Spherical Bilayer Research Articles

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations.

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.

Polarity decrease at the adhesive junction between two model membranes containing gangliosides

The increased electrical conductance previously observed between two model membranes containing gangliosides suggests the creation of a new environment in the adhesive junction [Brewer, G. J., & Thomas, P.D. (1984) Biochim. Biophys. Acta 776, 279]. In order to provide a mechanism for this novel finding, we now report an investigation of the micropolarity in the adhesive junction. Emission from the fluorescent probe PRODAN is a sensitive measure of polarity of the probe environment. A bimodal linear relationship correlates the emission wavelength from PRODAN with the inverse of solvent dielectric constant (1/epsilon). A better single linear relationship is obtained using Reichardt's relative polarity measure (RPM). Creation of two macroscopic spherical lipid bilayers from phosphatidylcholine, brain gangliosides, and PRODAN allowed selective excitation and observation of fluorescence from either a single bilayer or the double bilayer in the adhesive junction. The reported PRODAN polarity of -0.57 in a single ganglioside-containing membrane was midway between the polarity of water and n-hexane, suggesting PRODAN localization near the lipid carbonyls. The adhesive junctional region exhibited two new less polar environments of PRODAN fluorescence, RPM = -0.45 and -0.29. These measures are consistent with a relatively dehydrated immobilized phase. These changes were not observed in the adhesion zone between two membranes made with phosphatidylcholine without gangliosides. The changes in molecular structure in the junction that could be responsible for the altered PRODAN emission are discussed. A decrease in the hydrocarbon thickness of junctional membranes or a decrease in the aqueous junctional polarity could be responsible for the polarity decrease reported by PRODAN.

Congregation of gangliosides at the junction between two model membranes

The diversity and distribution of gangliosides in vertebrate tissue suggests an important role in cellular recognition. Two types of experiments are reported to test the hypothesis that gangliosides can congregate to form an adhesive junction between two membranes. First, to monitor ganglioside distribution and mobility in different regions of two large spherical bilayer membranes, fluorescent derivatives of natural gangliosides were synthesized. Second, the cation carrier nonactin was used as a conductance probe to measure the membrane surface potential, which would be altered if there were a redistribution of the charged gangliosides. These studies were conducted in large spherical artificial membranes made from egg phosphatidylcholine or oleoylpalmitoylphosphatidylcholine with 0-12 mol % bovine brain gangliosides dissolved in n-decane. The fluorescent gangliosides utilized were lucifer yellow adducts to the sialic acids (LY-gangliosides) or a cis-paranaric acid substitution of the N-acyl moiety in the ceramide portion of gangliosides GM1 and GD1a (paranaryl-GM1 and paranaryl-GD1a). The polarized fluorescence from the adhesive junction between two membranes containing LY-gangliosides or either paranarylganglioside was compared to that in nonadhesive regions. For LY-gangliosides, total fluorescence in the junction decreased with time, possibly due to electrostatic repulsion of this highly charged derivative. For paranarylgangliosides, fluorescence in the junction increased 7-fold with time, suggesting congregation of this ganglioside. In both cases, a measure of rotational mobility, fluorescence anisotropy, increased dramatically, about 2-fold, as expected for restricted mobility of adhesive compounds. Independent evidence for congregation of charge-bearing gangliosides was found with the conductance probe nonactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-sensitive dyes: measurement of membrane potentials induced by DC and AC electric fields.

Dye indicators of membrane potential have been available for the past 15 years and have been employed in numerous studies of cell physiology. Since the cell membrane is a likely primary site for the cascade of events resulting in a biological response to electromagnetic fields, methodologies for monitoring the membrane voltage will be critical. This laboratory has developed a series of dyes by using theoretical molecular orbital calculations to predict electrochromic structures. The spectra of electrochromic dyes are altered via a direct coupling of the molecular electronic states with an electric field. This mechanism has the advantage of providing both high temporal and high spatial resolution because the effect is instantaneous and is localized to the level of individual indicator molecules. It therefore can have significant advantages over traditional microelectrode techniques because fast changes in potential can be monitored simultaneously over many different regions of a biological preparation. We have used these dyes to monitor membrane potentials induced by both DC and AC electric fields. In a series of studies with a model membrane system, a spherical lipid bilayer, we showed that the potential develops on the membrane in good agreement with a time-dependent solution to Laplace's equation. Cell membranes can also be stained with voltage sensitive dyes. In experiments with dc fields, we are able to map the variation of the induced membrane potential along the surface of the cell by employing digital video fluorescence microscopy. We can also use the fluorescence microscope to detect membrane potential induced by ac fields by using a phase-sensitive detection scheme to extract the corresponding change in the light intensity from the fluorescent indicator. The technology can be extended to more complex biological preparations and can be used in conjunction with other optical techniques such as those which monitor intracellular ion concentrations. It may, therefore, prove highly valuable for the elucidation of biological responses to electromagnetic fields.

