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1.
In contrast to the widely used method of electroporation, the method of soft perforation of lipid bilayers is proposed. It is based on the structural rearrangement of the lipid bilayer formed from disaturated phospholipids at the temperature of the phase transition from the liquid crystalline state to the gel state. This allows us to obtain a lipid pore population without the use of a strong electric field. It is shown that the planar lipid bilayer membrane (pBLM) formed from dipalmitoylphosphatidylcholine in 1 M LiCl aqueous solution exhibits the appearance of up to 50 lipid pores per 1 mm2 of membrane surface, with an average single pore conductivity of 31±13 nS. The estimation of a single pore radius carried out with water-soluble poly(ethylene glycol)s (PEGs) showed that the average pore radius ranged between 1.0–1.7 nm. It was found experimentally that PEG-1450, PEG-2000, and PEG-3350 should be in a position to block the single pore conductivity completely, while PEG-6000 fully restored the ionic conductivity. The similarity of these PEG effects to ionic conductivity in protein pores makes it possible to suggest that the partition of the PEG molecules between the pore and the bulk solution does not depend on the nature of the chemical groups located in the pore wall. 相似文献
2.
Ralf W. Glaser Sergei L. Leikin Leonid V. Chernomordik Vasili F. Pastushenko Artjom I. Sokirko 《生物化学与生物物理学报:生物膜》1988,940(2)
The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated. 相似文献
3.
Reversible electrical breakdown of lipid bilayers: formation and evolution of pores 总被引:14,自引:0,他引:14
R W Glaser S L Leikin L V Chernomordik V F Pastushenko A I Sokirko 《Biochimica et biophysica acta》1988,940(2):275-287
The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated. 相似文献
4.
Electric impulses (1-20 kV cm-1, 1-5 microseconds) cause transient structural changes in biological membranes and lipid bilayers, leading to apparently reversible pore formation ( electroporation ) with cross-membrane material flow and, if two membranes are in contact, to irreversible membrane fusion ( electrofusion ). The fundamental process operative in electroporation and electrofusion is treated in terms of a periodic lipid block model, a block being a nearest-neighbour pair of lipid molecules in either of two states: (i) the polar head group in the bilayer plane or (ii) facing the centre of a pore (or defect site). The number of blocks in the pore wall is the stochastic variable of the model describing pore size and stability. The Helmholtz free energy function characterizing the transition probabilities of the various pore states contains the surface energies of the pore wall and the planar bilayer and, if an electric field is present, also a dielectric polarization term (dominated by the polarization of the water layer adjacent to the pore wall). Assuming a Poisson process the average number of blocks in a pore wall is given by the solution of a non-linear differential equation. At subcritical electric fields the average pore size is stationary and very small. At supercritical field strengths the pore radius increases and, reaching a critical pore size, the membrane ruptures (dielectric breakdown). If, however, the electric field is switched off, before the critical pore radius is reached, the pore apparently completely reseals to the closed bilayer configuration (reversible electroporation ). 相似文献
5.
Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation 总被引:1,自引:0,他引:1
Electropermeabilization, an electric field-induced modification of the barrier functions of the cell membrane, is widely used
in laboratories and increasingly in the clinic; but the mechanisms and physical structures associated with the electromanipulation
of membrane permeability have not been definitively characterized. Indirect experimental observations of electrical conductance
and small molecule transport as well as molecular dynamics simulations have led to models in which hydrophilic pores form
in phospholipid bilayers with increased probability in the presence of an electric field. Presently available methods do not
permit the direct, nanoscale examination of electroporated membranes that would confirm the existence of these structures.
To facilitate the reconciliation of poration models with the observed properties of electropermeabilized lipid bilayers and
cell membranes, we propose a scheme for characterizing the stages of electropore formation and resealing. This electropore
life cycle, based on molecular dynamics simulations of phospholipid bilayers, defines a sequence of discrete steps in the
electric field-driven restructuring of the membrane that leads to the formation of a head group-lined, aqueous pore and then,
after the field is removed, to the dismantling of the pore and reassembly of the intact bilayer. Utilizing this scheme we
can systematically analyze the interactions between the electric field and the bilayer components involved in pore initiation,
construction and resealing. We find that the pore creation time depends strongly on the electric field gradient across the
membrane interface and that the pore annihilation time is at least weakly dependent on the magnitude of the pore-initiating
electric field and, in general, much longer than the pore creation time. 相似文献
6.
Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations
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Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a palmitoyl-oleoyl-phosphatidylcholine bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows us to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four-state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid headgroups on both leaflets, and a second intermediate (prepore), where a polar chain across the bilayer is formed by 3-4 lipid headgroups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 ± 0.15 nm is seen, in favorable agreement with conductance measurements and electrooptical experiments of lipid vesicles. 相似文献
7.
The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer. 相似文献
8.
B. I. Medvedev T. S. Azarashvily Ju. V. Evtodienko A. I. Luk'yanenko L. S. Yagushinskij 《Molecular and cellular biochemistry》1991,48(1):19-23
Summary From the mitochondrial Ca2+-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca2+-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of
CaCl2 is increased 150-200 times. At 10-fold CaCl2 gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms
separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed It is
assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca2−, whereas the function of the lipid component consists in forming channels in the bilayer. 相似文献
9.
B I Medvedev T S Azarashvily A I Evtodienko JuVLuk'yanenko L S Yagushinskij 《Molecular and cellular biochemistry》1982,48(1):19-23
From the mitochondrial Ca2+-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca2+-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl2 is increased 150-200 times. At 10-fold CaCl2 gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed. It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca2+, whereas the function of the lipid component consists in forming channels in the bilayer. 相似文献
10.
Dependence of channel parameters formed by gramicidin A (conductivity and mean life time) on thickness, composition and tension of planar bilayer lipid membranes (BLM) was studied. BLM were obtained from solutions of alpha-monoglycerides of fatty acids in n-alkanes. It has been shown that channel conductivity depends on the length of lipid radical hydrocarbon and is insensitive to the isomerization of lipid and to the change of solvent. There was no direct relationship between the life time, thickness and composition of BLM. Logarithm tau for all the systems studied is proportional to BLM tension, which points to a significant role of surface phenomena in the formation by grammicidine A of a conducting pore in the lipid bilayer. 相似文献
11.
V F Antonov E V Shevchenko E T Kozhomkulov A A Mol'nar 《Biochemical and biophysical research communications》1985,133(3):1098-1103
The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm. 相似文献
12.
D. Bhattacharyya R. Basu A. Ghosh A. Manna A. Nandy P. Nandy 《Biophysical journal》1993,64(2):550-552
The current-voltage characteristics of bilayer lipid membranes of oxidized cholesterol separating two bathing solutions have already been extensively studied under a DC electric field. The observed deviation from linearity at high field has been explained by field-induced pore formation, which then act as ion channels in the membrane. Using thin films of oxidized cholesterol and of dipalmitoyl phosphatidylcholine, we have reported for the first time similar deviation from nonlinearity in the DC I-V characteristics when the applied field is above 40 V/cm. Upon application of an AC field, the conductivity increases as square of frequency, while the nonlinear nature of the I-V characteristic curve is still retained at all frequencies up to 5,000 Hz. Our results indicate that besides pore formation, the intrinsic electrical properties of the constituent lipid molecules are also responsible for the observed nonlinearity. 相似文献
13.
S. A. Akimov V. V. Aleksandrova T. R. Galimzyanov P. V. Bashkirov O. V. Batishchev 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2017,11(3):193-205
Theoretical model of a through pore formation in lipid bilayer membrane under applied lateral tension was developed. In the framework of elastic theory of liquid crystals adapted to lipid membranes, we calculated a continuous trajectory from intact bilayer through a hydrophobic defect to a through pore. It was shown that the major energetic characteristic of membrane stability with respect to the pore formation, i. e., line tension, depends both on the pore radius and on the value of the applied lateral tension. This leads to a non-monotonous dependence of the average waiting time of the pore formation on the lateral tension: at low tensions the waiting time was large, then there was a local minimum, after which the average waiting time was increasing again. For membranes formed from stearoyl oleoyl phosphatidylcholine, the local minimum corresponded to the lateral tension of 7 mN/m; the calculated value of the edge line tension of a large pore was 16.5 pN. These results are consistent with available experimental data. 相似文献
14.
Sarah Y. Kim Ana-Nicoleta Bondar William C. Wimley Kalina Hristova 《Biophysical journal》2021,120(4):618-630
Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ~6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved. 相似文献
15.
