Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.     

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

In studying of conductivity of single supernarrow pores (varying 1 to 15 nm in diameter), formed in thin membranes (10-12 microm in the thickness) from polyethylene terephthalate (PETP), there were revealed discrete changes of currents passing through such pores when applied from external source of potential difference from 200 to 1000 mV. By several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical the so-cold current of single ionic channels in the cell membranes. Supernarrow pores which properties are describes in the present work were obtained as a result of alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). Alkaline etching leads to formation of negative fixed charges on the walls of the pores compensated by positive counterions. When setting potential difference onto the PETP membrane, the latter cation layer is able to transfer the current and this transfer was called the surface conductance. In the case of nanometer pores, such surface conductance may be dominating. We have shown that these discrete changes of currents passing through nanometer pores are associated with metastability of the surface conductance. In the case of highly cation-selective channels in the cell membranes it is inevitable, that at least a part of these channels should have dominating cation surface conductance and mentioned above conductance metastability as well. Our findings allow us to propose a new explanation of the origin of the characteristic discreteness of the currents of cation-selective ionic channels in the cell membranes.     

The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes

Summary Alamethicin induces a conductance in black lipid films which increases exponentially with voltage. At low conductance the increase occurs in discrete steps which form a pattern of five levels, the second and third being most likely. The conductance of each level is directly proportional to salt concentration, inversely proportional to solution viscosity, and nearly independent of voltage.The probability distribution of the five steps is not a function of voltage, but as the voltage is increased, more levels begin to appear. These can be explained as super-positions of the original five, both in position and relative probability.This suggests that the five levels are associated with a physical entity which we call a pore. This point of view is confirmed by the following measurements. The kinetic response of the current to a voltage step is first order, and shows an exponential increase in rate of pore formation and an exponential decrease in rate of pore disappearance with voltage. If these rates are statistical, the number of pores should fluctuate about a voltage-dependent mean. High conductance current fluctuations are too large to be explained by fluctuation in the number of pores alone. But if fluctuations among the five levels are included, the magnitude of the fluctuations at high conductance is accurately predicted.Alamethicin adsorbs reversibly to the membrane surface, and the conductance at a fixed voltage depends on the ninth power of alamethicin concentration and on the fourth power of salt concentration, in the aqueous phase. In our bacterial phosphatidyl ethanolamine membranes, alamethicin added to one side of the membrane produces elevated conductance only when the voltage on that side is increased.On leave of absence from the Facultad de Ciencias, Universidad de Chile, Santiago de Chile.     

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

Summary The three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10^–12 m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris⁺, glucosamine⁺, Hepes^–_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.     

Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin

Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV.     

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

The Outer Membrane Protein VhOmp of <Emphasis Type="Italic">Vibrio harveyi</Emphasis>: Pore-Forming Properties in Black Lipid Membranes

Vibrio harveyi is known to cause fatal vibriosis in marine animals. Here, an outer membrane protein from V. harveyi, namely, VhOmp, was isolated and functionally characterized in terms of pore-forming contact with artificial lipid membranes. The native VhOmp exists as a trimer of a molecular weight similar to that of the porin OmpF from Escherichia coli. Reconstitution of VhOmp into black lipid membranes demonstrated its ability to form ion channels. The average pore conductance of VhOmp was revealed to be about 0.9 and 2 nS in 0.2 and 1 M KCl, respectively. Within transmembrane potentials of ±100 mV, VhOmp pores behaved as ohmic conduits, and their conductance scaled linearly with voltage. Nonlinear plots of the pore conductance versus symmetrical salt concentrations at either side of the protein-incorporating membrane suggested the influence of interior channel functionalities on the passage of charged species. In the presence of Omp-specific polyclonal antibodies, the pore-forming property of VhOmp was modulated so that the usual step-like current increments were replaced by random transitory current fluctuations. VhOmp exhibited a strong biological activity by causing hemolysis of human red blood cells, indicating that VhOmp may act as a crucial determinant during bacterial infection to animal host cells.     

