Semiconductor theory of ion transport in thin lipid membranes: II. Surface recombination

Following the theory of surface recombination in semiconductors, we have derived an expression for the rate of ion recombination at the membrane surface. The surface recombination rate is used in the boundary conditions of current flows at the interfaces. Expressions for the ion fluxes are then derived as functions of environmental variables and membrane parameters. Our analysis strongly suggests that the ion flow through a thin lipid membrane consists of two major components: the surface barrier jumping current and the surface recombination current that are controlled decisively by surface barrier height, surface trap density and surface recombination rates. These general formulations are useful not only for the calculation but also for the understanding of ion transport in thin lipid membranes under a variety of experimental conditions. The implications of this theory to biological membranes and its possible extensions are discussed.     

A theory of ion permeation through membranes with fixed neutral sites

Summary Some model membranes and biological membranes behave as if ion permeation were controlled by fixed neutral sites, i.e., by groups that are polar but lack net charge. By solving the boundary conditions and Nernst-Planck flux equations, this paper derives the expected properties of four types of membranes with fixed neutral sites: model 1, a membrane thick enough that microscopic electroneutrality is obeyed; model 2, same as model 1 but with a free-solution shunt in parallel; model 3, a membrane thin enough that microscopic electroneutrality is violated; and model 4, same as model 3 but with a free-solution shunt in parallel. The conductance-concentration relation and the current-voltage relation in symmetrical solutions are approximately linear for all four models. Partial ionic conductances are independent of each other for a thin membrane but not for a thick membrane. Sets of permeability ratios derived from conductances, dilution potentials, or biionic potentials agree with each other in a thin membrane but not in a thick membrane. The current-voltage relation in asymmetrical single-salt solutions is linear for a thick membrane but nonlinear for a thin membrane. Examples of potential and concentration profiles in a thin membrane are calculated to illustrate the meaning of space charge and the electroneutrality condition. The experimentally determined properties (by A. Cass, A. Finkelstein & V. Krespi) of thin lipid membranes containing “pores” of the anion-selective antibiotic nystatin are in reasonable agreement with model 3. Tests are suggested for deciding if a membrane of unknown structure has neutral sites, whether it is thick or thin, and whether the sites are fixed or mobile.     

二甲基亚砜对生物膜的作用机理

二甲基亚砜被广泛应用于生物、化学和药学领域,这些应用大多与其增加生物膜的通透性、促进活性分子跨膜传输的作用密切相关。本文对二甲基亚砜增加生物膜通透性的理论及实验研究做简要综述,主要强调二甲基亚砜在生物膜中诱导水性孔道形成的分子动力学模拟及其相关的实验研究。     

Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.     

Model answers to lipid membrane questions

Ever since it was discovered that biological membranes have a core of a bimolecular sheet of lipid molecules, lipid bilayers have been a model laboratory for investigating physicochemical and functional properties of biological membranes. Experimental and theoretical models help the experimental scientist to plan experiments and interpret data. Theoretical models are the theoretical scientist's preferred toys to make contact between membrane theory and experiments. Most importantly, models serve to shape our intuition about which membrane questions are the more fundamental and relevant ones to pursue. Here we review some membrane models for lipid self-assembly, monolayers, bilayers, liposomes, and lipid-protein interactions and illustrate how such models can help answering questions in modern lipid cell biology.     

Thylakoid membrane landscape in the sixties: a tribute to Andrew Benson

Prior to the 1960s, the model for the molecular structure of cell membranes consisted of a lipid bilayer held in place by a thin film of electrostatically-associated protein stretched over the bilayer surface: (the Danielli–Davson–Robertson “unit membrane” model). Andrew Benson, an expert in the lipids of chloroplast thylakoid membranes, questioned the relevance of the unit membrane model for biological membranes, especially for thylakoid membranes, instead of emphasizing evidence in favour of hydrophobic interactions of membrane lipids within complementary hydrophobic regions of membrane-spanning proteins. With Elliot Weier, Benson postulated a remarkable subunit lipoprotein monolayer model for thylakoids. Following the advent of freeze fracture microscopy and the fluid lipid-protein mosaic model by Singer and Nicolson, the subunits, membrane-spanning integral proteins, span a dynamic lipid bilayer. Now that high resolution X-ray structures of photosystems I and II are being revealed, the seminal contribution of Andrew Benson can be appreciated.     

Photoemf at a Single Membrane-Solution Interface specific to Lipid Bilayers containing Magnesium Porphyrins

THE study of ionic effects in Rudin-Mueller bimolecular lipid membranes containing ionophoric agents has increased the understanding of ion transport across biological membranes. The study of photoreactions in these membranes containing light sensitive chromophores promises to be equally informative. Several teams have reported on such studies^1–9, mostly using continuous light. The mixture of photoemf and conductance changes, however, is difficult to separate and it is not known whether the photoresponses originate from the thin bimolecular region or from the thick annular region (Plateau-Gibbs border) of the membrane. We report here evidence that fast photoemfs across a single interface of such membranes are specific to the bilayer.     

