On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of phase transitions of lipid mixtures from bilayer to non-bilayer structures: Model,experimental validation and implication on membrane fusion

Lipid bilayers provide a solute-proof barrier that is widely used in living systems. It has long been recognized that the structural changes of lipids during the phase transition from bilayer to non-bilayer have striking similarities with those accompanying membrane fusion processes. In spite of this resemblance, the numerous quantitative studies on pure lipid bilayers are difficult to apply to real membranes. One reason is that in living matter, instead of pure lipids, lipid mixtures are involved and there is currently no model that establishes the connection between pure lipids and lipid mixtures. Here, we make this connection by showing how to obtain (i) the short-range repulsion between bilayers made of lipid mixtures and, (ii) the pressure at which transition from bilayer phase to non-bilayer phases occur. We validated our models by fitting the experimental data of several lipid mixtures to the theoretical data calculated based on our model. These results provide a useful tool to quantitatively predict the behavior of complex membranes at low hydration.     

The functions of polarized water and membrane lipids: a rebuttal

In 1973, experimental evidence was reported in support of the view that water polarized in multilayers rather than simple lipid is responsible for the semipermeable properties of living cell membranes. The present article presents a full rebuttal to a subsequent attack on that view.     

The lipid bilayer and aquaporins: parallel pathways for water movement into plant cells

The plant plasma membrane is the the major barrier to water flow between cells and their surroundings. Water movement across roots involves pathways comprising many cells and their walls. There are three possible pathways which water can follow, (i) a trans-cellular pathway, which involves serial movement into and out from radial files of cells, (ii) a symplasmic pathway through the plasmodesmata, which creates a cytoplasmic continuum and (iii) a tortuous, extracellular pathway through the cell walls, the apoplasmic pathway. In each of these pathways water movement across cell membranes occurs at some stage. The possible role of water-channels in membranes is discussed in relation to this movement. The molecular identity of water-channel proteins in plasma membranes of plants has been confirmed but there remain a number of unresolved questions about their role in cell and tissue water relations, their interaction with the lipid components of membranes and the relationship between water movement through membranes by diffusion in the bilayer.     

Membrane Transport in Primitive Cells

Although model protocellular membranes consisting of monoacyl lipids are similar to membranes composed of contemporary diacyl lipids, they differ in at least one important aspect. Model protocellular membranes allow for the passage of polar solutes and thus can potentially support cell-to functions without the aid of transport machinery. The ability to transport polar molecules likely stems from increased lipid dynamics. Selectively permeable vesicle membranes composed of monoacyl lipids allow for many lifelike processes to emerge from a remarkably small set of molecules.Lipid bilayer membranes are an integral component of living cells, providing a permeability barrier that is essential for nutrient transport and energy production. It is reasonable to assume that a similar boundary structure would be required for the origin of cellular life (Szostak et al. 2001). Even though bilayer membranes are a cellular necessity, they also pose a significant obstacle to early cellular functions, the most obvious being that the permeability barrier would inhibit chemical exchange with the environment. Such an exchange is important not only for acquiring nutrient substrates for primitive metabolic processes, but also for the release of inhibitory side-products.Contemporary cells circumvent the permeability problem by incorporating complex transmembrane protein machinery that provides specific transport capabilities. It is unlikely that Earth’s first cells assembled bilayer membranes together with specific membrane protein transporters. Rather, intermediate evolutionary steps must have existed in which simple lipid molecules provided many of the characteristics of contemporary membranes without relying on advanced protein machinery. What seems to have been necessary was the appearance of a simple membrane system capable of retaining and releasing specific molecules. In short, a protocell needed to be selectively permeable.     

Membrane Partitioning of Anionic,Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling,Local Disordering,and Cholesterol Depletion

Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.     

