Ion transport across a bilayer lipid membrane facilitated by valinomycin

The facilitated ion transport from one aqueous phase, W1, to another, W2, across a bilayer lipid membrane, BLM, containing valinomycin, Val, as an ionophore was investigated by voltammetry. Cyclic voltammograms for the ion transfer were symmetrical about the origin (0 V, 0 A) and the magnitude of the ion transfer current increased with an increase in the absolute value of the applied potential. The magnitude of the ion transfer current at a definite potential in the voltammograms depended on the cation species added to W1 and W2 and was proportional to the concentration of Val in the BLM. The magnitude of the ion transfer current at a definite potential also varied in proportion to the hydrophobicity of the counter anion in W1 and W2. Taking into account the conjugated ion transfers at the W1|BLM and BLM|W2 interfaces, the positive current that flowed from W1 to W2 across the BLM was attributable to both the transfer of the complex-forming cation from W1 to the BLM and the transfer of the anion, which was distributed in the BLM as the counter ion from W2 to W1. The transfer from the BLM to W1 occurred at the W1|BLM interface and both the transfer of the cation from the BLM to W2 and the transfer of the anion from W2 to the BLM at the BLM|W2 interface. The negative current was then attributed to the opposite reaction. The voltammograms were asymmetrical with the origin when the ion components in W1 and W2 were different.     

Potassium ion transport by valinomycin across a Hg-supported lipid bilayer

A biomimetic membrane consisting of a lipid bilayer tethered to a mercury electrode via a hydrophilic spacer was investigated in aqueous KCl by potential-step chronocoulometry and electrochemical impedance spectroscopy, both in the absence and in the presence of the ionophore valinomycin. Impedance spectra, recorded from 1 x 10(-2) to 1 x 10(5) Hz over a potential range of 0.8 V, are satisfactorily fitted to a series of four RC meshes, which are straightforwardly related to the different substructural elements of the biomimetic membrane. The frequency-independent resistances and conductances of both the lipid bilayer and the hydrophilic spacer show a maximum when plotted against the applied potential. This behavior is interpreted on the basis of a general approximate approach that applies the concepts of impedance spectroscopy to a model of the electrified interphase and to the kinetics of potassium ion transport assisted by valinomycin across the lipid bilayer.     

Influence of inhalation anesthetics on ion transport across a planar bilayer lipid membrane

Ion transport from one aqueous phase (W1) to another (W2) across a planar bilayer lipid membrane (BLM) in the presence of inhalation anesthetics was electrochemically investigated. In the absence of inhalation anesthetics in the BLM system, no ion transport current flowed between W1 and W2 across the BLM. When inhalation anesthetics such as halothane, chloroform, diethyl ether and trichloroethylene were added to the two aqueous phases or the BLM, the ion transport current quite clearly appeared. When the ratio of the concentration of KCl or NaCl in W1 to that in W2 was varied, the zero current potential across the BLM was shifted. By considering the magnitude of the potential shift, we concluded that the ion transport current can be predominantly ascribed to the transport of Cl(-) across the BLM. Since the dielectric constants of these anesthetics are larger than that of the inner hydrophobic domain of the BLM, the concentration of hydrophilic electrolyte ions in the BLM increases with the increase in the dielectric constant of the inner hydrophobic domain caused by addition of these anesthetics. These situations lead to an increase in the ion permeability coefficient.     

Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: dependence on type of lipid bilayer

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC  PC/PS ≤ PC/SM  PC/PE. When the constant voltage of −100 or −70 mV was applied through the lipid bilayer membranes in the presence of positively charged tetracaine, the fluctuating current pulses with the large amplitude generated, but not appeared in the absence of tetracaine. The addition of uncharged benzocaine generated the fluctuating currents with the small amplitude. Both charged tetracaine and uncharged benzocaine facilitated electrophoretically the transport of small ions such as KCl in the buffer solution through the fluctuating pores in the lipid bilayer membranes formed by interaction with the local anesthetic under the negative applied membrane potential. The current pulses also contained actual transport of charged tetracaine together with the transport of the small ions. The amplitude and the duration time of the electrical current generated by adding the local anesthetics were dependent on the type of the lipid, the applied voltage and its voltage polarity.     

Proton transport into a tethered bilayer lipid membrane

Tethered bilayer lipid membranes (tBLMs) are increasingly used to study biological membranes, membrane proteins and a variety of related topics. A tBLM is formed by binding a lipid bilayer to a metal surface (usually gold) via a hydrophilic tether (usually an ethyleneoxy chain). In this report we present an electrochemical study on ubiquinone in a tBLM which has provided insights into the properties of this hydrophilic layer, which has a very limited capability of storing and releasing protons. It is concluded that the often observed decrease in tBLM resistance upon addition of ionophores (or protonophores) could be due to the penetration of ions (or protons) into the membrane rather than transport through the membrane.     

Arachidonic acid-induced channel- and carrier-type ion transport across planar bilayer lipid membranes.

