Permeability of small nonelectrolytes through lipid bilayer membranes

Summary Diffusion of small nonelectrolytes through planar lipid bilayer membranes (egg phosphatidylcholine-decane) was examined by correlating the permeability coefficients of 22 solutes with their partition coefficients between water and four organic solvents. High correlations were observed with hexadecane and olive oil (r=0.95 and 0.93), but not octanol and ether (r=0.75 and 0.74). Permeabilities of the seven smallest molecules (mol wt <50) (water, hydrofluoric acid, hydrochloric acid, ammonia, methylamine, formic acid and formamide) were 2- to 15-fold higher than the values predicted by the permeabilities of the larger molecules (50相似文献   

Monocarboxylic acid permeation through lipid bilayer membranes

Summary The membrane permeability coefficients for the homologous monocarboxylic acids, formic through hexanoic, as well as benzoic and salicylic, were determined for egg phosphatidylcholine-decane planar bilayer membranes. The permeabilities of formic, acetic and propionic acid were also determined for solvent-free phosphatidylethanolamine bilayers. Permeability coefficients were calculated from tracer fluxes measured under otherwise symmetrical conditions, and precautions were taken to ensure that the values were not underestimated due to unstirred layer effects. The relation between the nonionic (HA) permeability (P ^m ) and the hexadecane/water partition coefficient (K _p ) was: log ^m =0.90 log K_p+0.87 (correlation coefficient=0.996). Formic acid was excluded from the analysis because its permeability was sixfold higher than predicted by the other acids. The permeabilities for solvent-free membranes were similar to those for decanecontaining membranes. The exceptionally high permeability of formic acid and the high correlation of the other permeabilities to the hexadecane/water partition coefficient is a pattern that conforms with other nonelectrolyte permeabilities through bilayers. Similarly, the mean incremental free energy change per methylene group (G-CH₂-) was –764 cal mol^–1, similar to other homologous solutes in other membrane systems. However, much less negative G values (–120, to –400 cal mol^–1) were previously reported for fatty acids permeating bilayers and biological membranes. These values are due primarily to unstirred layer effects, metabolism and binding to membranes and other cell components.     

Proton/hydroxide conductance through lipid bilayer membranes

Summary A simple method of measuring proton/hydroxide conductance (G _H/OH) through planar lipid bilayer membranes is described. First the total conductance (G _m ) is measured electrically. Then the H⁺/OH^– transference number (T _H/OH) is estimated from the diffusion potential (V _m ) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H⁺ and OH^–. Thus,V _m is due entirely to H⁺/OH^– diffusion andG _H/OH can be calculated from the relations,V _m =T _H/OH E _H/OH andG _H/OH=T _H/OH G _m , whereE _H/OH is the equilibrium potential for H⁺ and OH^–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G _H/OH is nearly independent of pH, ranging from about 10^–9 S cm^–2 at pH 1.6 to 10^–8 S cm^–2 at pH 10.5. BecauseG _H/OH is nearly independent of pH, the calculated permeability coefficients to H⁺ and/or OH^– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G _H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG _H/OH.G _H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H⁺/OH^– conductance is not known, the relation betweenG _H/OH and water activity suggests that several water molecules are involved in the H⁺/OH^– transport process.     

On the interaction of hemocyanin with lipid bilayer membranes

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.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - DOC sodium deoxycholate     

The breakdown of bilayer lipid membranes by dendrimers

The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.     

Electrically silent anion transport through bilayer lipid membrane induced by tributylin and triethyllead

Summary The method of the measurement of the nonelectrogenic fluxes of hydrogen (or hydroxyl) ions (J _H) based on the local proton gradients formation in the unstirred layers near a bilayer lipid membrane (BLM) is applied for recording the nonelectrogenic anion/OH^– exchange on BLM induced by tributyltin (TBT) and a novel carrier (Hager, A., Moser, I., & Berthold, W. 1987.Z. Naturforsch.,42C1116–1120), triethyllead (TEL). This method has been used previously for measuring the cation fluxes through BLM. TBT and TEL are shown to be equally efficient in the induction of Cl^–/OH^– exchange.J _H induced by TBT is constant at 4J _H decreases at pH<4 and pH>7. Both ionophores have a transport sequence: I^–> Br^–>Cl^–>F^–. The quatitative measurements reveal that TEL better discriminates these four anions than TBT. It is concluded that this method may prove helpful in a search and study of anion/OH^–-exchangers isolated from natural membranes.     

Effect of weak inorganic acids and lower carboxylic acids on the conductivity of bilayer lipid membranes

The ability of weak inorganic acids (H₂S, HCN) and lower carboxylic acids to interact with bilayer lipid membranes, change their conductivity, and act as protonophores has been investigated. The mechanism of changes in membrane conductivity was studied. Factors influencing the interaction of acids with model lipid membranes were determined. Maximum changes in conductivity were observed at pH values equal to the dissociation constants of weak acids and correlated with the octanol-water partition coefficients.     

Electrical conductivity of bilayer lipid membranes in electrolytic solution

The electrical conductivity of bilayer lipid membranes (BLM) of oxidized cholesterol has been measured separately in bathing solutions of sodium sulphate, sodium chloride, sodium bromide, sodium iodide and also in bathing solutions of iodine and iodine containing these salts. An attempt has been made to explain the conduction of electric current across the membranes.     

