Interaction of substance P with phospholipid bilayers: A neutron diffraction study.

Neutron diffraction has been used to study the membrane-bound structure of substance P (SP), a member of the tachykinin family of neuropeptides. The depth of penetration of its C-terminus in zwitterionic and anionic phospholipid bilayers was probed by specific deuteration of leucine 10, the penultimate amino acid residue. The results show that the interaction of SP with bilayers, composed of either dioleoylphosphatidylcholine (DOPC), or a 50:50 mixture of DOPC and the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), takes place at two locations. One requires insertion of the peptide into the hydrophobic region of the bilayer, the other is much more peripheral. The penetration of the peptide into the hydrophobic region of the bilayer is reflected in a marked difference in the water distribution profiles. SP is seen to insert into DOPC bilayers, but a larger proportion of the peptide is found at the surface when compared to the anionic bilayers. The positions of the two label populations show only minor differences between the two types of bilayer.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.     

Hybrid bilayer membranes in air and water: infrared spectroscopy and neutron reflectivity studies.

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 A(-1), and provided a sensitivity to changes in total layer thickness on the order of 1-2 A. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.     

Hexane dissolved in dioleoyllecithin bilayers has a partial molar volume of approximately zero

Neutron diffraction has been used to measure the amount and distribution of hexane incorporated from the vapor phase into oriented dioleoylphosphatidylcholine bilayers at 66% relative humidity. We reported earlier that hexane at low concentrations is located largely in a zone 10 A wide at the center of the bilayer [White, S. H., King, G. I., & Cain, J. E. (1981) Nature (London) 290, 161-163]. Extending these studies to high hexane concentrations, we find no readily apparent change in the volume of the hydrocarbon region of the bilayer even though more than one hexane molecule per lipid enters the region. The hexane partial molar volume in the bilayer hydrocarbon region is thus approximately zero. Within our statistical confidence limits, the partial molar volume is certainly no greater than one-third the molecular volume of the hexane. Further, analysis of the data suggests that the mass density of the bilayer is considerably less than 1 in the absence of hexane. These findings are in conflict with the assumptions usually made about lipid bilayers and their interaction with nonpolar hydrophobic molecules. In the course of these experiments, we found that standard methods of interpreting diffraction results were not suitable for our purposes. We thus developed several new methods which are summarized in the text and two appendixes. One of these methods allows us to define with precision the width of the hydrocarbon core of the bilayer. The other provides a means of calculating the effects of changes in the absolute scaling of the bilayers with changes in composition without placing the structures on an absolute scattering length density scale.     

Crystallization of antimicrobial pores in membranes: magainin and protegrin

Membrane pores spontaneously formed by antimicrobial peptides in membranes were crystallized for the first time by manipulating the sample hydration and temperature. Neutron diffraction shows that magainins and protegrins form stable pores in fully hydrated fluid membranes. At lower hydration levels or low temperature, the membrane multilayers crystallize. In one crystalline phase, the pores in each bilayer arrange in a regular hexagonal array and the bilayers are stacked into a hexagonal ABC lattice, corresponding to the cubic close-packed structure of spheres. In another crystalline phase, the bilayers are modulated into the rippled multilamellae, corresponding to a 2D monoclinic lattice. The phase diagrams are described. Crystallization of the membrane pores provides possibilities for diffraction studies that might provide useful information on the pore structures.     

Effects of lipid structure on peptide-lipid interactions. Complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon calcitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin- DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol . There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Effects of lipid structure on peptide-lipid interactions complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon cacitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin-DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol. There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Location of chlorhexidine in DMPC model membranes: a neutron diffraction study

Chlorhexidine (CHX) is an effective anti-bacterial agent whose mode of action is thought to be the disruption of the cell membrane. It is known to partition into phospholipid bilayers of aqueous model-membrane preparations. Neutron diffraction data taken at 36 °C on the location of CHX in phosphatidylcholine (PC) bilayers is presented. The center of mass of the deuterated hydrocarbon chain of CHX is found to reside 16 Å from the center of the bilayer in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (14:0–14:0 PC). This places the drug near the glycerol backbone of the lipid, and suggests a mode of action whereby the molecule is bent in half and inserts wedge-like into the lipid matrix. This mechanism is distinct from detergent-like mechanisms of membrane disruption and more similar to some anti-microbial peptide action, where peptides insert obliquely into the bilayer headgroup region to disrupt its structure.     

Determining bilayer hydrocarbon thickness from neutron diffraction measurements using strip-function models.