Asymmetrie Reconstitution of the Erythrocyte Anion Transport System in Vesicles of Different Curvature: Implications for the Shape of the Band 3 Protein

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposomes of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be "right-side-out" in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein's intramembrane domain and that this domain has the form of a truncated cone or pyramid.

Magnetic susceptibility effects in quadrupolar echo spectroscopy of spin-1 nuclei

The influence of magnetic susceptibility. and chemical shifts on Fourier-transform quadrupolar echo spectra is described. The effects of local magnetic fields on the real and imaginary components of the echo signal are discussed and strategies required for the treatment of the quadrature Fourier transform are addressed. The influence of magnetic susceptibility is illustrated using a particularly striking example, that of single spherical bilayer membranes on spherical glass beads of diameter approximately one micrometer.

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.

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. The sample was dialyzed overnight against detergent-free buffer. Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30 degrees C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100 mM, inhibition of the flux by H2DIDS and flufenamate (approx. KI-values at 30 degrees C: 0.1 and 0.7 microM, respectively), and "right-side-out" orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.

Extraction of mitochondrial membrane proteins into organic solvents in a functional state

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1–79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4°C in hexane some enzymes loose less than 50% of their activity.

On the dielectrically observable consequences of the diffusional motions of lipids and proteins in membranes. 1. Theory and overview.

A system consisting of any array of cylindrical, polytopic membrane proteins (or protein complexes) possessed of a permanent dipole moment and immersed in a closed, spherical phospholipid bilayer sheet is considered. It is assumed that rotation of the protein (complex) in a plane normal to the membrane, if occurring, is restricted by viscous drag alone. Lateral diffusion is assumed either to be free and random or to be partially constrained by barriers of an unspecified nature. The dielectric relaxation times calculated for membrane protein rotation in a suspension of vesicles of the above type are much longer than those observed with globular proteins in aqueous solution, and fall in the mid-to-high audio frequency range. If the long range lateral diffusion of (charged) membrane protein complexes is essentially unrestricted, as in the "fluid mosaic" membrane model, dielectric relaxation times for lateral motions will lie, except in the case of the very smallest vesicles, in the sub-audio (ELF) range. If, in contrast, the lateral diffusion of membrane protein complexes is partially restricted by "barriers" or "long-range" interactions (of unspecified nature), significant dielectric dispersions may be expected in both audio- and radio-frequency ranges, the critical (characteristic) frequencies depending upon the average distance moved before a barrier is encountered. Similar analyses are given for rotational and translational motions of phospholipids. At very low frequencies, a dispersion due to vesicle orientation might in principle also be observed; the dielectrically observable extent of this rotation will depend, inter alia, upon the charge mobility and disposition of the membrane protein complexes, as well as, of course, on the viscosity of the aqueous phase. The role of electroosmotic interactions between double layer ions (and water dipoles) and proteins raised above the membrane surface is considered. In some cases, it seems likely that such interactions serve to raise the dielectric increment, relative to that which might otherwise have been expected, of dispersions due to protein motions in membranes. Depending upon the tortuosity of the ion-relaxation pathways, such a relaxation mechanism might lead to almost any characteristic frequency, and, even in the absence of protein/lipid motions, would cause dielectric spectra to be much broader than one might expect from a simple, macroscopic treatment.

