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1.
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. 相似文献
2.
Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes
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alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores. 相似文献
3.
Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV
o
(voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F
(v). For a given voltage,F
(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel. 相似文献
4.
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 order kinetics. This model is able to fit all our experimental curves obtained in single channel experiments. 相似文献
5.
6.
Characterization of the ion channels formed by poliovirus in planar lipid membranes. 总被引:7,自引:5,他引:2
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The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids. 相似文献
7.
Fumio Hayashi Masahiro Sokabe Masayuki Takagi Kohei Hayashi Uichiro Kishimoto 《生物化学与生物物理学报:生物膜》1978,510(2):305-315
A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ > Cs+ > Na+ > K+ > Li+. The conductance of the membrane was increased up to a value of about 10−2 ohm−1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn2+ > Cd2+ > Ca2+ > Sr2+ > Mg2+. 相似文献
8.
Transport of hemolysin by Escherichia coli 总被引:25,自引:0,他引:25
Michael Hrtlein Sigrid Schießl Wilma Wagner Ursula Rdest Jürgen Kreft Werner Goebel 《Journal of cellular biochemistry》1983,22(2):87-97
The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported. 相似文献
9.
F Hayashi M Sokabe M Takagi K Hayashi U Kishimoto 《Biochimica et biophysica acta》1978,510(2):305-315
A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+. 相似文献
10.
The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 μm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments. 相似文献
11.
Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of external hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 20 degrees C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool of preformed hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis. 相似文献
12.
Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC. 相似文献
13.
Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol 总被引:2,自引:0,他引:2
Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition. 相似文献
14.
15.
Summary When present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10–10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K+ and Na+ than to Cl– and SO
4
=
, but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K+ with respect to SO
4
=
, whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added. 相似文献
16.
Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins 总被引:2,自引:0,他引:2
Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined. 相似文献
17.
Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes 总被引:23,自引:0,他引:23
The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties. 相似文献
18.
19.
Description of modal gating of the cardiac calcium release channel in planar lipid membranes. 总被引:1,自引:1,他引:0
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Single channel activity of the cardiac ryanodine-sensitive calcium-release channel in planar lipid membranes was studied in order to elucidate the calcium-dependent mechanism of its steady-state behavior. The single channel kinetics, observed with Cs+ as the charge carrier at different activating (cis) Ca2+ concentrations in the absence of ATP and Mg2+, were similar to earlier reports and were extended by analysis of channel modal behavior. The channel displayed three episodic levels of open probability defining three gating modes: H (high activity), L (low activity), and I (no activity). The large difference in open probabilities between the two active modes resulted from different bursting patterns and different proportions of two distinct channel open states. I-mode was without openings and can be regarded as the inactivated mode of the channel; L-mode was composed of short and sparse openings; and H-mode openings were longer and grouped into bursts. Modal gating may explain calcium-release channel adaptation (as transient prevalence of H-mode after Ca2+ binding) and the inhibitory effects of drugs (as stabilization of mode I), and it provides a basis for understanding the regulation of calcium release. 相似文献
20.
Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes. 总被引:10,自引:5,他引:5
Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed. 相似文献