Neutron diffraction methods provide information about the distribution of matter in biological and model membrane systems. The information is derived from plots (profiles) of scattering length density along an axis normal to the membrane plane. Without the use of specific deuteration, the generally low resolution of the profiles limits their interpretation in terms of specific chemical constituents (e.g., lipid headgroup, lipid hydrocarbon, protein, and water). A fundamental and useful structural assignment to make is the boundary between the headgroup and hydrocarbon regions of bilayers. We demonstrate here that strip-function model representations of neutron scattering length density profiles of bilayers are sufficient to determine accurately the position of the headgroup-hydrocarbon boundary. The resulting hydrocarbon thickness of the bilayer is useful for determining the area per lipid molecule and consequently the molecular packing arrangements of the membrane constituents. We analyze data obtained from dioleoylphosphatidylcholine (DOPC) bilayers at 66% RH using standard Fourier profile analyses and from DOPC deuterated specifically at the C-2 carbon of the acyl chains using difference Fourier analysis. We demonstrate that strip-function models accurately define the positions of the C-2 carbons and thus the hydrocarbon thickness (dhc) of the bilayer. We then show, using quasi-molecular models, that the strip-model analysis probably provides an accurate measure of dhc because of the exceptionally high scattering length density difference between the carbonyl and methylene groups.     

Soluble amyloid beta-oligomers affect dielectric membrane properties by bilayer insertion and domain formation: implications for cell toxicity

It is well established that Alzheimer's amyloid beta-peptides reduce the membrane barrier to ion transport. The prevailing model ascribes the resulting interference with ion homeostasis to the formation of peptide pores across the bilayer. In this work, we examine the interaction of soluble prefibrillar amyloid beta (Abeta(1-42))-oligomers with bilayer models, observing also dramatic increases in ion current at micromolar peptide concentrations. We demonstrate that the Abeta-induced ion conductances across free-standing membranes and across substrate-supported "tethered" bilayers are quantitatively similar and depend on membrane composition. However, characteristic signatures of the molecular transport mechanism were distinctly different from ion transfer through water-filled pores, as shown by a quantitative comparison of the membrane response to Abeta-oligomers and to the bacterial toxin alpha-hemolysin. Neutron reflection from tethered membranes showed that Abeta-oligomers insert into the bilayer, affecting both membrane leaflets. By measuring the capacitance of peptide-free membranes, as well as their geometrical thicknesses, the dielectric constants in the aliphatic cores of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers were determined to be epsilon = 2.8 and 2.2, respectively. The magnitude of the Abeta-induced increase in epsilon indicates that Abeta-oligomers affect membranes by inducing lateral heterogeneity in the bilayers, but an increase in the water content of the bilayers was not observed. The activation energy for Abeta-induced ion transport across the membrane is at least three times higher than that measured for membranes reconstituted with alpha-hemolysin pores, E(a) = 36.8 vs. 9.9 kJ/mol, indicating that the molecular mechanisms underlying both transport processes are fundamentally different. The Abeta-induced membrane conductance shows a nonlinear dependence on the peptide concentration in the membrane. Moreover, E(a) depends on peptide concentration. These observations suggest that cooperativity and/or conformational changes of the Abeta-oligomer particles upon transfer from the aqueous to the hydrocarbon environment play a prominent role in the interaction of the peptide with the membrane. A model in which Abeta-oligomers insert into the hydrophobic core of the membrane-where they lead to a local increase in epsilon and a concomitant reduction of the membrane barrier-describes the experimental data quantitatively.     

Structure of Sphingomyelin Bilayers: A Simulation Study

We have carried out a molecular dynamics simulation of a hydrated 18:0 sphingomyelin lipid bilayer. The bilayer contained 1600 sphingomyelin (SM) molecules, and 50,592 water molecules. After construction and initial equilibration, the simulation was run for 3.8 ns at a constant temperature of 50°C and a constant pressure of 1 atm. We present properties of the bilayer calculated from the simulation, and compare with experimental data and with properties of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The SM bilayers are significantly more ordered and compact than DPPC bilayers at the same temperature. SM bilayers also exhibit significant intramolecular hydrogen bonding between phosphate ester oxygen and hydroxyl hydrogen atoms. This results in a decreased hydration in the polar region of the SM bilayer compared with DPPC. Since our simulation system is very large we have calculated the power spectrum of bilayer undulation and peristaltic modes, and we compare these data with similar calculations for DPPC bilayers. We find that the SM bilayer has significantly larger bending modulus and area compressibility compared to DPPC.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

Quantitative comparison of two types of planar lipid bilayers--folded and painted--with respect to fusion with vesicles

Two major types of planar lipid bilayers, painted and folded, were compared with respect to vesicle fusion using one chamber for the preparation of both bilayers. Liposomes containing ion channels composed of nystatin and ergosterol were used as the vesicle sample. Fusion of the liposome to either bilayer elicited a spike-like current change, which corresponds to a fusion event. The lag time between the first fusion event and the addition of the vesicles is an index of the ease with which the vesicles fuse with the bilayers. The lag time in the painted bilayer at a KCl concentration (cis) of 450 mM was 1.58+/-1.18 min, similar to that in the folded bilayer (1.65+/-0.64 min). The lag time decreased with increase of the osmotic difference across the painted bilayer, whereas this change was small in the folded bilayer. The fusion of the liposomes to the painted bilayer was markedly reduced by stopping the stirring in the cis compartment, whereas the fusion to the folded bilayer was not affected significantly. These results imply that no practical difference exists in the ability of vesicles to fuse with the painted and folded bilayers. For the study of single channel behavior, the painted bilayer could have an advantage because simply stopping the stirring prevents excess fusion.     

