Determination of rate constants in carrier-mediated diffusion through lipid bilayers

In this work data are presented on the relaxation current, under a voltage step, through soybean lipid bilayers in the presence of the carrier valinomycin. Measurements of voltage-dependent steady-state conductance have also been performed. These measurements are sufficient to calculate the full set of kinetic parameters determining the transport.The data are analyzed according to the kinetic model, based on an Eyring treatment of the carrier-mediated diffusion. Complementary measurements of conductance as a function of antibiotic concentration have also been reported. These data allow one to calculate the membrane-solution partition coefficient of the carrier and the surface charge density of the membrane. The results are compared with those previously obtained with membranes of different lipid composition.     

A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers

The transmembrane profile of oxygen solubility and diffusivity in a lipid bilayer was assessed by (13)C NMR of the resident lipids (sn-2-perdeuterio-1-myristelaidoyl-2-myristoyl-sn-glycero-3-phosphocholine) in combination with molecular dynamics (MD) simulations. At an oxygen partial pressure of 50 atm, distinct chemical shift perturbations of a paramagnetic origin were observed, spanning a factor of 3.2 within the sn-1 chain and an overall factor of 10 from the headgroup to the hydrophobic interior. The distinguishing feature of the (13)C NMR shift perturbation measurements, in comparison to ESR and fluorescence quenching measurements, is that the local accessibility of oxygen is achieved for nearly all carbon atoms in a single experiment with atomic resolution and without the use of a probe molecule. MD simulations of an oxygenated and hydrated lipid bilayer provided an immersion depth distribution of all carbon nuclei, in addition to the distribution of oxygen concentration and diffusivity with immersion depth. All oxygen-induced (13)C NMR chemical shift perturbations could be reasonably approximated by simply accounting for the MD-derived immersion depth distribution of oxygen in the bilayer, appropriately averaged according to the immersion depth distribution of the (13)C nuclei. Second-order effects in the paramagnetic shift are attributed to the collisionally accessible solid angle or to the propensity of the valence electrons in the vicinity of a given nuclear spin to be polarized or delocalized by oxygen. A method is presented to measure such effects. The excellent agreement between MD and NMR provides an important cross-validation of the two techniques.     

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.     

Quasielastic neutron scattering measurements of fast local translational diffusion of lipid molecules in phospholipid bilayers

Quasielastic incoherent neutron scattering has been used to investigate the rate of local translational diffusion of lipid molecules in phospholipid bilayers of dipalmitoyl-phosphatidylcholine. The measured translational diffusion constants (4 x 10(-10) m2 s-1 at 63 degrees C and 1.4 x 10(-11) m2 s-1 at 30 degrees C) are considerably faster than those deduced using other less direct methods, but are in agreement with those measured in soap-water lyotropic liquid crystals, and with calculated values. This disagreement is attributed to differences in the time and distance scales characterising the various measurements. Quasielastic neutron scattering experiments observe fast motions over molecular distances, whereas other methods tend to measure a rate of diffusion which is averaged over macroscopic distances, and may thus contain contributions from long distance slow diffusive motions such as diffusion between the bilayers.     

Chain length and pressure dependence of lipid translational diffusion

The translational diffusion of pyrene, pyrene butyric acid and pyrene decanoic acid has been determined in phosphatidylcholine bilayers of different chain length and under pressure up to 200 bars. In the liquid crystalline phase and at a given temperature the diffusion decreases with increasing chain length. At a constant reduced temperature, T _red (about 10 K above the transition temperature), long chain lipids exhibit the fastest diffusion which is in disagreement with hydrodynamic models but favours free volume models for diffusion in lipid bilayers. The volume of activation, V _act, calculated from the decrease of the diffusion coefficient with pressure, ln D/P, depends on lipid chain length. V _act decreases with decreasing lipid chain length at a given temperature, T=65°C, and increases at the reduced temperature. These results are again in agreement with the dependence of the diffusion on lipid chain length and therefore with the free volume model.Abbreviations DLPC Dilauroylphosphatidylcholine - DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmitoylphosphatidylcholine - DSPC Distearoylphosphatidylcholine - LUV Large unilamellar vesicles - SUV Small unilamellar vesicles - Tris Tris(hydroxymethyl)aminomethan     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

Lateral diffusion in oriented bilayers of saturated cholesterol-containing phosphatidylcholines, dipalmitoylphosphatidylcholine and dimyrilstoylphosphatidylcholine upon their limiting hydration has been studied by NMR with impulse gradient of magnetic field. For both systems, similar dependences of the coefficient of lateral diffusion on temperature and cholesterol concentration were observed, which agree with the phase diagram showing the presence of regions of ordered and unordered liquid-crystalline phases and a two-phase region. Under similar conditions, the coefficient of lateral diffusion for dipalmytoylphosphatidylcholine has lower values, which is in qualitative agreement with its greater molecular mass. A comparison of data for dipalmytoylphosphatidylcholine with the results obtained earlier for dipalmytoylsphyngomyelin/cholesterol under the same conditions shows, despite a similarity in phase diagrams, greater (two- to threefold) differences in the values of the coefficient of lateral diffusion and a different mode of dependence of the coefficient on cholesterol concentration. A comparison of data for dimyrilstoylphosphatidylcholine with the results obtained previously shows that the values of the coefficient of lateral diffusion and the mode of its dependence on cholesterol concentration coincide in the region of higher concentrations (more than 15 mole %) and differ in the region of lower concentrations (below 15 mole %). The discrepancies may be explained by different contents of water in the systems during the measurements. At a limiting hydration (more than 35%) of water, the coefficient of lateral diffusion decreases with increasing cholesterol concentration. If the content of water is about 25% (as a result of equilibrium hydration from vapors), the coefficient of lateral diffusion of phosphatidylcholine is probably independent of cholesterol concentration. This results from a denser packing of molecules in the bilayer at a lower water concentration, an effect that competes with the ordering effect of cholesterol.     