Formation of a bilayer lipid membrane on rigid supports: an approach to BLM-based biosensors

Sensory transduction in living cells is thought to involve a change of electrical parameters at the receptor membrane following specific binding events at the membrane surface. Because of the complexity of the biomembrane structure and the environmental factors associated with it, experimental bilayer lipid membranes (BLMs) have been employed for elucidation of processes at the membrane level. This is because the BLM system can be easily probed by a host of powerful and sensitive electrochemical methods. Further, recent advances in microelectronics and biotechnology suggest that the development of a BLM-based electrochemical biosensor may be possible. This paper describes the use of bilayer lipid membranes on solid substrates for analysis of sensor development problems, with relevance to a possible novel type of biomolecular device. Some electrical parameters of the new structure were measured and compared to usual BLM results. The advantages of the self-assembled structure, together with the measuring system, are discussed in terms of stability and sensitivity.     

Aquaporin Membrane Channels: Biophysics, Classification, Functions, and Possible Biotechnological Applications

Water represents the major component of all living organisms. To make the cell to adapt to the surrounding environment and carry out its biological functions, water has to move into and out of the cell interior driven by osmotic forces. For over a century, scientists studying the movement of fluid into and out of the cell struggled with a difficult biophysical question: how does water pass through the cell? Movement of water across the membrane was indicated almost as soon as the lipid bilayer was recognized as being the plasma membrane of cells, back in the 1920s. Simple diffusion of water across the lipid bilayer occurs through all biological membranes. However, its low velocity and finite extent soon became apparent, suggesting the existence of additional pathways for water moving through the membrane. In spite of the enormous amount of work carried out in this area, the precise and complete answer only came relatively recently with the discovery of aquaporins, transmembrane channel proteins making the membrane tenfold to 100-fold more permeable to water than membranes lacking such pores. The water conductance featured by the aquaporins is astonishing: in fact, each single aquaporin pore can conduct billions of water molecules per second. A branch of the aquaporin family, the aquaglyceroporins, features conductance to small neutral solutes in addition to water. This review summarizes recent updates on the molecular structure, regulation, biophysics, and biological functions of aquaporins. Possible biotechnological applications of aquaporins are also described.     

DMSO Induces Dehydration near Lipid Membrane Surfaces

Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (X_DMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at X_DMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At X_DMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.     

Lycopene,but not zeaxanthin,serves as a skeleton for the formation of an orthorhombic organization of intercellular lipids within the lamellae in the stratum corneum: Molecular dynamics simulations of the hydrated ceramide NS bilayer model

Carotenoids play an important role in the protection of biomembranes against oxidative damage. Their function depends on the surroundings and the organization of the lipid membrane they are embedded in. Carotenoids are located parallel or perpendicular to the surface of the lipid bilayer. The influence of carotenoids on the organization of the lipid bilayer in the stratum corneum has not been thoroughly considered. Here, the orientation of the exemplary cutaneous carotenoids lycopene and zeaxanthin in a hydrated ceramide NS24 bilayer model and the influence of carotenoids on the lateral organization of the lipid bilayer model were studied by means of molecular dynamics simulations for 32 °C and 37 °C. The results confirm that lycopene is located parallel and zeaxanthin perpendicular to the surface of the lipid bilayer. The lycopene-loaded lipid bilayer appeared to have a strong orthorhombic organization, while zeaxanthin-loaded and pure lipid bilayers were organized in a disordered hexagonal-like and liquid-like state, respectively. The effect is stronger at 32 °C compared to 37 °C based on p-values. Therefore, it was assumed that carotenoids without hydroxyl polar groups in their structure facilitate the formation of the orthorhombic organization of lipids, which provides the skin barrier function. It was shown that the distance between carotenoid atoms matched the distance between atoms in the lipids, indicating that parallel located carotenoids without hydroxyl groups serve as a skeleton for lipid membranes inside the lamellae. The obtained results provide reasonable prediction of the overall qualitative properties of lipid model systems and show the importance of parallel-oriented carotenoids in the development and maintenance of the skin barrier function.     

Insertion of short amino-functionalized single-walled carbon nanotubes into phospholipid bilayer occurs by passive diffusion

Carbon nanotubes have been proposed to be efficient nanovectors able to deliver genetic or therapeutic cargo into living cells. However, a direct evidence of the molecular mechanism of their translocation across cell membranes is still needed. Here, we report on an extensive computational study of short (5 nm length) pristine and functionalized single-walled carbon nanotubes uptake by phospholipid bilayer models using all-atom molecular dynamics simulations. Our data support the hypothesis of a direct translocation of the nanotubes through the phospholipid membrane. We find that insertion of neat nanotubes within the bilayer is a "nanoneedle" like process, which can often be divided in three consecutive steps: landing and floating, penetration of the lipid headgroup area and finally sliding into the membrane core. The presence of functional groups at moderate concentrations does not modify the overall scheme of diffusion mechanism, provided that their deprotonated state favors translocation through the lipid bilayer.     

