Proton permeation of lipid bilayers

Proton permeation of the lipid bilayer barrier has two unique features. First, permeability coefficients measured at neutral pH ranges are six to seven orders of magnitude greater than expected from knowledge of other monovalent cations. Second, proton conductance across planar lipid bilayers varies at most by a factor of 10 when pH is varied from near 1 to near 11. Two mechanisms have been proposed to account for this anomalous behavior: proton conductance related to contaminants of lipid bilayers, and proton translocation along transient hydrogen-bonded chains (tHBC) of associated water molecules in the membrane. The weight of evidence suggests that trace contaminants may contribute to proton conductance across planar lipid membranes at certain pH ranges, but cannot account for the anomalous proton flux in liposome systems.Two new results will be reported here which were designed to test the tHBC model. These include measurements of relative proton/potassium permeability in the gramicidin channel, and plots of proton flux against the magnitude of pH gradients. (1) The relative permeabilities of protons and potassium through the gramicidin channel, which contains a single strand of hydrogenbonded water molecules, were found to differ by at least four orders of magnitude when measured at neutral pH ranges. This result demonstrates that a hydrogen-bonded chain of water molecules can provide substantial discrimination between protons and other cations. It was also possible to calculate that if approximately 7% of bilayer water was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. (2) The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result that is consistent with one of three alternative tHBC models for proton conductance described by Nagle elsewhere in this volume.     

Faster-than-anticipated Na/Cl diffusion across lipid bilayers in vesicles

Maintenance of electrochemical potential gradients across lipid membranes is critical for signal transduction and energy generation in biological systems. However, because ions with widely varying membrane permeabilities all contribute to the electrostatic potential, it can be difficult to measure the influence of diffusion of a single ion type across the bilayer. To understand the electrodiffusion of H⁺ across lipid bilayers, we used a pH-sensitive fluorophore to monitor the lumenal pH in vesicles after a stepwise change in the bulk pH. In vesicles containing the ion channel gramicidin, the lumenal pH rapidly approached the external pH. In contrast, the lumen of intact vesicles showed a two stage pH response: an initial rapid change occurred over ~ 1 min, followed by a much slower change over ~ 24 h. We provide a quantitative interpretation of these results based on the Goldman–Hodgkin–Katz ion fluxes discharging the electrical capacitance of the bilayer membrane. This interpretation provides an estimate of the permeability of the membranes to Na⁺ and Cl⁻ ions of ~ 10^− 8 cm/s, which is ~ 3 orders of magnitude faster than previous reports. We discuss possible mechanisms to account for this considerably higher permeability in vesicle membranes.     

Lateral diffusion and percolation in two-phase, two-component lipid bilayers. Topology of the solid-phase domains in-plane and across the lipid bilayer.

Fluorescence recovery after photobleaching (FRAP) has recently been used to examine the percolation properties of coexisting phases in two-component, two-phase phosphatidylcholine bilayers [Vaz, W. L. C., Melo, E. C. C., & Thompson, T. E. (1989) Biophys. J. 56, 869-876]. We now report the use of FRAP to study two additional problems in similar systems. The first is the effect of solid-phase obstacles on the lateral diffusion in the fluid phase. The second is the question of whether or not, in a single bilayer, solid-phase domains in one monolayer are exactly superimposed on solid domains in the apposing monolayer. To address the first problem, the lateral diffusion of N-(7-nitrobenzoxa-2,3-diazol-4-yl)-1-palmitoyl-2-oleoylphosp hatidylethanolamine (NBD-POPE), a probe soluble only in the fluid phase when solid and fluid phases coexist, has been studied in the mixture N-lignoceroyldihydrogalactosylceramide (LigGalCer)/dipalmitoylphosphatidylcholine (DPPC). Percolation of the fluid phase occurs at a high mass fraction of solid phase. This indicates that the solid domains have a centrosymmetric shape, a characteristic which makes this a good experimental system to test theoretical simulations of diffusion in an archipelago. It is shown that agreement between theory and experiment is poor, a result that had already been observed when the obstacles were integral membrane proteins. We develop an effective-medium model for diffusion in two-phase systems which explains both our results and those obtained with integral proteins. The distinctive feature of the model is the consideration of an annular region around the obstacles where the lipids are more ordered than in the bulk fluid phase. The diffusion coefficient is then calculated by extending the free area model to two-phase systems, taking these annuli into account. The second question, the organization of the solid-phase domains across the lipid bilayer, is examined in the systems LigGalCer/DPPC and dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) by comparing the diffusion of a fluid-phase-soluble, gel-phase-insoluble lipid derivative which spans the two monolayers of a bilayer (NBD-membrane-spanning-phosphatidylethanolamine, NBD-msPE) with that of a probe which is restricted to a single monolayer. In LigGalCer/DPPC, 20:80, the distribution of solid domains in one of the monolayers is independent of the distribution in the apposing monolayer. In contrast, in DMPC/DSPC, 50:50, the solid domains in one monolayer are exactly superimposed upon the solid domains existing in the apposing monolayer.     

