Dielectric properties of synaptosomes isolated from rat brain cortex

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37 % of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 ΜF cm⁻²) remained constant irrespective of nature and concentration of the univalent salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically provided that some intra-synaptosomal structure (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50 % of the particulate volume, the remainder being in equilibrium with the external salt medium.     

Dielectric analysis of mitochondria isolated from rat liver. I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer, L. and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589-601, and ibid. 7, 603-612) is presented. The suspensions of 'mitoplasts' prepared from rat liver mitochondria by a hyposmotic (10 mM KCl) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1-100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after scrutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297-305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 microF/cm2, (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6-0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Dielectric analysis of mitochondria isolated from rat liver I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer L., and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589–601, and ibid. 7, 603–612) is presented. The suspensions of ‘mitoplasts’ prepared from rat liver mitochondria by a hyposmotic (10 mM KCI) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1–100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after serutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297–305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 μF/cm², (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6–0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

Dielectric Properties and Ion Mobility in Erythrocytes

The impedance of erythrocytes of man, cattle, sheep, dog, cat, rabbit, and chicken was measured in the range from 0.5 to 250 Mc. The dielectric constant of the red cell interior is 50 at 250 Mc, varies but little with species, and can readily be accounted for by the cells' hemoglobin content. The electrical conductivity of the red cell interior was determined between 70 and 100 Mc. The values differ from species to species within the rather limited range from 4.4 to 5.3 mmho/cm. Removal of the cell membranes does not affect the conductivity. Hence, the cell interior behaves, from an electrical point of view, like a highly concentrated hemoglobin solution. A theoretical value for the electrical conductivity of erythrocyte interiors, which is calculated on the basis of the salt content of the cell, ion mobility, and the volume concentration of the hemoglobin, is roughly twice as large as the measured value. This discrepancy is typical not only of the red blood cell. Pertinent measurements show that it is probably caused by hydrodynamic and possibly by electrostatic effects also, which lower the mobility of the ions. From the lower electrical mobility it appears that a lowered diffusion constant of the electrolytes and nonelectrolytes within the cell is indicated.     

The Relationship of Internal Conductance and Membrane Capacity to Mitochondrial Volume

A study was made of the effect of mitochondrial size on the electrical properties of the membrane and the internal conductivity of mitochondria. The dielectric constant and electrical conductivity of suspensions of guinea pig heart mitochondria were examined in the frequency range 5 x 10⁵ to 2.5 x 10⁸ C.P.S. Membrane capacity was calculated to be 1.1 to 1.3 µf./cm.² and was virtually the same in mitochondria whose surface area was made to vary by a factor of 4 by osmotic means. This finding suggested that some mechanism must exist for the transfer of mitochondrial material into membrane structure during fluctuations in mitochondrial size. The electrical capacity of the membrane was unaffected by a 33-fold change in potassium chloride concentration. The internal conductance of swollen mitochondria was 2 to 3 times lower than that of the external medium. In shrunken mitochondria the internal conductance was virtually independent of the conductivity of the external medium. These results were discussed in relation to current concepts of mitochondrial structure.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behaviour of a suspension of “shell spheres” as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 Μm in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), internal phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.     

A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance.

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behavior of a suspension of "shell spheres" as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 mum in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), interval phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy.Voltage-dependent Ca²⁺ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive ⁴⁵Ca²⁺ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K⁺] medium. Time course studies indicated that voltage-dependent Ca²⁺ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase.ATP-dependent Ca²⁺ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported ⁴⁵Ca²⁺ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated ⁴⁵Ca²⁺ from the vesicles. The maximal rate of ATP-dependent Ca²⁺ transport occurred at a [Ca²⁺ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca²⁺ ions.     

The redox properties of cytochromes b imposed by the membrane electrostatic environment.

The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.     

Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. I. Dielectric behavior of cell suspensions in 0.01-500 MHz and its simulation with a single-shell model

