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  • 1
    ISSN: 1432-1424
    Keywords: Key words: HlyA — RTX toxins — Leukotoxicity — Pore Formation — Pore properties — Human macrophages — Patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: Key words: Pore-forming toxin — Sea anemone — Secondary structure — Chemical modification — Sulfhydryl reagents — Pore size — Ion selectivity —Actinia equina
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 71 (1983), S. 173-182 
    ISSN: 1432-1424
    Keywords: hemocyanin channel ; planar bilayer membrane ; multistate transitions ; channel kinetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The kinetics of the hemocyanin channel conductance transitions in black lipid membranes have been studied in two different ways. In one method, voltagejump current-relaxation experiments were performed with membranes containing many channels (102÷104). The steady-state conductance-voltage curves obtained are S-shaped and the slow kinetic processes (1–100 sec) for the approach to steady-state can be fitted by three exponentials. The three time constants were found to depend on the actual applied voltages, but not on the past history (preconditioning voltages). In the other method, membranes with one single channel were used and the spontaneous discrete fluctuations of conductance at constant voltage were analyzed. Slow fluctuations occur between four conductance levels with the lower ones preferred at high positive voltages. The voltage dependence of the conductance of each level and of the transition rates has been measured. A tentative model for the gating mechanism is suggested, which associates each conductance level to a configurational state of the channel and considers the transitions between them as a Markow jump process.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 10 (1983), S. 169-184 
    ISSN: 1432-1017
    Keywords: Hemocyanin channel ; Planar lipid membrane ; Conductance ; Current fluctuations ; Voltage dependence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Helix pomatia hemocyanin forms ion-conducting channels in planar lipid bilayer membranes when added at mg/ml concentration. These channels have several original features. They fluctuate between one conducting and some poorly conducting states and fluctuations can be grouped in bursts. Different channels can have widely different conductance amplitudes. Both channel conductance and burst lifetime are dependent on the applied voltage. Fluctuations within a burst show a complex kinetic behaviour which has been explained developing a multistate model. The model calls for one single open state and six different closed states. Transitions are allowed only between one of the closed states and the open one and obey first oder kinetics. This model is able to fit all our experimental curves obtained in single channel experiments.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 12 (1985), S. 33-41 
    ISSN: 1432-1017
    Keywords: Hemocyanin ; lipid vesicles ; planar bilayer ; ion permeability ; osmotic jump ; membrane fusion ; ionic channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Osmotic jump experiments were used to measure the ionic permeability induced in lipid vesicles by Megathura crenulata hemocyanin. It was found that this protein strongly increases the conductance of K+ and Cl- through these membranes but not that of SO 4 = . These effects were attributed to the formation of ionic channels in the vesicles. We have found that a simple first-order binding model can explain the dependence of the number of pore-containing vesicles both on the time after exposure to hemocyanin and on the protein concentration. Milder effects were attributed to a non-specific adhesion of the protein to the membrane surface. Consistent with the hypothesis of reversible association, vesicles which retained hemocyanin after step sucrose density gradient centrifugation at low ionic strength, lost most of the protein upon recentrifugation at high ionic strength. Consistent with the hypothesis of channel formation bot the above vesicle preparations transferred voltage-dependent hemocyanin channels into planar bilayers when they were made to fuse with them. It is concluded that hemocyanin can interact both specifically, by forming pores within the hydrophobic core of lipid membranes, and non-specifically, probably by means of electrostatic interaction with the surface of the same membrane.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 14 (1987), S. 349-358 
    ISSN: 1432-1017
    Keywords: Staphylococcus aureus ; α-toxin ; ionic channel ; activation energy ; oligomerization ; fluorescence ; lipid vesicles ; planar lipid membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Staphylococcus aureus α-toxin forms ionic channels of large size in lipid bilayer membranes. We have developed two methods for studying the mechanism of pore formation. One is based on measurement of the ionic current flowing through a planar lipid membrane after exposure to the toxin; the other is based on measuring the release of the fluorescent complex Tb-Dipicolinic acid from large unilamellar vesicles under similar conditions. Both methods indicate that the pore formation process is complex, showing an initial delay followed by non-linear kinetics. The power dependence of the pore formation rate on the toxin concentration in planar bilayers indicates that an aggregation mechanism underlies the channel assembly. Arrhenius plots, obtained with both techniques, show no deviation from linearity up to 50°C and the derived activation energies are found to be comparable to those for the binding and the lysis of rabbit erythrocytes by the same toxin. The temperature dependence of the conductance induced in planar bilayers by a large number of toxin channels indicates that the pores are filled with aqueous solution. The analysis of single conductance events shows that a heterogeneous population of pores exist and that smaller channels are preferred at low temperature. We attribute this heterogeneity to the existence of pores resulting from the aggregation of different numbers of monomers.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 10 (1983), S. 143-168 
    ISSN: 1432-1017
    Keywords: Lipid bilayer ; Haemocyanin channel ; Terbium ; Current saturation ; Ion binding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Megathura crenulata haemocyanin can be incorporated in phosphatidylcholine planar bilayers, forming ionic channels. Ca2+ and Tb3+ are known to bind to the protein and to compete for some common sites, here their effects on the electrical properties of the channel have been studied. Both cations have similar characteristics: they diffuse through the channel, linearize and shift its instantaneous current voltage curve and induce inactivation at negative voltages. Two-salt experiments are described in which small amounts of Ca2+ or Tb3+ are added to a KCl solution, allowing titration of these effects on the channel. S-shaped conductance voltage curves and strongly saturating conductance-concentration curves are explained on the basis of a two-state gating mechanism, coupled with the influence of a titratable fixed charge near the pore entrance. The model requires non-specific screening of the negative charge by counter-ions in solution and specific binding to it by divalent and small monovalent cations. Dissociation constants for the binding of Ca2+ and Tb3+ to the M. crenulata haemocyanin channel are calculated and found to be in good agreement with published data on binding of the same cations to other haemocyanins in physiological conditions.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Biochimica et Biophysica Acta (BBA)/Biomembranes 1153 (1993), S. 53-58 
    ISSN: 0005-2736
    Keywords: (G. vaginalis) ; Cholesterol ; Divalent cation ; Erythrocyte ; Exotoxin ; Hemolysin ; Lipid vesicle
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology , Medicine , Physics
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Biochimica et Biophysica Acta (BBA)/Biomembranes 856 (1986), S. 672-687 
    ISSN: 0005-2736
    Keywords: Hemocyanin ; Ion channel ; Ion selectivity ; Phospholipid bilayer ; Voltage gating ; pH control
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology , Medicine , Physics
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Biochimica et Biophysica Acta (BBA)/Biomembranes 643 (1981), S. 616-625 
    ISSN: 0005-2736
    Keywords: (Lipid bilayer) ; Hemocyanin channel ; Ion transport ; Titration
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology , Medicine , Physics
    Type of Medium: Electronic Resource
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