a milliammeter. The voltage across the membrane was detected by a voltmeter connected by the two salt bridges S to two Calomel electrodes as shown The results obtained in this type of measurements are shown in fig. 6. This method did not clamp the current or voltage during the measurement. The second method used a current clamp circuit to apply a given voltage Y across the membrane with a nanosecond time constant.Fig. 7 shows the circuit diagram with the operational amplifier in place. The current flowing was detected across a 10 k ohm resistance in series with the membrane. 178 ELECTRICAL OSCILLATORY PHENOMENA IN PROTEIN MEMBRANES Both the current and voltage measurements were carried out with Ag/AgCl electrodes connected with salt bridges across the membrane. A characteristic curve for the membrane was directly plotted onto an oscilloscope screen adjusted to display the voltage and current on the x and y axes respectively. All Ag/AgCl electrodes used were converted to chloride form immediately before each measurement. Fig. 8 shows a photograph of the oscilloscope screen for data of a polylysine -polyglutamic acid membrane system.RESULTS AND DISCUSSION Table 1 lists the various c ++ a junctions membranes studied to date and the polarizations applied to the membranes to initiate electrical oscillatory phenomena. Fig. 1 and 4 show the types of results obtained. In general random oscillatory patterns were more common. The constant frequency oscillations obtained ranged TABLE 1.-COMPOSITIONOF CATION f-) ANION JUNCTION MEMBRANES polycationic phase polyanionic phase polylysine HBr polyglutamic acid polydimethylaminoethyl acrylate polyacrylic acid Ca2+ yeast RNA BaZf dextran sulphate (m.w. 2x lo6) cytochrome c acrylic acid/acrylamide copolymer (50/50) Ca2+ acrylic acid/methylacryIate copolymer (50/50) poly L-sarcosine polyglutamic acid poly L-lysine HBr DNA During the oscillatory mode the positive and negative electrodes were connected to the polycations and polyanionic compartnients respectively of the c f-) a junctioii membranes.WLYGLUTAMIC-Ca" c 1234 .a2 E Lrolll) A B C FIG.6.-Current-voltage measurements of different types of c-a junction membranes ;electrolyte was 0.12 N NaCI + 1 mM CaCl for A and B and 0.12 N NaCI for C. Ec is the initial voltage at which electrical oscillations are observed. from a low range of 13 Hz for a RNA f-) Ca2+to about 100 Hz for a polyglutamic acid c+ Caz+ membrane. While it is not yet possible to specify the detailed procedures required for producing constant frequency patterns we believe that uniform loading and a narrow distribution of pore sizes in the matrix membrane may be among the critical factors.Fig. 6 shows the current-voltage characteristics of three c f-) a membranes measured under conditions of no current clamping. All three membranes show V. E. SHASHOUA characteristics of rectifiers with a ‘‘negative ” resistance region. The polyglutamic acid t)Ca++ membrane in fig. 6A has three regions of linear-current-voltage properties corresponding to resistance values of 888 ohm/cm2 45 ohm/cm2 and 110 ohm/cm2. The resistance of the unloaded matrix membrane was 12 ohm/cm2 considerably lower than for each of results for loaded membranes. Fig. 6C depicts ,111 10K 200 pf OP AMP +=-FIG.7.-Diagram of the current-clamp circuit used for obtaining (I V) curves for the co membranes.a more complicated (current voltage) curve obtained for an acrylic acid/acrylamide (50/50)c.)cytochrome c membrane. In all the graphs in fig. 6 the oscillatory pro-perties of the membranes are obtained when the membranes are polarized at the critical voltage E, i.e. at the negative resistance region of the membrane. The shift from non-oscillatory to oscillatory behavior can actually be seen on an oscilloscope. Experimentally the scope is set for a.c. recording then as the voltage is raised to a critical region there is a rapid transition and the oscilloscope trace shifts from one stable mode to another accompanied by the onset of “ spike ” generation. This transition in membrane properties occurs within a few seconds and is better illustrated in current-clamp type of measurements.Fig. 8 shows the current-clamp data for a polylysine t.)polyglutamic acid mem-brane. The photograph of the oscilloscope trace was obtained by a point by point setting of each voltage. It is seen that when a voltage of +3 V was applied across the membrane that a sudden decrease of voltage occurred at a constant current of -0.12 mA down to + 1.0 V. From then on the properties of the membrane had ohmic resistance characteristics. The only way to restore the membrane to its original state was to reverse the voltage and obtain (I V) characteristics as shown in the photograph. It is clear that the membrane behaves like a typical “ tunnel-diode ” with “ negative resistance ” characteristics. Katchalsky 15* l6 has proposed a molecular mechanism for the properties of these c -+ a membranes.It is based on the concept that polyelectrolytes undergo a phase transition at certain critical salt concentrations. Thus current