Membrane molecule reorientation in an electric field recorded by attenuated total reflection Fourier-transform infrared spectroscopy

Electric fields play an important role in the physiological function of macromolecules. Much is known about the role that electric fields play in biological systems, but membrane molecule structure and orientation induced by electric fields remain essentially unknown. In this paper, we present a polarized attenuated total reflection (ATR) experiment we designed to study the effect of electric fields on membrane molecule structure and orientation by Fourier-transform infrared (FTIR) spectroscopy. Two germanium crystals used as the internal reflection element for ATR-FTIR experiments were coated with a thin layer of polystyrene as insulator and used as electrodes to apply an electric field on an oriented stack of membranes made of dioleylphosphatidylcholine (DOPC) and melittin. This experimental set up allowed us for the first time to show fully reversible orientational changes in the lipid headgroups specifically induced by the electric potential difference.     

Calculation of deformation energies and conformations in lipid membranes containing gramicidin channels.

In this paper we calculate surface conformation and deformation free energy associated with the incorporation of gramicidin channels into phospholipid bilayer membranes. Two types of membranes are considered. One is a relatively thin solvent-free membrane. The other is a thicker solvent-containing membrane. We follow the approach used for the thin membrane case by Huang (1986) in that we use smectic liquid crystal theory to evaluate the free energy associated with distorting the membrane to other than a flat configuration. Our approach is different from Huang, however, in two ways. One is that we include a term for surface tension, which Huang did not. The second is that one of our four boundary conditions for solving the fourth-order differential equation describing the free energy of the surface is different from Huang's. The details of the difference are described in the text. Our results confirm that for thin membranes Huang's neglect of surface tension is appropriate. However, the precise geometrical form that we calculate for the surface of the thin membrane in the region of the gramicidin channel is somewhat different from his. For thicker membranes that have to deform to a greater extent to accommodate the channel, we find that the contribution of surface tension to the total energy in the deformed surface is significant. Computed results for the shape of the deformed surface, the total energy in the deformed surface, and the contributions of different components to the total energy, are presented for the two types of membranes considered. These results may be significant for understanding the mechanisms of dimer formation and breakup, and the access resistance for ions entering gramicidin channels.     

Formation of Flat Lamellar Intramembrane Lipid Structures in Microorganisms

Analysis of freeze-fracture replicas and thin sections of cells of the bacteria Sulfobacillus thermosulfidooxidans and Anaerobacter polyendosporus showed that their cytoplasmic membranes contain some regions in the form of flat lamellar inverted lipid membranes a few tenths of nanometers to a few microns in size. The specific features of these membrane structures are as follows: (i) they contain no familiar intramembrane particles commonly present on freeze-fracture replicas; (ii) in cross thin sections, intramembrane structures are bifurcate on the periphery and look like thylakoids; and (iii) the leaflets of intramembrane structures in S. thermosulfidooxidans cells are corrugated. These structures were revealed in bacterial cells cultivated under normal growth conditions. The data obtained suggest the occurrence of a complex type of compartmentalization in biological membranes. Received: 17 July 2000/Revised: 22 November 2000     

Multispectral imaging of ion transport in neutral carrier-based cation-selective membranes.

BACKGROUND: High-resolution spectroscopic imaging of the cross section of ion-selective membranes during real-time electrochemical measurements is termed spectroelectrochemical microscopy (SpECM). SpECM is aimed for optimizing the experimental conditions in mass transport controlled ion-selective electrode (ISE) membranes for improved detection limit. METHODS: The SpECM measurements are performed in a thin layer electrochemical cell. The key element of the cell is a membrane strip spacer ring assembly which forms a two compartment electrochemical cell. The cell is placed onto the stage of a microscope and the membrane strip is positioned in the center of the field of view. A slice of the image is focused onto the entrance slit of the imaging spectrometer. RESULTS: SpECM has been used for the determination of the diffusion coefficients of different membrane ingredients and for the quantitative assessment of the charged site concentrations in ISE membranes and membrane plasticizers. In addition, changes in the concentration profiles of the ionophore (free and complexed) and charged mobile sites inside the ISE membranes are documented upon the application of large external voltages. CONCLUSIONS: This account demonstrates the power and advantages of SpECM, a multispectral imaging method for investigations of mass transport processes in ISE membranes during electrochemical measurements.     

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Mechanics and Dynamics of Bacterial Cell Lysis

Membrane lysis, or rupture, is a cell death pathway in bacteria frequently caused by cell wall-targeting antibiotics. Although previous studies have clarified the biochemical mechanisms of antibiotic action, a physical understanding of the processes leading to lysis remains lacking. Here, we analyze the dynamics of membrane bulging and lysis in Escherichia coli, in which the formation of an initial, partially subtended spherical bulge (“bulging”) after cell wall digestion occurs on a characteristic timescale of 1 s and the growth of the bulge (“swelling”) occurs on a slower characteristic timescale of 100 s. We show that bulging can be energetically favorable due to the relaxation of the entropic and stretching energies of the inner membrane, cell wall, and outer membrane and that the experimentally observed timescales are consistent with model predictions. We then show that swelling is mediated by the enlargement of wall defects, after which cell lysis is consistent with both the inner and outer membranes exceeding characteristic estimates of the yield areal strains of biological membranes. These results contrast biological membrane physics and the physics of thin, rigid shells. They also have implications for cellular morphogenesis and antibiotic discovery across different species of bacteria.     