Osmotic control of bilayer fusion

We have used photography and capacitance measurement to monitor the steps in the interaction and eventual fusion of optically black lipid bilayers (BLMs), hydrostatically bulged to approximately hemispherical shape and pushed together mechanically. A necessary first step is drainage of aqueous solution from between the bilayers to allow close contact of the bilayers. The drainage can be controlled by varying the osmotic difference across the bilayers. If the differences are such as to remove water from between the bilayers, fusion occurs after a time that depends on the net osmotic difference and the area of contact. If there is an osmotic flow of water into the space between the bilayers, fusion never occurs. In the fusion process, a single central bilayer forms from the original apposed pair of bilayers. The central bilayer may later burst to allow mixing of the two volumes originally bounded by the separate bilayer; the topological equivalent of exocytosis.     

Interactions between charged, uncharged, and zwitterionic bilayers containing phosphatidylglycerol.

Pressure vs. distance relationships have been obtained for phosphatidylglycerol bilayers, in both charged and uncharged states. Water was removed from the lipid multilayers by the application of osmotic pressures in the range of 0-2.7 x 10(9) dyn/cm2, and the distance between adjacent bilayers was obtained from Fourier analysis of lamellar x-ray diffraction data. For phosphatidylglycerol bilayers made electrically neutral either by lowering the pH or by adding equimolar concentrations of the positively charged lipid stearylamine, the pressure-distance data could be fit with a single exponential. The measured decay lengths were 1.1 A at low pH and 1.5 A with stearylamine, which are similar to decay lengths of the hydration pressure found for gel phases of other neutral bilayers. In addition, the magnitude of this repulsive pressure was proportional to the square of the Volta potential (equivalent to the dipole potential for electrically neutral bilayers) measured in monolayers in equilibrium with bilayers, in agreement with results previously found for the hydration pressure between phosphatidylcholine bilayers. For charged phosphatidylglycerol bilayers, the pressure-distance relation had two distinct regions. For bilayer separations greater than 10 A, the pressure-distance data had an exponential decay length (11 A) and a magnitude consistent with that expected for electrostatic repulsion from double-layer theory. For bilayer separations of 2-10 A, the pressure decayed much more rapidly with increasing bilayer separation (decay length less than 1 A). We interpret these data at low bilayer separations in terms of a combination of hydration repulsion and steric hindrance between the lipid head groups and the sodium ions trapped between apposing bilayers.     

A new mechanism of model membrane fusion determined from Monte Carlo simulation

We have carried out extensive Monte Carlo simulations of the fusion of tense apposed bilayers formed by amphiphilic molecules within the framework of a coarse-grained lattice model. The fusion pathway differs from the usual stalk mechanism. Stalks do form between the apposed bilayers, but rather than expand radially to form an axial-symmetric hemifusion diaphragm of the trans leaves of both bilayers, they promote in their vicinity the nucleation of small holes in the bilayers. Two subsequent paths are observed. 1) The stalk encircles a hole in one bilayer creating a diaphragm comprised of both leaves of the other intact bilayer, which ruptures to complete the fusion pore. 2) Before the stalk can encircle a hole in one bilayer, a second hole forms in the other bilayer, and the stalk aligns and encircles them both to complete the fusion pore. Both pathways give rise to mixing between the cis and trans leaves of the bilayer and allow for transient leakage.     

The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes

This review details how bilayer structural/elastic properties impact three distinct areas of biological significance. First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition. The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage. Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains (“rafts”) enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer. This is likely due to the large area compressibility modulus of SM:cholesterol bilayers. Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side. We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol’s strong interaction with saturated chains.     

Association of Model Neurotransmitters with Lipid Bilayer Membranes

Aimed at reproducing the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely tethered bilayer lipid membranes, reveal the structural aspects of neurotransmitters’ association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts nonspecifically with the membrane at physiologically relevant concentrations, whereas γ-aminobutyric acid does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity, and neutron reflectometry shows that it penetrates the membrane deeply, whereas γ-aminobutyric is excluded from the bilayer.     

Optical and electrical properties of thin monoolein lipid bilayers

Summary Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K⁺ and valinomycin-Rb⁺ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K⁺ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb⁺ complex.