Amphotericin B channel conductance inactivation

Effects induced in bilayer lipid membranes by amphotericin B and its alkyl derivatives was analysed. Inactivation of the antibiotic-dependent multichannel membrane conductance was discovered. Kinetics of membrane conductivity was shown to depend on the antibiotic concentration in the membrane. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to the transient. We suggest that the phenomenon of biphasic kinetics of membrane conductance is the result of a consecutive transformation of polyene channels in the membrane: half-pores are assembled on either side of membrane-nonconducting 1; two half-pores combine to build up a conducting channels-conducting 2, and the conducting channels are disassemled to monomers and nonconducting self-associated forms inside the membrane-disassembled state (nonconducting 3). To explain the transient characteristics of the induced conductance, it is proposed that the antibiotic, present in the solution under self-associated form, binds the membrane and forms pores, then dissociates in the bilayer in a non-active monomeric form. The existence of definite monomers and nonconducting self-associated forms of amphotericin B molecules inside the membrane was estimated from the dependence of kinetic conductance of lipid membranes of amphotericin B and its alkyl derivatives, when the antibiotics are washed out from aqueous medium. Equilibrium between different antibiotic assemblies inside the membrane was demonstrated by the kinetics of conductance decrease following washing the antibiotic. Using circular dichroism measurements, we observed that amphotericin B alkyl derivatives were in self-associated form being susceptible to form pores across cholesterol-containing membranes. The phenomenon of biophasic kinetics was observed only in the cholesterol-containing membrane. The substitution of membrane cholesterol for ergosterol provides monotonic kinetics of membrane conductance at any antibiotic concentration.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Cell-attached patch clamp study of the electropermeabilization of amphibian cardiac cells.

Potential gradients imposed across cell or lipid membranes break down the insulating properties of these barriers if an intensity and time-dependent threshold is exceeded. Potential gradients of this magnitude may occur throughout the body, and in particular in cardiac tissue, during clinical defibrillation, ablation, and electrocution trauma. To study the dynamics of membrane electropermeabilization a cell-attached patch clamp technique was used to directly control the potential across membrane patches of single ventricular cells enzymatically isolated from frog (Rana pipiens) hearts. Ramp waveshapes were used to reveal rapid membrane conductance changes that may have otherwise been obscured using rectangular waveshapes. We observed a step increase (delta t less than 30 microseconds) or breakdown in membrane conductance at transmembrane potential thresholds of 0.6-1.1 V in response to 0.1-1.0 kV/s voltage ramps. Conductance kinetics on a sub-millisecond time scale indicate that breakdown is preceded by a period of instability during which the noise and amplitude of the membrane conductance begin to increase. In some cells membrane breakdown was observed to be fully reversible when using an intershock interval of 1 min (20-23 degrees C). These findings support energetic models of membrane electropermeabilization which describe the formation of membrane pores (or growth of existing pores) to a conducting state (instability), followed by a rapid expansion of these pores when the energy barrier for the formation of hydrophilic pores is overcome (breakdown).     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

Bacterial membrane injuries induced by lactacin F and nisin

The combined action of nisin and lactacin F, two bacteriocins produced by lactic acid bacteria, is additive. In this report, the basis of this effect is examined. Channels formed by lactacin F were studied by experiments using planar lipid bilayers, and bactericidal effects were analyzed by flow cytometry. Lactacin F produced pores with a conductance of 1 ns in black lipid bilayers in 1 mM KCl at 10 mV at 20 °C. Pore formation was strongly dependent on voltage. Although lactacin F formed pores at very low potential (10 mV), the dependence was exponential above 40 mV. The injuries induced by nisin and lactacin F in the membranes of Lactobacillus helveticus produced different flow cytometric profiles. Probably, when both bacteriocins are present, each acts separately; their cooperation may be due to an increase in the number of single membrane injuries. Electronic Publication     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Voltage-induced nonconductive pre-pores and metastable single pores in unmodified planar lipid bilayer

Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150-550 mV to monitor transient single pores for a long period of time. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. Although mean lifetime of the pores was 3 +/- 0.8 ms (250 mV), some pores remained open for up to approximately 1 s. The mean amplitude of conductance fluctuations (approximately 500 pS) was independent of voltage and close for bilayers of different area (40,000 and 10 microm(2)), indicating the local nature of the conductive defects. The distribution of pore conductance was rather broad (dispersion of approximately 250 pS). Based on the conductance value and its dependence of the ion size, the radius of the average pore was estimated as approximately 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. These data indicate that opening and closing of lipidic pores proceed through some electrically invisible (silent) pre-pores. Similar pre-pore defects and metastable conductive pores might be involved in remodeling of cell membranes in different biologically relevant processes.