Water permeability of gramicidin A-treated lipid bilayer membranes

In membranes containing aqueous pores (channels), the osmotic water permeability coefficient, P f, is greater than the diffusive water permeability coefficient, P d. In fact, the magnitude of P f/P d is commonly used to determine pore radius. Although, for membranes studied to date, P f/P d monotonically declines with decreasing pore radius, there is controversy over the value it theoretically assumes when that radius is so small that water molecules cannot overtake one another within the channel (single-file transport). In one view it should equal 1, and in another view it should equal N, the number of water molecules in the pore. Gramicidin A forms, in lipid bilayer membranes, narrow aqueous channels through which single-file transport may occur. For these channels we find that P f/P d approximately 5. In contrast, for the wider nystatin and amphotericin B pores, P f/P d approximately 3. These findings offer experimental support for the view that P f/P d = N for single-file transport, and we therefore conclude that there are approximately five water molecules in a gramicidin A channel. A similar conclusion was reached independently from streaming potential data. Using single-channel conductance data, we calculate the water permeability of an individual gramicidin A channel. In the Appendix we report that there is a wide range of channel sizes and lifetimes in cholesterol-containing membranes.     

Water and nonelectrolyte permeability of lipid bilayer membranes

Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n- butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility- diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).     

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 interaction of anionic detergents with lipid bilayer membranes

Summary The time course of the reaction of anionic surfactants with lipid bilayers is followed and analyzed. The distribution of detergents in the membrane phase gives rise to an asymmetry potential followed by a diffusion potential. Detergent-doped membranes are cation-permselective. It is postulated that a variable profile of mobile charges in the membrane account for the cation-permselectivity, the intercation selectivity, and the voltage-dependent gating phenomena observed in excitable membranes.Supported by a grant from the Medical Research Council of Canada.I thank Mr. G. Beauchesme for his technical assistance in part of this work.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

Influence of dolichyl phosphate on permeability and stability of bilayer lipid membranes

The ionic permeability coefficients, ionic transference numbers, activation energy of ion transport and breakdown voltage of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl 12-phosphate have been studied. The electrical measurements showed that dolichyl phosphate in phospholipid bilayers decreases membrane permeability, changes membrane ionic selectivity and increases membrane stability. These results are discussed in light of the aggregation behavior and the intramolecular clustering of a dolichyl phosphate molecule in phospholipid membranes. From our data we suggest that the hydrophilic part of dolichyl phosphate molecules regulates their behavior in membranes.     

The interaction of n-alkanols with lipid bilayer membranes: a 2H-NMR study

The interaction of eight n-alkanols with bilayers of dimyristoylphosphatidylcholine (DMPC) has been studied by deuterium nuclear magnetic resonance (2H-NMR). At comparable temperatures and concentrations of solute in the bilayer, order parameters measured at the 1-methylene segment of the n-alkanols show a maximum for n-dodecanol. For both n-dodecanol and n-tetradecanol, orientational ordering shows a maximum at the C-4 to C-7 methylene segments, with labels at both ends of the n-alkanol exhibiting reduced order. These observations are consistent with earlier findings for n-octanol and n-decanol. Unlike the longer chain n-alkanols, ordering in n-butanol decreases from the hydroxyl group end to the methyl group end of the molecule. Orientational ordering at nine inequivalent sites in DMPC, has also been measured as a function of temperature, for bilayers containing n-butanol, n-octanol, n-dodecanol and n-tetradecanol. At the 3R,S sites on the glycerol backbone, for comparable temperatures and solute concentrations, n-butanol produces a larger disordering than the other n-alkanols. This result probably reflects the greater fraction of time spent by the hydroxyl group of n-butanol in the vicinity of the lipid polar head group compared with the hydroxyl group in longer chain n-alkanols. It was found that n-octanol orders the acyl chains of DMPC, unlike n-butanol which disorders them, and the longer chain n-alkanols which have little effect. Within experimental error, the effect of n-dodecanol on order at all sites in DMPC is the same as n-tetradecanol. The influence of n-alkanols on DMPC ordering at twelve sites has been compared with that of cholesterol which is shown to interact with DMPC bilayers in a distinctly different manner from the n-alkanols.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

The action of gramicidin S on the ionic permeability of bilayer lipid membranes

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.     