The ionic permeability of model bilayer membranes prepared from total lipids of the brain, heart, liver, and muscles of four species of higher vertebrates was studies. The electrical conductivity and potential of membranes breakdown were measured in KCl, NaCl, and LiCl solutions. It was found that the permeability for potassium ions of bilayer lipid membranes from the lipids of nervous tissue decreases in the row of poikilothermal vertebrates and increases in mammals. A reverse regularity was observed for membrane stability, which manifested itself as an increase in membrane breakdown in the order fish-amphibian-reptile and a decrease in membrane breakdown in mammalian membranes. It was shown that, in most cases, the the permeability of bilayer lipid membranes for K+ ions is higher than for Na+ and Li+. 相似文献
16.
Durba Sengupta Hari Leontiadou Alan E. Mark Siewert-Jan Marrink 《生物化学与生物物理学报:生物膜》2008,1778(10):2308-2317
A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism. 相似文献
17.
M I Agadzhanov S A Badzhinian V G Mkhitarian 《Biulleten' eksperimental'no? biologii i meditsiny》1979,87(5):422-425
The membrane conductivity for K+ and Ca+2 ions was studied on bilayer phospholipid membranes formed by phospholipids extracted from the rat liver 1 hour, 1, 3, 7, and 15 days after burning. A pronounced increase in the membrane conductivity was noted. The most potent effects were seen by the 1t, 3d and 7th day of the experiment and the conductivity value varied depending on the medium pH. The process was accompanied by lipid peroxidation increase. Intraperitoneal administration of alpha-tocopherol at 1 mg/kg dose immediately after burning, followed by injections on the 3d, 7th and 12th day, normalized the characteristics studied. Bilayer membranes formed by healthy rat liver phospholipids previously added with an appropriate amount of methyl oleate or cumylhydroperoxide demonstrated higher conductivity as compared to the normal. 相似文献
18.
Fast digital imaging was used to study the deformation and poration of giant unilamellar vesicles subjected to electric pulses. For the first time the dynamics of response and relaxation of the membrane at micron-scale level is revealed at a time resolution of 30 micros. Above a critical transmembrane potential the lipid bilayer ruptures. Formation of macropores (diameter approximately 2 microm) with pore lifetime of approximately 10 ms has been detected. The pore lifetime has been interpreted as interplay between the pore edge tension and the membrane viscosity. The reported data, covering six decades of time, show the following regimes in the relaxation dynamics of the membrane. Tensed vesicles first relax to release the acquired stress due to stretching, approximately 100 micros. In the case of poration, membrane resealing occurs with a characteristic time of approximately 10 ms. Finally, for vesicles with excess area an additional slow regime was observed, approximately 1 s, which we associate with relaxation of membrane curvature. Dimensional analysis can reasonably well explain the corresponding characteristic timescales. Being performed on cell-sized giant unilamellar vesicles, this study brings insight to cell electroporation. The latter is widely used for gene transfection and drug transport across the membrane where processes occurring at different timescales may influence the efficiency. 相似文献
19.
Albrecht Ludwig Angela Schmid Roland Benz Werner Goebel 《Molecular & general genetics : MGG》1991,226(1-2):198-208
Summary By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9–37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI–DIII) located between amino acids 238–410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498–830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670–830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed. 相似文献
20.
O V Krasilnikov R Z Sabirov V I Ternovsky P G Merzliak B A Tashmukhamedov 《General physiology and biophysics》1988,7(5):467-473
Polyethylene glycols (PEG) with molecular weight less than or equal to 3000 were shown to effectively protect human erythrocytes from osmotic lysis induced by alpha-staphylotoxin (ST). PEG with MW less than 3000 do not change the conductivity of ion channels induced by ST in bilayer lipid membranes (BLM). Changing the bilayer from a pure phosphatidylcholine (PC) to a negatively charged phosphatidylserine (PS) film results in an asymmetry of the current-voltage characteristics. This is evidenced by the asymmetrical position of the ST-channel pore in bilayer membranes. The results obtained allow to conclude that the ST-channel is an interprotein pore filled with water (with an inner diameter of 2.5-3 nm and a length of approximately 10 nm). It is composed of six molecules of alpha-toxin from Staphylococcus aureus. The ST-channel incorporates into a membrane with only one mouth in contact with the polar lipid heads and the other one protruding 4.5-5 nm from the bilayer plane in water solution. 相似文献