Computer simulation of small molecule permeation across a lipid bilayer: dependence on bilayer properties and solute volume, size, and cross-sectional area

Cell membrane permeation is required for most drugs to reach their biological target, and understanding this process is therefore crucial for rational drug design. Recent molecular dynamics simulations have studied the permeation of eight small molecules through a phospholipid bilayer. Unlike experiments, atomistic simulations allow the direct calculation of diffusion and partition coefficients of solutes at different depths inside a lipid membrane. Further analyses of the simulations suggest that solute diffusion is less size-dependent and solute partitioning more size-dependent than was commonly thought.     

State of water and its diffusion across lipid bilayers: effect of hydration degree

The state of adsorbed water (estimated from the dependence of the shape of the 1H NMR spectrum on the angle between the normal to the bilayers and the direction of the magnetic field) and the diffusion of water molecules in the direction of the normal to the bilayers (estimated by 1H NMR spectroscopy with the impulse gradient of magnetic field) in microscopically oriented dioleoylphosphatidylcholine bilayers have been studied depending on hydration. The dependences of the shape of the NMR spectrum on angle differ qualitatively only at concentrations of water greater and less than the concentration that is achieved upon hydration from saturated vapors chi(eq) (about 23 weight %). At concentrations below chi(eq), all water present in samples enters the hydrate shells of polar "heads" of lipids or is in the state of "rapid exchange" with the water of hydrate shells, with the result that the signal of spin echo for water is observed only in a narrow range of angles close to the "magic angle", 54 degrees C. At concentrations above xhi(eq), the signal of spin echo for water is retained at all orientations, indicating probably that part of water between the bilayers ("quasi-free water") is in the state of a "slow exchange" with water "bound" to polar "heads". It was found that the coefficient of self-diffusion of water across the system of bilayers inversely depends on the degree of hydration, which is described in the Tanner model with consideration of the self-diffusion of water molecules in the hydrophobic moiety of the bilayer. The permeability of the bilayer, the coefficient of distribution of molecules between the water and lipid phases, and the coefficient of self-diffusion of water in the hydrophobic moiety of the bilayer were estimated.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

Real-time measurement of solute partitioning to lipid monolayers

The interaction of a peripheral protein with a lipid-water interface can show a pronounced dependence on the composition and two-dimensional packing density of the lipids that comprise the interface. We report a novel optical method for measuring the adsorption of macromolecules, such as proteins and nucleic acids, and smaller solutes, such as drugs, to lipid monolayers at the gas-liquid interface. Using fluorescence emission from proteins and a small molecule, we demonstrate that the emissions from these solutes when in the aqueous phase and when associated with the monolayer can be temporally separated. Such separation allows measurement of the extent of solute adsorption, spectral characterization of the adsorbed solute, and characterization of lipid organization using adsorption kinetics. The method does not require, but is compatible with, the solute having different spectral properties in the bulk and surface phases. Indeed, if optical signals from adsorbed and soluble solute are the same or their relationship is known, absolute surface excess of adsorbed solute can be calculated without independent calibration. With appropriate instrumental configuration, the method should be adaptable for screening solutes for interaction with planar monolayers having both well-defined composition and adjustable lipid packing density.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.     

Ionic selectivity of melittin-modified flat lipid bilayers]

It has been established in experiments with the bilayer lipid membranes (BLM) that at pH greater than 6.6 the melittin pores are cation-selective and at lower pH they are more selective by anions. The property of melittin pores is shown to be provided by the amino group of the N-terminal glycine residue. The selectivity of melittin-containing membranes may be controlled by the transmembrane potential, the cross-section of water pores being changed. The data obtained are explained within the alimethicine-like model.     