Structural rearrangements induced by glycerol increase the permeability of bilayer lipid membranes for amphotericin

It has been shown that structural rearrangements induced by glycerol in bilayer lipid membranes (BLM) containing cholesterol facilitate the transmembrane transport of amphotericin B molecules in the direction of glycerol gradient. The addition of amphotericin B to the same side with glycerol results in a change in bilayer selectivity from the cation to the anion one. Besides, the final conductivity is blocked by tetraethylammonium from the solution with no amphotericin B added. It testifies to the transport of amphotericin molecules to the opposite side of the membrane. The transport effect depends on the cholesterol content in bilayer, ionic strength of the medium and slightly depends on temperature. It is concluded that transport of amphotericin B in such conditions differs from the diffusive one and is due to the formation of intermediate lipid phases in the course of structural rearrangements of bilayers.     

Study of the interaction of myoglobin with lipid bilayer membranes by potentiodynamic method

For modeling the interaction of myoglobin with mitochondrial membranes, the adsorption of different ligand forms, the physiologically active reduced MbO₂ and inactive oxidized met-Mb, on one of the surfaces of artificial bilayer lipid membrane (BLM) was studied using a potentiodynamic technique known as the “capacity minimization” method. As mitochondrial membranes are negatively charged, BLM of the negatively charged palmitoyl-2-oleyl-phosphatidyl glycerol (POPG) and neutral soybean phosphatidylcholine (lecithin) were used. It is shown that both myoglobins strongly interact with BLM in the pH range 6–8. The dependence of the potential difference between cis-and trans-surfaces of the lipid membrane (ΔE, mV) on the protein concentration is characteristic of the Langmuir adsorption isotherm, and the saturation level (ΔE _max, mV) corresponds to monolayer of myoglobin. The protein adsorption is essentially electrostatic in nature, as adsorption activity increases sharply in the case of the membrane from POPG: ∼15-fold in the case of MbO₂ and ∼2.5 times for met-Mb. The parameters of the MbO₂ and met-Mb adsorption on BLM of lecithin and POPG do not change in the pH 6–8 range. It can be assumed that the anionic groups of phospholipids associate with the cationic groups of the protein, the charge state of those does not change in the pH 6–8 range. The most likely candidates for interaction with phospholipids of BLM are invariant lysines and arginines in the environment of the myoglobin heme cavity.     

Study of the interaction of myoglobin with lipid bilayer membranes by potentiodynamic method

For modeling the interaction of myoglobin with mitochondrial membranes, the adsorption of different ligand forms, the physiologically active reduced MbO2 and inactive oxidized met-Mb, on one of the surfaces of artificial bilayer lipid membrane (BLM) was studied using potentiodynamic technique known as the "capacity minimization" method. As mitochondrial membranes are negatively charged, BLM from the negatively charged palmitoyl-2-oleil-phosphatidyl glycerol (POPG) and neutral soybean phosphatidylcholine (lecithin) were used. It is shown that both myoglobins strongly interact with BLM in the pH range 6-8. The dependence of the potential difference between cis-and trans-surfaces of the lipid membrane (deltaE, mV) on the protein concentration is characteristic for the Langmuir adsorption isotherm, and the saturation level (deltaEmax, mV) corresponds to monolayer of myoglobin. The protein adsorption is essentially electrostatic in nature, as adsorption activity increases sharply in the case of the membrane from POPG: in a approximately 15-fold in the case of MbO2 and in a approximately 2.5 times for the met-Mb. The parameters of the MbO2 and met-Mb adsorption on BLM from lecithin and POPG do not change in the pH 6-8 range. It can be assumed that the anionic groups of phospholipids associate with the cationic groups of the protein, the charge state of those does not change in the pH 6-8 range. The most likely candidates for interaction with phospholipids of BLM are invariant lysines and arginines in the environment of the myoglobin heme cavity.