Do sterols reduce proton and sodium leaks through lipid bilayers?

Proton and/or sodium electrochemical gradients are critical to energy handling at the plasma membranes of all living cells. Sodium gradients are used for animal plasma membranes, all other living organisms use proton gradients. These chemical and electrical gradients are either created by a cation pumping ATPase or are created by photons or redox, used to make ATP. It has been established that both hydrogen and sodium ions leak through lipid bilayers at approximately the same rate at the concentration they occur in living organisms. Although the gradients are achieved by pumping the cations out of the cell, the plasma membrane potential enhances the leakage rate of these cations into the cell because of the orientation of the potential. This review proposes that cells use certain lipids to inhibit cation leakage through the membrane bilayers. It assumes that Na(+) leaks through the bilayer by a defect mechanism. For Na(+) leakage in animal plasma membranes, the evidence suggests that cholesterol is a key inhibitor of Na(+) leakage. Here I put forth a novel mechanism for proton leakage through lipid bilayers. The mechanism assumes water forms protonated and deprotonated clusters in the lipid bilayer. The model suggests how two features of lipid structures may inhibit H(+) leakage. One feature is the fused ring structure of sterols, hopanoids and tetrahymenol which extrude water and therefore clusters from the bilayer. The second feature is lipid structures that crowd the center of the bilayer with hydrocarbon. This can be accomplished either by separating the two monolayers with hydrocarbons such as isoprenes or isopranes in the bilayer's cleavage plane or by branching the lipid chains in the center of the bilayers with hydrocarbon. The natural distribution of lipids that contain these features are examined. Data in the literature shows that plasma membranes exposed to extreme concentrations of cations are particularly rich in the lipids containing the predicted qualities. Prokaryote plasma membranes that reside in extreme acids (acidophiles) contain both hopanoids and iso/anteiso- terminal lipid branching. Plasma membranes that reside in extreme base (alkaliphiles) contain both squalene and iso/anteiso- lipids. The mole fraction of squalene in alkaliphile bilayers increases, as they are cultured at higher pH. In eukaryotes, cation leak inhibition is here attributed to sterols and certain isoprenes, dolichol for lysosomes and peroxysomes, ubiquinone for these in addition to mitochondrion, and plastoquinone for the chloroplast. Phytosterols differ from cholesterol because they contain methyl and ethyl branches on the side chain. The proposal provides a structure-function rationale for distinguishing the structures of the phytosterols as inhibitors of proton leaks from that of cholesterol which is proposed to inhibit leaks of Na(+). The most extensively studied of sterols, cholesterol, occurs only in animal cells where there is a sodium gradient across the plasma membrane. In mammals, nearly 100 proteins participate in cholesterol's biosynthetic and degradation pathway, its regulatory mechanisms and cell-delivery system. Although a fat, cholesterol yields no energy on degradation. Experiments have shown that it reduces Na(+) and K(+) leakage through lipid bilayers to approximately one third of bilayers that lack the sterol. If sterols significantly inhibit cation leakage through the lipids of the plasma membrane, then the general role of all sterols is to save metabolic ATP energy, which is the penalty for cation leaks into the cytosol. The regulation of cholesterol's appearance in the plasma membrane and the evolution of sterols is discussed in light of this proposed role.     

Permeability of lipid bilayers to water and ionic solutes

The lipid bilayer moiety of biological membranes is considered to be the primary barrier to free diffusion of water and solutes. This conclusion arises from observations of lipid bilayer model membrane systems, which are generally less permeable than biological membranes. However, the nature of the permeability barrier remains unclear, particularly with respect to ionic solutes. For instance, anion permeability is significantly greater than cation permeability, and permeability to proton-hydroxide is orders of magnitude greater than other monovalent inorganic ions. In this review, we first consider bilayer permeability to water and discuss proposed permeation mechanisms which involve transient defects arising from thermal fluctuations. We next consider whether such defects can account for ion permeation, including proton-hydroxide flux. We conclude that at least two varieties of transient defects are required to explain permeation of water and ionic solutes.     

Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance

Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.     

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.     

Membrane actions of water-soluble fusogens: Effect of dimethyl sulfoxide,glycerol and sucrose on lipid bilayer order and fluidity

The effect of three water-soluble fusogens: dimethyl sulfoxide (DMSO), glycerol and sucrose on the structural properties of model lipid membranes has been studied by electron spin resonance (ESR) using 5-doxylstearic acid as a spin probe and by fluorescence spectroscopy using pyrene as an excimer forming fluorescent probe. All three fusogens tested produce a marked increase in the order parameter of the region close to the polar surface of the lipid bilayer. The ordering effect of DMSO, but not of glycerol and sucrose, is much stronger with respect to membranes prepared from acidic than from neutral phospholipids. The membrane-perturbing action of glycerol and sucrose manifests itself also in the reduced lateral mobility of membrane incorporated pyrene, indicating thus a decreased fluidity of the bilayer hydrophobic region. The structural perturbations produced in model membranes by DMSO, glycerol and sucrose are discussed in relation to the mechanism by which these substances promote cell fusion.     

Properties of a self-assembled phospholipid membrane supported on lipobeads

The overall objective of our work was to make a hydrogel-supported phospholipid bilayer that models a cytoskeleton-supported cell membrane and provides a platform for studying membrane biology. Previously, we demonstrated that a pre-Lipobead, consisting of phospholipids covalently attached to the surface of a hydrogel, could give rise to a Lipobead when incubated with liposomes because the attached phospholipids promote self-assembly of a phospholipid membrane on the pre-Lipobead. We now report the properties of that Lipobead membrane. The lateral diffusion coefficient of fluorescently labeled phosphatidylcholine analogs in the membrane was measured by fluorescence recovery after photobleaching and was found to decrease as the surface anchor density and hydrogel crosslinking density increased. Results from the quenching of phosphatidylcholine analogs suggest that the phospholipid membrane of the Lipobead was composed mostly of a semipermeable lipid bilayer. However, the diffusional barrier properties of the Lipobead membrane were demonstrated by the entrapment of 1.5-3.0 K dextran molecules in the hydrogel core after liposome fusion. This hydrogel-supported bilayer membrane preparation shows promise as a new platform for studying membrane biology and for high throughput drug screening.     

Constant normal pressure, constant surface tension, and constant temperature molecular dynamics simulation of hydrated 1,2-dilignoceroylphosphatidylcholine monolayer

A constant normal pressure, constant surface tension, and constant temperature (NP(N)gammaT) molecular dynamics (MD) simulation of the liquid condensed phase of a 1,2-dilignoceroylphosphatidylcholine (DLGPC) monolayer has been performed at 293.15 K. A DLGPC molecule has two saturated 24-carbon acyl chains, giving the hydrocarbon core thickness of the monolayer approximately 28 A, which is close to the hydrocarbon core thickness of a membrane of a living system. NP(N)gammaT ensemble was used to reproduce the experimental observations, such as area/lipid, because surface tension is an essential factor in determining the monolayer structure. Data analysis on DLGPC/water monolayer shows that various liquid condensed-phase properties of the monolayer have been well reproduced from the simulation, indicating that surface tension 22.9 mN/M used in the simulation is an appropriate condition for the condensed-phase NP(N)gammaT simulation. The simulation results suggest that this long-chain phospholipid monolayer shares many structural characteristics with typical short-chain 1,2-diacylphosphatidylcholine systems, such as DPPC/water monolayer in the condensed phase and DPPC/water bilayer in the gel phase. Furthermore, it was found that DLGPC/water monolayer has almost completely rotationally disordered acyl chains, which have not been observed so far in short-chain 1,2-diacylphosphatidylcholine/water bilayers. This study indicates the good biological relevance of the DLGPC/water monolayer which might be useful in protein/lipid studies to reveal protein structure and protein/lipid interactions in a membrane environment.