flow through the membrane causes cations to move into the polyanionic phase but when they pass into the polyampholyte layer they suddenly encounter the polycation phase. This represents a very highly charged positive layer so the cations are repelled and they accumulate at the polyampholyte interface. Similarly anions arrive through the polycation phase. The net result is that the NaCl (or electrolyte) concentration builds up at the interface. At a critical concentration there is a sudden shrinkage of the polyelectrolyte and the membrane produces a “ breakdown ” region.This 180 ELECTRICAL OSCILLATORY PHENOMLNA IN PROTEIN MEMBRANES shrinkage is the result of the well known conformation change of polyelectrolytes at high ionic strength. The result is a conductance change and electrolyte rushes through to wash out the excess salt to regenerate the original membrane state. An electrical analogy to the properties of C-a junction membranes can be obtained from comparison of the c t)a structure to that of an n-p semiconductor. Fig. 9 shows a diagram of this type of consideration. Essentially the polycation and i-+lq JUNCTION --I--I--__I CATION INTERACTION ANION ZONE @ FIXED IONS -+ MOBILE IONS FIG.9.-Diagram of distribution of charges in a c-a junction membrane. The diagram indicates the presence of fixed charges provided by the polyelectrolyte and the mobile charges (current carrying) provided by the electrolyte.polyanion phases represent (in cross-section) regions of fixed ions. The poly- ampholyte zone is the interaction zone and the current carrying species are the mobile ions Na+ and C1-. Additional similarities are to be found from a consideration of the dynamic properties of c -a junction membranes. Katchalsky and Spangler l6 derived a theoretical equation for the frequency of the oscillations of c -a membranes. where v = frequency do = membrane thickness Lp = the filtration coefficient w = the salt permeability of the neutral zone and Cp = the concentration of the polymer in mol at the membrane surface.One remarkable aspect of this equation is that it predicts a square root relationship to concentrations of the polymer at the interface zone i.e. for the concentration of fixed ions at the interaction zone. The equation for the self-resonant frequency of a tunnel diode also has a square root relationship where R,,Cj and L are the junction resistance junction capacitance and series inductance of an equivalent circuit of a tunnel diode. At present we are not able suficiently to control the parameters for preparing the membranes SO as to test various aspects of the "tunnel diode " and the dynamic polyampholyte models. The general concept of a ce,a junction membrane and its analogy to semi- conductors can be applied to a number of membrane models. The requirements are that a two phase system with fixed ions should be present to act as a barrier for the current carrying mobile ions.In the simplest example (see table 1) both the cationic and anionic phases are fixed as polymeric components. It is also possible to have one fixed phase and one "pseudo " fixed phase. For example Ca2.+ ions can be used V. E. SHASHOUA as the polycationic phase. These ions are introduced into a matrix membrane already loaded with the polyanionic component to give a c c-)a membrane in which the Caz+ ions cross-link the polyanion to form a graded ‘‘ polycationic ” structure. Thus a preponderance of Ca2+ ions are present at the outer surface of the membrane and a “ neutral ’’ phase is developed in the centre of the polyanionic component.In such a system the polycationic phase is dynamic in nature and it is necessary to have extra Ca2+ ions present in the polycationic compartment so that any removal of the Caz+ by ion exchange can be replaced from the electrolyte. Examples of c -a membranes can also be prepared in which the polyanionic phase is derived from labile components. Thus polylysine can be used as the poly- cationic phase to provide the fixed cations and a bilayer of lipids (such as lecithin) can act as the anionic phase. If the lipid bilayer is stabilized as a smectic phase then the outer monolayer facing the electrolyte acts as the anionic (negatively charged) component while the inner monolayer interacts with the polycationic component to form the neutral zone of the c ++ a junction membrane The integrity of such a membrane can be maintained as long as the bilayer remains intact and extra lipid molecules are available to replace molecules lost by diffusion and electrophoresis.Such membrane systems have recently been prepared by Montal lo and earlier in some of the experiments of Mueller et a2.l7 using lipid bilayers with the addition of polylysine and protamine respectively. In both these examples the c t)a junction model can be used to provide a mechanism for the generation of the excitability and semiconductor properties of the membranes. This paper is dedicated to the memory of Aharon Katchalsky for his enthusiastic encouragement of the research. 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