Histamine,theophylline and tryptamine transport through lipid bilayer membranes

Diffusion of histamine, theophylline and tryptamine through planar lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine plus cholesterol (1 : 1 mol ratio) in tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to nonionic and ionic species. Only the nonionic forms crossed the membrane at a significant rate. The membrane permeabilities to the nonionic species were: histamine, $\text{3.5 · 10}{}^{\mathrm{?5}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; theophylline, $\text{2.9 · 10}{}^{\mathrm{?4}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; and tryptamine, $\text{1.8 · 10}{}^{\mathrm{?1}}\text{cm}\text{· s}{}^{\mathrm{?1}}$. Chemical reactions in the unstirred layers are important in the transport of tryptamine and theophylline, but not histamine. For example, as pH decreased from 10.0 to 7.5 the ratio of nonionic (B) to ionic (BH⁺) tryptamine decreased by 300-fold, but the total tryptamine permeability decreased only 3-fold. The relative insensitivity of the total tryptamine permeability to the ratio, [B]/[BH⁺], is due to the rapid interconversion of B and BH⁺ in the instirred layers. Our model describing diffusion and reaction in the unstirred layers can explain some ‘anomalous’ relationships between pH and weak acid/base transport through lipid bilayer and biological membranes.     

On the transport noise of hydrophobic ions in lipid bilayer membranes

The effect of transit time on the electrical transport noise of a closed one-barrier model at equilibrium as proposed by Kolb and Läuger [6] is studied using the master-equation approach. A transit time is the time for an ion to cross the energy barrier (membrane interior) when the energy of the ion reaches the barrier height. Both the time correlation function and the noise power spectrum are obtained as functions of the transit time of the ions. Possible effects of transit time on the time correlation function of transport of dipicrylamine ions in lipid bilayers as reported by Bruner and Hall [13] and on the noise power spectrum as reported by Kolb and Läuger [6] are discussed.     

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.     

The induction by protons of ion channels through lipid bilayer membranes

The appearance of ion channels was induced in phospholipid bilayers by acidification of the bulk solution on one side of the bilayer. by addition of HCl. acetic acid or by hydrolytic production of protons using purified acetylcholinesierase. Further acidification below an apparent critical pH range led to restoration of a low conductance state similar to that seen at neutral pH. Such experiments were performed with a heterogeneous soybean lecithin extract, with homogeneous synthetic di-phytanoylphosphatidylcholine, and with a mixture of cholesterol and synthetic dioleoylphosphatdylcholine. It is proposed that the physical mechanism for this phenomenon involves fluctuations of lipid order induced by fluctuations in protnation of phospholipid head groups within a critical pH range; these, in turn, create conductive defect in the two-dimensional lattice of the lipid bilayer.     

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.     

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Pressure variation of the lateral diffusion in lipid bilayer membranes

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.     

Millimeter microwave effect on ion transport across lipid bilayer membranes

The effects of millimeter microwaves in the frequency range of 54–76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB_?). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% ± 0.5%. At the same time, membrane current induced by TPhB transport increased by 5% ± 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% ± 0.4%). No “resonance-like” effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 °C. © 1995 Wiley-Liss, Inc.     

Effects of the plant alkaloid sanguinarine on cation transport by human red blood cells and lipid bilayer membranes

Summary The plant alkaloid, sanguinarine, inhibits the ouabain-sensitive K–Na pump and increases the downhill, ouabain-insensitive movements of K and Na in human red cells. These two effects have different temporal and concentration dependencies and are mediated by two different chemical forms of sanguinarine. The oxidized, charged form (5×10^–5 m) promptly inhibits the pump but does not affect leakage of K and Na. The reduced, uncharged form of sanguinarine causes lysis of red cells but does not inhibited the pump. Sanguinarine also increases the conductance of bilayers formed from sheep red cell lipids. The effect is produced by the uncharged but not by the charged form of sanguinarine. Bilayer conductance increases as the fourth power of sanguinarine concentration when the compound is present on both sides of the membrane and as the second power of concentration when present on only one side. Conductance also increasee-fold for each 34 mV increase in the potential difference imposed across the membrane. The results suggest that the uncharged forms of sanguinarine produce voltage-dependent channels in bilayers.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

Temperature dependence of water self-diffusion through lipid bilayers assessed by NMR

The temperature dependence of the coefficient of water self-diffusion across plane-parallel multib-ilayers of dioleoylphosphatidylcholine oriented on a glass support was studied in the 20–60°C range by pulsed field gradient NMR. The coefficient for transbilayer diffusion of water proved almost four orders of magnitude smaller than for bulk water, and 10 times smaller than that for lateral diffusion of lipid under the same conditions. The temperature dependence obeyed the Arrhenius law with apparent activation energy of 41 kJ/mol, much higher than that for bulk water (18 kJ/mol). The experimental data were analyzed using the “dissolution-diffusion” model, by simulating water passage through membrane channels, and by examining water exchange in states with different modes of translational mobility, including pore channels and bilayer defects. Each approach could take into account the role of bilayer permeability and assess the apparent activation energy for water diffusion in the hydrophobic part of the bilayer, which proved close to the value for bulk water. Estimates were obtained for water diffusion coefficients in the system, coefficients of bilayer permeability for water, and the influence of bilayer defects on the lateral and transverse diffusion coefficients.