Intracytoplasmic membrane formation and increased oxidation of glycerol growth of Gluconobacter oxydans.

Gluconobacter oxydans is well known for the limited oxidation of compounds and rapid excretion of industrially important oxidation products. The dehydrogenases responsible for these oxidations are reportedly bound to the cell's plasma membrane. This report demonstrates that fully viable G. oxydans differentiates at the end of exponential growth by forming dense regions at the end of each cell observed with the light microscope. When these cells were thin sectioned, their polar regions contained accumulations of intracytoplasmic membranes and ribosomes not found in undifferentiated exponentially growing cells. Both freeze-fracture-etched whole cells and thin sections through broken-cell envelopes of differentiated cells demonstrate that intracytoplasmic membranes occur as a polar accumulation of vesicles that are attached to the plasma membrane. When cells were tested for the activity of the plasma membrane-associated glycerol dehydrogenase, those containing intracytoplasmic membranes were 100% more active than cells lacking these membranes. These results suggest that intracytoplasmic membranes are formed by continued plasma membrane synthesis at the end of active cell division.     

Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS

The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.     

A genomically/chemically complete module for synthesis of lipid membrane in a minimal cell

A minimal cell is a hypothetical cell defined by the essential functions required for life. We have developed a module for the synthesis of membrane precursors for a mathematical minimal cell model. This module describes, with chemical and genomic detail the production of the constituents required to build a cell membrane and identifies the corresponding essential genes. Membranes allow selective nutrient passage, harmful substance exclusion, and energy generation. Bacterial membrane components range from lipids to fatty acids with embedded proteins and are structurally similar to eukaryotic cell membranes. Membranes are dynamic structures and experimental analyses show great variations in bacterial membrane composition. The flexibility of the model is such that different membrane compositions could be obtained in response to simulated changes in culture conditions. The model's predictions are in close agreement with the observed biological trends. The model's predictions correspond well with the experimental values of total lipid content in cells grown in chemostat culture, but less well with data from batch growth. Cell shape and size results agree especially well for data for growth rate relative to maximum growth rate larger than 0.5; and DNA, RNA, and protein predictions are consistent with experimental observations. A better understanding of the simplest bacterial membrane should lead to insights on the more complex behavior of membranes of higher species as well as identification of potential targets for antimicrobials.     

Ultrastructural Properties of the Extra Membranes of Escherichia coli O111a as Revealed by Freeze-Fracturing and Negative-Staining Techniques

Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.     

Flip-Flop-Induced Relaxation of Bending Energy: Implications for Membrane Remodeling

Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids—they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.     

Dielectric Spectroscopy and Membrane Organisation

The possible bases for field-mediated effects on cellular processes are reflected in the passive electrical properties of biological systems. The historical, present and prospective utility of dielectric spectroscopy in assessing the static and dynamic organisation of biological membranes is reviewed within this context. The basis for the view that the static capacitance of bioraembranes is as great as 1 fiF/cm² is doubted; contributions from the (partially restricted) motions of membrane components, and of double-layer ions, probably contribute to this apparent value in bioraembrane vesicle suspensions. The importance of improving our knowledge of the static electrical capacitance of energy coupling membranes is stressed. Theoretical and experimental procedures for assessing the contribution of rotational and translational motions of membrane components, and of double-layer/membrane interactions, to dielectric spectra in the approximate frequency range 10 to 10⁷ Hz are described. Finally, three outstanding and generally unsolved problems requiring further work are detailed.     

A New Proposal for the Action of Vasopressin, Based on Studies of a Complex Synthetic Membrane

The total osmotic flow of water across cell membranes generally exceeds diffusional flow measured with labeled water. The ratio of osmotic to diffusional flow has been widely used as a basis for the calculation of the radius of pores in the membrane, assuming Poiseuille flow of water through the pores. An important assumption underlying this calculation is that both osmotic and diffusional flow are rate-limited by the same barrier in the membrane. Studies employing a complex synthetic membrane show, however, that osmotic flow can be limited by one barrier (thin, dense barrier), and the rate of diffusion of isotopic water by a second (thick, porous) barrier in series with the first. Calculation of a pore radius is meaningless under these conditions, greatly overestimating the size of the pores determining osmotic flow. On the basis of these results, the estimation of pore radius in biological membranes is reassessed. It is proposed that vasopressin acts by greatly increasing the rate of diffusion of water across an outer barrier of the membrane, with little or no accompanying increase in pore size.