Voltage-dependent gating properties of the channel formed by E. coli hemolysin in planar lipid membranes

1989 ◽  
Vol 9 (4) ◽  
pp. 465-473 ◽  
Author(s):  
Gianfranco Menestrina ◽  
Monica Ropele
Keyword(s):  
Lipid Membranes ◽  
Ph Dependence ◽  
Phospholipid Bilayer ◽  
Bilayer Membranes ◽  
E Coli ◽  
Simple Physical Model ◽  
Voltage Dependent ◽  
Planar Lipid Membranes ◽  
Escherichia Coli Hemolysin ◽  
Gating Process

Escherichia coli hemolysin forms cation selective, ion-permeable channels of large conductance in planar phospholipid bilayer membranes. The pore formation mechanism is voltage dependent resembling that of some colicins and of diphtheria toxin: pores open when negative voltages are applied and close with positive potentials. The pH dependence of this gating process suggests that it is mediated by a negative fixed charge present in the lumen of the pore. A simple physical model of how the channel opens and closes in response to the applied voltage is given.

Electrical Impedance Analysis of Phospholipid Bilayer Membranes for Enabling Engineering Design of Bio-based Devices

2007 ◽  
Vol 1061 ◽  
Author(s):  
Stephen A. Sarles ◽  
Vishnu B. Sundaresan ◽  
Donald J. Leo
Keyword(s):  
Lipid Membranes ◽  
Electrical Impedance ◽  
Lipid Membrane ◽  
Electrical Power ◽  
Impedance Analysis ◽  
Electrical Impedance Spectroscopy ◽  
Phospholipid Bilayer ◽  
Silicon Substrates ◽  
Bilayer Membranes ◽  
Single Aperture

ABSTRACTRecent research at Virginia Tech have shown that active transporter proteins reconstituted into suspended bilayer lipid membranes (BLMs) formed across an array of pores in synthetic substrates can convert chemical energy available in adenosine triphosphate (ATP) into electricity. Experimental results from this work show that this system—called BioCell—is capable of 1.7μW of electrical power per square centimeter of BLM area and per 15μL of ATPase enzyme. In support of such a system, the lipid membrane, as host to active biological proteins and channels, must be formed evenly across a porous substrate, remain stable and yet fluid-like for protein folding and activation, and provide sufficient electrical insulation. We report on the formation and characterization using electrical impedance spectroscopy (EIS) of BLMs formed across two types of porous substrates: polycarbonate filters and single-aperture silicon substrates. Equivalent electrical circuits describing the lipid membranes and their supporting substrates are approximated to fit the measured responses. The results show that BLMs formed in some but not all of the 400nm pores of the filters, while the formation of BLMs on the single-aperture silicon substrates was much more consistent.

scholarly journals Conductive Properties and Gating of Channels Formed by Syringopeptin 25A, a Bioactive Lipodepsipeptide from Pseudomonas syringae pv. syringae, in Planar Lipid Membranes

1999 ◽  
Vol 12 (5) ◽  
pp. 401-409 ◽  
Author(s):  
Mauro Dalla Serra ◽  
Ivonne Bernhart ◽  
Paola Nordera ◽  
Domenico Di Giorgio ◽  
Alessandro Ballio ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Lipid Membranes ◽  
Pore Radius ◽  
Maximal Conductance ◽  
Channel Opening ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Planar Lipid Membranes ◽  
Opening And Closing ◽  
Conductive Properties

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.

Colicin K acts by forming voltage-dependent channels in phospholipid bilayer membranes

Nature ◽  
1978 ◽  
Vol 276 (5684) ◽  
pp. 159-163 ◽  
Author(s):  
Stanley J. Schein ◽  
Bruce L. Kagan ◽  
Alan Finkelstein
Keyword(s):  
Phospholipid Bilayer ◽  
Bilayer Membranes ◽  
Voltage Dependent

scholarly journals Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes.

1990 ◽  
Vol 96 (5) ◽  
pp. 943-957 ◽  
Author(s):  
R O Blaustein ◽  
A Finkelstein
Keyword(s):  
Rate Constant ◽  
Phospholipid Bilayer ◽  
Anthrax Toxin ◽  
Diffusion Control ◽  
Dependent Manner ◽  
Diffusion Limitation ◽  
Entry Rate ◽  
Bilayer Membranes ◽  
Tetraalkylammonium Ions ◽  
Voltage Dependent

Current flow through the channel formed in planar phospholipid bilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (kcis1) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (kcis-1) of Bu4N+ from the blocking site to the cis solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (= kcis-1/kcis1), is not altered by the dextran-induced viscosity increase of the cis solution. The entry rate constants (kcis1) for tetrapentylammonium (Pe4N+), tetrahexylammonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of kcis1 for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusion-limitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., approximately 12 A.

scholarly journals An Abnormally High Closing Potential of the OMPF Porin Channel from Yersinia Ruckeri: The Role of Charged Residues and Intramolecular Bonds