Frequency dependence of relative permittivity (dielectric constant) and conductivity, or the 'dielectric dispersion', of cultured cells (RBL-1 line) in suspension was measured using a fast impedance analyzer system capable of scanning 92 frequency points over a 10 kHz-500 MHz range within 80 s. Examination of the resulting dispersion curves of an improved reliability revealed that the dispersions consisted of at least two separate components. The low-frequency component (dispersion 1) had a permittivity increment (delta epsilon) of 10(3)-10(4) and a characteristic frequency (fc) at several hundred kHz; for the high-frequency component (dispersion 2), delta epsilon was smaller by a factor of 10(2) and fc = 10-30 MHz. Increments delta epsilon for both components increased with the volume fraction of cell suspension, while fc did not change appreciably as long as the conductivity of suspending medium was fixed. By fitting a model for shelled spheres (the 'single-shell' model) to the data of dispersion 1, the dielectric capacity of the plasma membrane phase (Cm) was estimated to be approx. 1.4 microF/cm2 for the cells in an isotonic medium. However, simulation by this particular shell model failed to reproduce the entire dispersion profile leaving a sizable discrepancy between theory and experiment especially at frequencies above 1 MHz where dispersion 2 took place. This discrepancy could not be filled up even by taking into consideration either the effect of cell size distribution actually determined or that of possible heterogeneity in the intracellular conductivity. The present data strongly indicate the need for a more penetrating model that effectively accounts for the behavior of dispersion 2.     

Molecular dynamics simulation of α-melanocyte stimulating hormone in a water-membrane model interface

The conformation of the tridecapeptide α-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant ɛ, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation. Received: 27 November 1998 / Revised version: 11 March 1999 / Accepted: 8 April 1999     

Dielectric properties of mouse lymphocytes and erythrocytes

In order to study the effect of the nucleus on dielectric behavior of the whole cell, permittivity (dielectric constant) and conductivity of mouse lymphocytes and erythrocytes were measured over a frequency range from 0.1 to 250 MHz. Erythrocytes (spherocytes) showed a single dielectric dispersion, which was explained by a single-shell model that is a conducting sphere covered with a thin insulating shell. On the other hand, lymphocytes showed a broad dielectric dispersion curve which was composed of two subdispersions. The high-frequency subdispersion, which was not found for erythrocytes, was assigned to the Maxwell-Wagner dispersion of the nucleus occupying about 65% of the total cell volume. Analysis of the lymphocyte dispersion was carried out by a double-shell model, in which a shelled sphere, i.e., nucleus, is incorporated into the single-shell model. The following electrical parameters were consequently estimated; the capacitance of the plasma membrane, 0.86 microF.cm-2; the conductivity of the cytoplasm, 3.2 mS.cm-1; the capacitance and conductance of the nuclear envelope are, respectively, 0.62 microF.cm-2 and 15 S.cm-2, and the permittivity and conductivity of the nucleoplasm are 52 and 13.5 mS.cm-1.     

Functional membrane vesicles from the nervous system of insects: I. Sodium- and chloride-dependent γ-aminobutyric acid transport

(1) A synaptosomal fraction obtained from locust nervous tissue has been shown to possess an active γ-aminobutyric acid transport mechanism. This activity is preserved and even enriched by the membrane vesicles derived from osmotically shocked synaptosomes. (2) Electron-microscopy examination indicates that the above membrane vesicles are derived predominantly from the neuronal plasma membrane and are devoid of any internal cellular organelles and components. Active transport of γ-aminobutyric acid into these vesicles has been demonstrated with artificially imposed ion gradients as the sole energy source. (3) γ-Aminobutyric acid transport can be driven by an Na⁺ gradient (out>in) and /or by a gradient of Cl^? (out>in). This process is absolutely dependent on the simultaneous presence of both types of ion in the external medium. The stimulation of the process by valinomycin indicates that γ-aminobutyric acid transport is an electrogenic process which is stimulated by a membrane potential (interior negative).     

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.     

Salt bridge interactions: Stability of the ionic and neutral complexes in the gas phase,in solution,and in proteins

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen-bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley-Liss, Inc.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

Model Hydrophobic Ion Exchange Membrane

Two models of hydrophobic ion exchange membranes were examined theoretically with regard to the characteristics of cellulose acetate-nitrate membranes saturated with hydrophobic solvents. The first model, consisting of fixed negative sites dispersed in a homogeneous medium of low dielectric constant, was shown to be invalid for the experimental membranes. The second model, consisting of fixed negative sites in an aqueous channel surrounded by a medium of low dielectric constant, explains many properties of the cellulose acetate-nitrate hydrophobic membranes and was analyzed in some detail. Organic cations can enter the membranes through the hydrophobic phase as well as through the aqueous channels. The mechanism of counterion movement in such a model is assumed to consist of exchange of vacancies and or double-occupied sites positions. The presence of the medium of low dielectric constant around the aqueous channel increases the “self”-energy of the ions in the channel and the electrostatic interaction between a fixed site and a counterion in the membrane. Both these factors can account for the marked dependence of ion mobility in the aqueous channels on the dielectric constant of the surrounding medium. The model predicts membrane preference for monovalent counterions over divalent ones.