Lateral diffusion of small solutes and partition of amphipaths in defect structures of lipid bilayers

The excimer formation technique has been applied to investigate the mechanism of the lateral diffusion in the crystalline P beta' phase of dipalmitoylphosphatidylcholine vesicles. This became possible at low pyrene concentrations. From the shape of phase-transition curves, from the effect of pressure up to 150 bar and from the perturbation induced by cholesterol, we conclude that the free volume model could also be applied to the P beta' phase. However, the diffusion is thought to occur in defect structures that are considered to form fluid pathways between domains of crystalline lipid. Partition coefficients of amphipaths provide a basis for testing for the role of defects. The amphipath chlorpromazine partitions into fluid membranes with a partition coefficient, kp, of 3200, into the crystalline phase with kp = 200 but into the P beta' phase with a value of kp = 800. This again gives rise to the assumption that the P beta' phase contains fluid domains that behave like the fluid L alpha phase and make up about 10-20% of the total amount of lipid in the bilayer. Cholesterol is known to interfere especially with defect structures in the P beta' phase. It fills up the gaps, and therefore reduces the partition coefficient to almost zero in the P beta' phase.     

Mesoscopic lateral diffusion in lipid bilayers

The lateral diffusion in bilayers is modeled with a multiscale mesoscopic simulation. The methodology consists of two simulations, where the first employs atomistic models to obtain exact results for the mesoscopic model. The second simulation takes the results obtained from the first to parameterize an effective force field that is employed in a new coarse-grained model. The multiscale aspect of this scheme occurs at the point where the microscopic time-averaged results of the first simulation are employed to parameterize the second simulation that operates in a higher spatial and temporal domain. The results of both simulation schemes give quantitative information on the details associated with lipid lateral diffusion.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Lateral diffusion of the phospholipid molecule in dipalmitoylphosphatidylcholine bilayers. An investigation using nuclear spin--lattice relaxation in the rotating frame

Measurements of the proton NMR spin--lattice relaxation time in the rotating frame (T 1rho) have permitted the explicit determination of the lateral diffusion coefficient of phospholipid molecules in the lamellar mesophase of dipalmitoylphosphatidylcholine at temperatures above the phase-transition temperature. The experimentally observed temperature and frequency dependence of T 1rho for the dipalmitoylphosphatidylcholine protons suggest that intermolecular dipole--dipole relaxation contributions are important. Proton T 1rho experiments involving dilution with deuterated dipalmitoylphosphatidylcholine support the premise that intermolecular dipolar interactions are significant and, concomitantly, that lateral diffusion is the motion modulating that interaction. The lateral diffusion coefficient is determined directly from the dependence of the rotating frame spin--lattice relaxation rate (1/T 1rho) on the strength of the applied radiofrequency field in the spin-locking experiment. A series of experiments with varying concentrations of dipalmitoylphosphatidylcholine in the lamellar mesophase indicates that the lateral diffusion coefficient varies as a function of phospholipid concentration.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol lipid bilayers: a free volume analysis.

The technique of fluorescence recovery after photobleaching is used to perform an extensive study of the lateral diffusion of a phospholipid probe in the binary mixture dimyristoylphosphatidylcholine/cholesterol, above the melting temperature of the phospholipid. In the regions of the phase diagram where a single liquid phase exists, diffusion can be quantitatively described by free volume theory, using a modified Macedo-Litovitz hybrid equation. In the liquid-liquid immiscibility region, the temperature dependence of the diffusion coefficient is in excellent agreement with current theories of generalized diffusivities in composite two-phase media. A consistent interpretation of the diffusion data can be provided based essentially on the idea that the primary effect of cholesterol addition to the bilayer is to occupy free volume. On this basis, a general interpretation of the phase behavior of this mixture is also proposed.     

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.     

The transmembrane homotrimer of ADAM 1 in model lipid bilayers

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin alpha and fertilin beta that plays an important role in sperm-egg fusion. Fertilin alpha and beta are members of the ADAM family, and contain each one transmembrane alpha-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane alpha-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate.     

Lateral diffusion of phospholipids in the lipid surface of human low-density lipoprotein measured with a pyrenyl phospholipid probe

Human low-density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1-palmitoyl-2-(1'-pyreneoctanoyl)-sn-glycero-3-phosphocholine by using phosphatidylcholine-specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling-crowd model (J. Eisinger et al. (1986) Biophys. J. 49, 987-1001] to obtain the short-range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein-free LDL-like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE = 1 for excimer production between nearest-neighbour probes gives the lower limits for f, the frequency of translational lipid--lipid exchanges of the probe of 0.62 x 10(8), 0.19 x 10(8) and 0.19 x 10(8)s-1 in LDL, LDL-like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 microns 2 s-1. The results suggest that the translational mobility of phospholipid molecules in the lipid--protein surface of LDL is not constrained by the apolipoprotein B-100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid--associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein-free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.