Shear stress-facilitated calcium ion transport across lipid bilayers.

Small unilamellar liposomes were used in this study of shear stress effects on the trans-bilayer flux of calcium ions (Ca2+). Liposome suspensions were prepared from 99% egg phosphatidylcholine by a microporous filter extrusion technique. The inner aqueous phase of the unilamellar liposomes contained indo-1(5-), a fluorescent indicator of free Ca2+. The external aqueous phase was composed of Hepes-buffered saline containing normal physiological levels of common ionic species. Calcium ion levels were set at 100 nM and 1 mM in the inner and outer aqueous phases, respectively. Liposome suspensions were exposed to graded levels of uniform shear stress in an optically modified rotational viscometer. Intraliposome Ca2+ concentration was estimated from continuous measurement of indo-1(5-) fluorescence. Electronically measured particle size distribution was used to determine liposome surface area for estimation of trans-bilayer Ca2+ flux. Trans-bilayer Ca2+ flux increased linearly with applied shear rate from 27 s-1 to 2700 s-1. Diffusional resistance of the lipid bilayer, not the convective resistance of the surrounding fluid, was the limiting step in the transport of Ca2+. Liposome permeability to Ca2+ increased by nearly two orders of magnitude over the physiologically relevant shear rate range studied. Solute transport in injectable liposome preparations may be dramatically influenced by cardiovascular fluid stress. Solute delivery rates determined in liposomes exposed to static conditions may not accurately predict in vivo, cardiovascular solute transport.     

Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers

The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.     

On the mechanism of non-electrolyte permeation through lipid bilayers and through biomembranes

The temperature effects on the permeation of polyhydroxy alcohols through the lipid bilayers of liposomes with a great variety in chemical composition were studied. Although important differences in the permeability of the various lipid bilayers were observed, Arrhenius plots demonstrated that the activation energy is independent of the degree of unsaturation or the presence of cholesterol in the paraffin barriers. The activation energies found for the penetration of a bilayer with a liquid paraffin core are 14.3 kcal for glycol, 19.4 kcal for glycerol, and 20.8 kcal for erythritol. These values are in agreement with the energies that can be expected for complete dehydration of the permeant molecules. The idea that the activation energy is determined by the number of hydrogen bonds with water is supported by the finding that a series of different diols did demonstrate practically identical activation energies. Studies on a number of biological membranes demonstrated the same activation energies for the penetration of glycerol and erythritol as found in the experiments with liposomes. These facts support the view that both the lipid bilayers and the biological membranes are penetrated by single fully dehydrated molecules.     

Equilibrium gel permeation: measurement of solute partitioning by direct fluorescence scanning

The feasibility of using fluorescence detection in quantitative gel permeation measurements has been explored. It is shown that the effect of scattering by the gel matrix can be evaluated in terms of pathlength-dependent turbidity functions for excitation and emission wavelengths. Experimental studies were carried out to evaluate these functions in cross-linked dextran gels (Sephadexes) and in agarose gels (Sepharoses). Empirical turbidity functions derived for these gels have a simple form, leading to accurate simplifying approximations for the scattering correction required in a fluorescence gel permeation measurement. Using this approach, partition cross-sections were estimated for dansyl-conjuaated β-gamma globuline and for dansyl-conjugated bovine serum albumin. The results establish feasibility of the method and clearly indicate the instrumentation requirements for its accurate implementation.     

Energetics and dynamics of SNAREpin folding across lipid bilayers

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).     

The transport of hydrophobic ions across lipid bilayers

The three-capacitor model for hydrophobic ion adsorption in lipid membranes (Andersen, O.S., Feldberg, S., Nakadomari, H., Levy, S. and McLaughlin, S. (1978) Biophys. J. 21, 35-70) is extended to ion transport whereby electrostatic effects from the interfacially adsorbed hydrophobic space charge have been encountered. The phenomena of current saturation with increasing concentrations of hydrophobic ions in the bulk electrolyte and the associated increase of the time constant of current relaxation can be quantitatively understood on the basis of space charge-limited currents as well as the nonexponential current decay.     

Interaction of the influenza hemagglutinin fusion peptide with lipid bilayers: area expansion and permeation.