Acta Naturae ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 89-98
Author(s):  
D. K. Chistyulin ◽  
O. D. Novikova ◽  
E. A. Zelepuga ◽  
V. A. Khomenko ◽  
G. N. Likhatskaya ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Gram Negative Bacteria ◽  
Amino Acid Residues ◽  
Yersinia Ruckeri ◽  
Ompf Porin ◽  
E Coli ◽  
Total Conductance ◽  
Voltage Dependent ◽  
Acidic Conditions

Electrophysiological experiments on bilayer lipid membranes showed that the isolated outer membrane major porin of Yersinia ruckeri (YrOmpF) exhibits activity typical of porins from Gram-negative bacteria, forming channels with a mean conductance of 230 pS (in 0.1 M KCl) and slight asymmetry with respect to the applied voltage. Under acidic conditions (up to pH = 3.0), there was no significant decrease in the total conductance of the YrOmpF channel reconstituted into the bilayer. The studied channel significantly differed from the porins of other bacteria by high values of its critical closing potential (Vc): Vc = 232 mV at pH = 7.0 and Vc = 164 mV at pH = 5.0. A theoretical model of the YrOmpF spatial structure was used for the analysis of the charge distribution in the mouth and inside the channel to explain these properties and quantitatively assess the bonds between the amino acid residues in the L3 loop and on the inner wall of the barrel. The parameters of YrOmpF were compared with those of the classical OmpF porin from E. coli. The results of electrophysiological experiments and theoretical analysis are discussed in terms of the mechanism for voltage-dependent closing of porin channels.

scholarly journals Voltage-Dependent Conductance Induced in Thin Lipid Membranes by Monazomycin

1972 ◽  
Vol 60 (3) ◽  
pp. 263-284 ◽  
Author(s):  
Robert U. Muller ◽  
Alan Finkelstein
Keyword(s):  
Steady State ◽  
Lipid Membranes ◽  
Membrane Surface ◽  
Potassium Conductance ◽  
Voltage Characteristic ◽  
Phospholipid Bilayer ◽  
Negative Slope ◽  
Nacl Concentration ◽  
Voltage Dependent ◽  
Salt Gradient

When present in micromolar amounts on one side of phospholipid bilayer membranes, monazomycin (a positively charged, polyene-like antibiotic) induces dramatic voltage-dependent conductance effects. Voltage clamp records are very similar in shape to those obtained from the potassium conductance system of the squid axon. The steady-state conductance is proportional to the 5th power of the monazomycin concentration and increases exponentially with positive voltage (monazomycin side positive); there is an e-fold change in conductance per 4–6 mv. The major current-carrying ions are univalent cations. For a lipid having no net charge, steady-state conductance increases linearly with KCl (or NaCl) concentration and is unaffected by Ca++ or Mg++. The current-voltage characteristic which is normally monotonic in symmetrical salt solutions is converted by a salt gradient to one with a negative slope-conductance region, although the conductance-voltage characteristic is unaffected. A membrane treated with both monazomycin and the polyene antibiotic nystatin (which alone creates anion-selective channels) displays bistability in the presence of a salt gradient. Thus monazomycin and nystatin channels can exist in parallel. We believe that many monazomycin monomers (within the membrane) cooperate to form a multimolecular conductance channel; the voltage control of conductance arises from the electric field driving monazomycin molecules at the membrane surface into the membrane and thus affecting the number of channels that are formed.

Bacterial hemolysins and leukotoxins affect target cells by forming large exogenous pores into their plasma membrane. Escherichia coli hemolysin a as a case example

1995 ◽  
Vol 15 (6) ◽  
pp. 543-551 ◽  
Author(s):  
Gianfranco Menestrina ◽  
Mauro Dalla Serra ◽  
Cecilia Pederzolli ◽  
Monica Bregante ◽  
Franco Gambale
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Plasma Membrane ◽  
Virulence Factors ◽  
Lipid Membranes ◽  
Model Membranes ◽  
Target Cells ◽  
Planar Lipid Membranes ◽  
Escherichia Coli Hemolysin

Many bacteria include among their virulence factors exoproteins which exert leukocidal and cytolytic functions and have the ability to form pores in model membranes. We show that, at least in the case of the RTX hemolysin produced by Escherichia coli (HlyA), formation of pores in planar lipid membranes is parallelled by opening of strikingly similar channels in the plasma membrane of exposed macrophages. Formation of such lesions in leukocytes can give rise to a variety of effects leading altogether to a diminished immune response towards the invasive bacteria.

scholarly journals Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance.

1990 ◽  
Vol 96 (5) ◽  
pp. 905-919 ◽  
Author(s):  
R O Blaustein ◽  
A Finkelstein
Keyword(s):  
Single Channel ◽  
Membrane Conductance ◽  
Phospholipid Bilayer ◽  
Anthrax Toxin ◽  
Dependent Manner ◽  
Cationic Form ◽  
Bilayer Membranes ◽  
Tetraalkylammonium Ions ◽  
Voltage Dependent ◽  
Pass Through

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.

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