Dielectric dispersion of a single spherical bilayer membrane in suspension

Single spherical bilayer membranes of the Pagano-Thompson type (Pagano, R. and Thompson, T.E. (1967) Biochim. Biophys. Acta 144, 666–669), formed from monooleyl phosphate and cholesterol dissolved in CHCl 3/CH 3OH/ n-decane, were subjected to a fast impedance analysis of high precision. Dielectric behavior of the whole system, as monitored from outside the spherical membrane, was sensitive to changes in the membrane state from the thick colored to the thin black state. With a spherical membrane 2–3 mm in diameter formed in the sample cavity containing 0.12 ml 10 mM NaCl, the former state was characterized by a dielectric dispersion having dielectric increment (Δϵ) of some 10 2 and characteristic frequency ( ƒ c ) around 10 6 Hz, while the latter had Δϵ ≅ 10 5 and ƒ c ≅ 10 3 Hz . Complex plane plots for both dispersions traced semicircles, indicating that the present system may be unequivocally analyzed to yield spherical radius and membrane capacity ( C m) on the basis of a well-established dielectric theory. C m for the thin membranes has thus been determined to be 0.54 μF · cm −2, in excellent agreement with a separate determination on planar membranes. The applicability of the present type of spherical membranes under dielectric monitoring to the study of membrane fusion or of exocytosis is suggested.

Transfer of insulin receptors and of glucose transport-inducing proteins onto phospholipid vesicles

Insulin receptors and glucose transport-inducing proteins have been extracted from rat liver membranes onto positively charged lipid bilayer vesicles. The extraction was carried out during the incubation of the vesicles with lipid vesicles caused an overall enhancement of specific insulin binding and of glucose transport inducement. The latter has been inferred from the oxidation rate of transported glucose through a spherical bilayer membrane entrapping the oxidizing glucose oxidase. Glucose transport is not enhanced by insulin binding, indicating that the two functions become dissociated when the proteins are transferred from the plasma membrane onto the bilayer vesicles.

Protein-induced conductivity changes in black lipid membranes and protein aggregation

Solubilized membrane proteins, especially the ‘integral’ ones, show a strong tendency towards aggregation. Little is known of the consequences this behaviour might have in experiments on protein-lipid interactions. Recently, however, it was briefly reported that protein association has an influence on lipid binding of apoprotein A-l from serum high-density lipoprotein [l] . It may be anticipated that similar effects might also occur with membrane apoproteins. During the last several years,we have investigated the interactions of the ‘strongly bound’ protein fraction from human erythrocyte membranes with spherical and planar lipid bilayers. This protein fraction essentially consists of the two main integral proteins of the erythrocyte membrane: a protein of molecular weight 100 000 (‘band 3-protein’ [2] and ‘glycophorin’, themembrane sialoglycoprotein; the band 3-pr~eing the dominating component [3,4]. During&r studies on the planar bilayers (black lipid membranes, BLM) we have found that the main peculiarity of the protein-BLM-system, the occurrence of large protein-induced changes in membrane conductivity [5,6], varies considerably with protein subfractions of different states of aggregation.

Pigment containing lipid vesicles. I. Preparation and characterization of chlorophyll a-lecithin vesicles.

Vesicles obtained by sonication of chlorophyll a-lecithin mixtures dispersed in anaqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophyll a. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophyll a molecule per 55 lecithins can be incorporated into membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102 +/- 5 A and independent of the chloropyll a content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).

Fusion in phospholipid spherical membranes. II. Effect of cholesterol, divalent ions and pH.

Effect of cholesterol, divalent ions and pH on spherical bilayer membrane fusion was studied as a function of increasing temperature. Spherical bilayer membranes were composed of natural [phosphatidylcholine (PC) and phosphatidylserine (PS)] as well as synthetic (dipalmitoyl-PC, dimyristoyl-PC and dioleoyl-PC) phospholipids. Incorporation of cholesterol into the membrane (33% by weight) suppressed the fusion temperature and also greatly reduced the percentage of membrane fusion. The presence of 1 mM divalent ions (Ca++, Mg++ or Mn++) on both sides or one side of the PC membrane did not affect appreciably its fusion characteristic with temperature, but the PS membrane fusion with temperature was greatly enhanced by the presence of divalent ions. The variation of pH of the environmental solution in the range of 5.5 approximately 7.0 did not affect the membrane fusion characteristic. However, at pH 8.5, the fusion with respect to temperature was shifted toward the lower temperature by approximately 3degreesC for PC and PS membranes, and at pH 3.0 the opposite situation was observed as the fusion temperature was increased by 6degreesC for PS membranes and by 4degreesC for PC membranes The results seem to indicate that membrane fluidity and structural instability in the bilayer are important for membrane fusion to occur.

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The preliminary characterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of "empty" virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not seen in cells infected with wild-type virus or with ts 1 at the permissive temperature. The "empty" appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just outside the viral membrane and normally sidiments with host and virus membranes. This protein made by mutant ts 5 does not pellet with these membranes; instead, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with ts 5 at the restrictive temperature produced inside the cell an abundance of virus-size membrane vesicles. Taken together, these results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.