Fusion is a crucial event in the infection of animal cells by enveloped viruses (e.g., HIV or influenza). Viral fusion is mediated by glycoproteins, spanning the viral envelope, which attach to a membrane surface and induce fusion of the viral envelope to the cellular membrane. Influenza fusion protein (hemagglutinin) contains an amino-terminal segment critical to fusion, referred to as the fusion peptide. We show here that the native fusion peptide (wt-20) of hemagglutinin destabilizes membranes formed of 99% 1 -stearoyl-2-oleoylphosphatidylcholine (SOPC). The first step in destabilization is rapid insertion of the peptide into the membrane, in which membrane area increases by as much as 11% in just seconds. We visualized and quantified the area expansion by using optical video microscopy combined with micropipette aspiration. This rapid membrane area expansion is followed by the formation of membrane defects in the size range of 0.5 nm, and results in membrane rupture. Both the rate of area increase and maximum area increase are significantly higher at a pH near 5.0 compared to pH 7.0. These results suggest that enhanced membrane insertion of wt-20 and accompanying area expansion at pH 5.0 are responsible for the relatively greater lytic activity at this pH. We show that a deletion of the N-terminal glycine of wt-20 results in a lack of area expansion or membrane perturbation at pH 5.0.     

The permeation of organic acids through lecithin bilayers. Resemblance to diffusion in polymers.

1. The fluxes of aliphatic acids and their derivatives through black lipid membranes made of egg lecithin in decane were measured by means of a proton titration method. 2. Permeability coefficients were calculated and these were divided by the partition coefficient of the diffusing solute in different solvent systems: n-decane, olive oil, ether and octanol. The logarithms of the diffusion coefficients thus obtained were plotted against the logarithm of the molecular weight. The data could not be fitted to a single regression line in any solvent system. 3. When the logarithm of the diffusion coefficients were correlated to the logarithm of the molecular volume (equals molecular weight/ specific gravity) all the diffusants could be fitted to the same regression line, indicating that the molecular volume is a better index of molecular size and shape than the molecular weight. 4. Analysis of the experimental results assuming a model of diffusion through soft polymers (Lieb, W.R. and Stein, W. D. (1971) Current Topics in Membranes and Transport, vol. 2, pp. 1-39, Academic Press, New York) showed that decane and olive oil are not adequate model solvents for planar lecithin membranes but ether and octanol are good models. 5. The differential mass selectivity coefficient was found to be similar to that for soft polymers and biological membranes, i.e. greater than 3.0. 6. Water could be fitted by the same regression line, thus emphasizing the generality of passive transfer and implying that water crosses lipid membranes as single molecules.     

Tributyltin-mediated exchange diffusion of halides in lipid bilayers

This paper describes the effect of tributyltin (TBT) on the inorganic anion permeability of lipid bilayers. When this compound is added in micromolar concentrations to one or both sides of a phosphatidyl ethanolamine (PE) membrane formed in 0.1 M NaCl or KCl (pH 7), there is no change in the electrical conductance. Under these circumstances, the Cl self-exchange flux measured with 36Cl (MCl) increases from a value of approximately 10(-12) mol.cm-2.s-1, to approximately 10(-8) mol.cm-2.s-1. It was further found that the relation between chloride flux and [TBT] and [Cl] can be described as: MCl = B[TBT] [Cl]. When chloride was replaced by an equimolar concentration of different univalent anions in the trans compartment, the heteroexchange flux of chloride followed the sequence: I greater than Br greater than Cl greater than F greater than NO3. Under all experimental conditions tested, the chloride flux was always more than 10(3) times the maximum flux predicted from the value of the membrane conductance, and at least 100 times higher than the expected fluxes of ion pairs (TBT-Cl) diffusing across the unstirred layers. Thus, the mechanism by which tributyltin increases anion permeability in bilayers seems to be that of an obligatory exchange diffusion, with the reaction between tributyltin and the halides occurring at the membrane surface. Measurements of interfacial potentials indicate that tributyltin chloride lowers the positive intrinsic dipole potential of PE membranes by approximately 70 mV (at a TBT concentration of 30 microM) without substantial alteration of other parameters of the bilayer. The estimated adsorption coefficient of TBT-Cl was found to be 3 x 10(-4) cm.