Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Studies of Electric Capacitance of Membranes: I. A Model Membrane Composed of a Filter Paper and a Lipid Analogue

A hydrophobic filter paper of a given pore size containing a synthetic lipid, i.e. dioleyl phosphate, was interposed between aqueous electrolyte solutions having the same chemical composition and temperature. The electric capacitance and conductance of the membrane immersed in various concentrations of KCl were measured in the frequency range from 20 to 3 × 10⁶ cycle/sec. The observed capacitance and conductance were found to be strongly dependent on the applied frequency. A theory is proposed to account for this dispersion of impedance observed in the present membrane-electrolyte system. The dispersion is attributed to the formation of bilayer membranes of the lipid inside the filter paper. The effects of the salt concentration, the adsorbed quantity of the lipid, and the pore size of the filter paper on the capacitance and conductance of the membrane are discussed in terms of the distribution function of bilayers formed within the filter paper.     

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Lateral diffusion in nuclear membranes

Chemical modification of rat liver nuclei with citraconic anhydride selectively removed outer nuclear membrane. This conclusion was based on (a) transmission electron microscopy, (b) lipid analysis, (c) lamin B as an inner membrane-associated marker, and (d) the demonstration of phospholipid lateral mobility on outer membrane-depleted nuclei as a criteria for inner membrane integrity. Addition of urea or N-ethylmaleimide resulted in the additional disruption of inner membrane. Fluorescence photobleaching was used to determine the long range (greater than 4 microns) lateral transport of lectin receptors and a phospholipid analog in both membranes. The diffusion coefficient for wheat germ agglutinin on whole nuclei was 3.9 X 10(-10) cm2/s whereas the diffusion coefficient for wheat germ agglutinin in outer membrane-depleted nuclei was less than or equal to 10(-12) cm2/s. Phospholipid mobilities were the same in whole and outer membrane-depleted nuclei (3.8 X 10(-9) cm2/s). The protein diffusion differences observed between whole and outer membrane-depleted nuclei may be interpreted in the context of two functionally different membrane systems that compose the double bilayer of the nucleus.     

Studies of electric capacitance of membranes

Summary The electric capacitance and conductance of a model membrane composed of a hydrophobic filter paper and a synthetic lipid analogue, i.e., dioleylphosphate, immersed in an electrolyte solution were observed with various frequencies ranging from 20 to 3×10⁶ Hz. With successive increase of salt concentration in the external solution, the capacitance and conductance of the membrane increased discontinuously at a certain critical value of the external salt concentration. This variation of the capacitance and conductance of the membrane with the salt concentration was found to be reversible, and the critical value of salt concentration was independent of the adsorbed quantity of the lipid, and of the pore size of the filter paper as far as the adsorbed quantity of the dioleylphosphate was large.A theoretical analysis based on the membrane model for the filter paper-phospholipid system proposed in Part I of this series revealed that the dioleylphosphate impregnated in the filter paper changed its conformation from oil droplets or globular micelles to a number of bilayer membranes when the salt concentration reached the critical value for a given pair of electrolyte species and the membrane. The conformational change of the lipid analogue in the filter paper is discussed in connection with the ability of formation and stability of a black bilayer membrane of the dioleylphosphate.     

Discrete conductance fluctuations in lipid bilayer protein membranes

Discrete fluctuations in conductance of lipid bilayer membranes may be observed during the initial stages of membrane interaction with EIM ("excitability inducing material"), during destruction of the EIM conductance by proteolysis, and during the potential-dependent transitions between low and high conductance states in the "excitable" membranes. The discrete conductance steps observed during the initial reaction of EIM with the lipid membranes are remarkably uniform, even in membranes of widely varying lipid composition. They range only from 2 to 6 x 10_-10 ohm_-1 and average 4 x 10_-10 ohm_-1. Steps found during destruction of the EIM conductance by proteolysis are somewhat smaller. The transition between high conductance and low conductance states may involve steps as small as 0.5 x 10_-10 ohm_-1. These phenomena are consistent with the formation of a stable protein bridge across the lipid membrane to provide a polar channel for the transport of cations. T6he uniform conductance fluctuations observed during the formation of these macromolecular channels may indicate that the ions in a conductive channel, in its open state, are largely protected from the influence of the polar groups of the membrane lipids. Potential-dependent changes in conductance may be due to configurational or positional changes in the protein channel. Differences in lipid-lipid and lipid-macromolecule interactions may account for the variations in switching kinetics in various membrane systems.     

Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers.

The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.     

Ion permeability of bilayer membranes formed from synthetic phospholipids in the phase transition region

Ion permeability of black lipid membranes formed from synthetic phospholipids has been studied. The resistance of BLM formed from phosphatidylcholine, tiophosphatidylcholine, threealkylphosphate and threealkyltiophosphate was 10(7)--10(8) Ohm.cm2. It was shown that the membrane potential of the 10--30 mV arised in KCl gradient indicating the preference cation conductance in synthetic lipid membranes. A sharp decrease of the membrane conductance near to the phase transition temperature was discovered. The change of conductance by phase transition temperature was sensitive to chemical nature of the polar head of phospholipids used.     

Two-dimensional electron transfer from cytochrome C to photosynthetic reaction centers

The arrangement and the electron transfer are studied for photosynthetic reaction centers (RC) of Rhodopseudomonas sphaeroides reconstituted into phospholipid vesicles. Freeze-etch electron micrographs of phase separated mixed vesicles reveal an RC enrichment in the phase containing the acidic lipid serine. It is demonstrated that the electron transfer from cytochrome c to RC involves a two-dimensional diffusion of the membrane bound electron donor with diffusion coefficients (D approximately 10(-9) cm2/sec) characteristic for membrane proteins.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Effect of amphotericin B added to one side of a membrane

Amphotericin B which was added to the one side of a membrane in aqueous solutions with low pH 3.0 sharply increases the membrane conductance by the mechanism of channel forming. Long living ionic channels (20 sec) with large conductance dispersion (2-20 Ps) were found at the concentration 2 X 10(-8) M. Amphotericin B concentration which is necessary for obtaining single channels does not depend on cholesterol concentration in the membrane-forming solution in the range from 2 to 10 mg/ml. The effect of amphotericin B on the membrane depends on the phospholipid concentration in the membrane. The one-side selective permeability is the same as one under the symmetric action of the antibiotic. It is assumed that amphotericin B induced the membrane conductance by the mechanism of half-pores formation.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

Voltage-dependent lipid flip-flop induced by alamethicin.

Alamethicin appears to allow voltage-dependent lipid exchange ("flip-flop") between leaflets of a planar bilayer. In membranes with one leaflet of phosphatidyl serine and one of phosphatidyl ethanolamine, the shape of the nonactin current-voltage curve accurately reports the difference in surface potential between the two sides of the membrane. The surface potential is itself a good measure of membrane asymmetry. Alamethicin added to the bathing solutions of an asymmetric membrane does not per se reduce the membrane asymmetry, but turning on the alamethicin conductance by application of a voltage pulse does. Immediately after application of a voltage pulse, large enough to turn on the alamethicin conductance, the asymmetry of the nonactin-K+ current voltage curve decreases, in some cases, nearly to zero. During the pulse, the alamethicin conductance activates if a decrease in surface potential favors turn-on of the alamethicin conductance or inactivates if a decrease in surface potential favors turn-off of the alamethicin conductance. After the pulse, the nonactin-K+ asymmetry returns to its original value if the alamethicin conductance is not turned on. The time-course of this return allows an estimate of the diffusion constant of lipid in the planar bilayer. The value obtained is 5.1 x 10(-8) cm2/s.