Nonelectrolyte diffusion across lipid bilayer systems.

The permeability coefficients of a homologous series of amides from formamide through valeramide have been measured in spherical bilayers prepared by the method described by Jung. They do not depend directly on the water:ether partition coefficient which increases regularly with chain length. Instead there is a minimum at acetamide. This has been ascribed to the effect of steric hindrance on diffusion within the bilayer which increases with solute molar volume. This factor is of the same magnitude, though opposite in sign to the effect of lipid solubility, thus accounting for the minimum. The resistance to passage across the interface has been compared to the resistance to diffusion within the membrane. As the solute chain length increases the interface becomes more important, until for valeramide it comprises about 90% of the total resistance. Interface resistance is also important in urea permeation, causing urea to permeate much more slowly than an amide of comparable size, after allowance is made for the difference in the water:ether partition coefficient. Amide permeation coefficients have been compared with relative liposome permeation data measured by the rate of liposome swelling. The ratios of the two measures of permeation vary between 3 and 16 for the homologous amides. The apparent enthalpy of liposome permeation has been measured and found to be in the neighborhood of 12 kcal mol-1 essentially independent of chain length. Comparison of the bilayer permeability coefficients with those of red cells shows that red cell permeation by the lipophilic solutes resembles that of the bilayers, whereas permeation by the hydrophilic solutes differs significantly.

Fusion in phospholipid spherical membranes. I. Effect of temperature and lysolecithin.

A study concerning membrane contact and fusion phenomena was made for phospholipid spherical bilayer systems with respect to temperature. Specific temperatures were obtained for the spherical bilayer membranes of phosphatidyl choline (PC) and phosphatidyl serine (PS) which indicated a greater degree of membrane fusion and were designated Tf (the fusion temperature -- PC: 43 degrees C, PS: 38 degrees C). These temperatures were reduced by about 10 degrees C for the membranes incorporated with 20% lysophosphatidyl choline. The results of the contact and fusion observed in the spherical membranes are compared and discussed with the conductance characteristics of the PC and PS planar bilayer membranes as well as dissolution study on the phospholipid monolayers formed at the air/water interface with respect to temperature. Also, a possible molecular mechanism of membrane fusion is discussed in terms of the fluidity and instability of the membrane.

31P NMR of Phospholipid membranes: Effects of chemical and anisotropy at high magnetic field strengths

The 31P NMR spectrum of sonicated dipalmitoyl lecithin vesicles consists of two chemically shifted resonances, separated by ∼0.15 ppm, which arise from phosphate groups in phospholipid molecules on the inside and the outside of the spherical bilayer vesicles. The widths of the resonances are remarkably sensitive to the crystalline-liquid crystalline phase transition, the magnetic field-strength, and the viscosity of the surrounding aqueous medium. The results are interpreted in terms of two phosphorus relaxation mechanisms: modulation of the anisotropic phosphorus chemical shift and dipolar interaction with protons on adjacent methylene groups. At high magnetic fields (7.5 T) the modulation of the phosphorus chemical shift anisotropy by the Brownian rotation of the intact spherical vesicles dominates the linewidth. The chemical shift anisotropy in the sonicated vesicles is compared with that in the unsonicated dispersions. It is concluded that the structure around the phosphate group is not significantly disrupted by the sonication process. The order parameter for the internal motion of the phosphate group is estimated, and a tentative model for the motion of the phosphate group in the membrane is proposed.

Chaotropic anions and the surface potential of bilayer membranes

The chaotropic anions perchlorate and thiocyanate adsorb to artificial phospholipid membranes. The negative electrostatic potential they produce at the surface of the membranes was measured by two independent techniques. The conductance produced by neutral carriers of cations and anions was measured to estimate changes in the surface potentials of planar black lipid films and the electrophoretic mobility of phospholipid vesicles was used to monitor changes in the zeta potentials of spherical bilayer membranes. Qualitatively similar results were obtained with the two techniques. The results, moreover, agreed with the change in surface potential produced by these anions at an air water interface, as measured directly with an ionizing electrode (Randles, J.E.B. (1957) Discuss. Faraday Soc. 24, 194–199). The results obtained with artificial bilayers may explain the observation (Wieth, J.O. (1970) J. Physiol. 207, 581–609) that thiocyanate increases the sodium or potassium and decreases the sulfate permeability of erythrocyte membranes.