Transmembrane ion transport by arachidonic acid (AA) through bilayer lipid membranes (BLMs) was investigated by means of electrochemical measurements to provide a basis for designing a sensor membrane. We found that AA induces a channel-type current, in addition to a carrier-type current, across planar BLMs. A linear relation between the logarithmic value of the AA concentration and the current responses (given as integrated currents) was observed for a carrier-type current, while a sigmoid relation was found for a channel-type current. Although AA transports Na+, Ca2+ and Mg2+ and exhibits ion selectivity between Na+ and Mg2+ for the carrier-type current, ion transport for the channel-type current was non-selective. It was found that ion transport via the channel mechanism occurs frequently for AA, while channel-type currents were only occasionally observed for y-linolenic acid and prostaglandin D2. No channel-type currents were induced by other fatty acids (oleic, linoleic, stearic, myristic, eicosapentanoic and docosahexanoic acids) and metabolites of AA (12-HETE and 5-HETE). The carrier-type ion transport occurs selectively to these compounds if the concentration is below 1.0 microM. These results suggest that AA selectively facilitates an ion flux through the BLMs, generating channel-type and/or carrier-type currents, which can be used as a measure of the AA concentration.     

Effects of palmitic acid and cholesterol on proton transport across black lipid membranes

We studied the effect of palmitic acid (PA) and cholesterol (approximately 17 wt.%) on proton translocation across asolectin (charged) and diphytanoylphosphatidylcholine (DPhPC, neutral) black lipid membranes (BLMs). Potential difference (PD), short circuit current (SCC), and conductance (G(total)) were measured with a digital electrometer. Membranes were exposed to pH gradients (0.4-2.0 units), followed by PA addition to bath (symmetrically, 40-65 microM). The membrane conductive pathway was subdivided into an unspecific and a proton-related routes. A computer program estimated the conductances (G(un) and G(H)) of the two pathways from the measured parameters. No significant differences in proton selectivity were found between DPhPC membranes and DPhPC/cholesterol membranes. By contrast, cholesterol incorporation into asolectin increases membranes selectivity to proton. Cholesterol dramatically reduced G(un) reflecting, probably, its ability of inducing order in lipid chains. In asolectin membranes, PA increases proton selectivity, probably by acting as a proton shuttle according to the model proposed by Kamp and et al. [Biochemistry 34 (1995) 11928]. Cholesterol incorporation into asolectin membranes eliminates the PA-induced increase in proton selectivity. In DPhPC and DPhPC/cholesterol membranes, PA does not affect proton selectivity. These results are discussed in terms of the presence of cardiolipin (CL) in asolectin, cholesterol/PA interactions, and cholesterol order-inducing effects on acyl-chains.     

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.     

Selective transport of ATP across a phospholipid bilayer by a molecular umbrella

Acylation of each primary group of spermidine with cholic acid, followed by acylation of the secondary amino group using an activated form of Nalpha,Ndelta,Nomega-tri-CBZ-l-arginine, and subsequent hydrogenolysis, afforded a conjugate (i.e., 1) which readily transports adenosine 5-triphosphate, but not glutathione, across phospholipid bilayers made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol.     

Voltammetric study on ion transport across a bilayer lipid membrane in the presence of a hydrophobic ion or an ionophore

This review describes voltammetric studies on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By addition of a small amount of hydrophobic ion into W1 and/or W2 containing a hydrophilic salt as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with the counter ion to maintain electroneutrality within the BLM phase. It was found that the counter ion was transferred between W1 and W2 across the BLM by applying a membrane potential. Facilitated transport of alkali ions across a BLM containing Val as an ion carrier compound, could be interpreted by considering not only the formation of the alkali metal ion–Val complex but also the distribution of both the objective cation and the counter ion. In the case of addition of GA as a channel-forming compound into the BLM, the facilitated transport of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transport of alkali ions across the BLM could be explained in terms of the hydrophobicity and the ionic radius of the counter ion.     

Stretching effects on the permeability of water molecules across a lipid bilayer

Using a coarse grained molecular dynamics model of a solvent-surfactant system, we study the effects of stretching on the permeability of water across a lipid bilayer. The density profile, free energy profile, diffusion profile, and tail ordering parameter were computed for a set of stretched membranes maintained at constant area. We computed the water permeability across each membrane using the inhomogeneous solubility-diffusion model first proposed by Marrink and Berendsen [J. Phys. Chem. 98, 4155 (1994)]. We find that even though the resistance to permeation profile shows a great deal of qualitative change as the membranes are stretched, the overall permeability remains nearly constant within the relevant range of stretching. This is explained by the fact that the main barrier to permeation, located in the densest section of the tails, is insensitive to increased area per lipid, as a result of competing effects. Expansion leads to thinning and a higher density in the tail region, the latter leading to an increase in the free energy barrier. However, this is compensated by the reduction in the transverse distance to cross and a larger diffusion coefficient due to increased disordering in the chains.     

Di‐ and tri‐phenyltin chlorides transfer across a model lipid bilayer

A compound's ability to penetrate the plasma membrane of a cell is the critical parameter that determines its potential to become a biologically potent factor. A well‐known group of organotin compounds that exhibit toxic properties in relation to biological systems are phenyltins. There are as yet no studies that in a direct manner have established whether organotin compounds such as diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT) diffuse, or not, through the lipid bilayer, although we know that at least some organotins absorb in both liposome and biological membranes. In this paper we present a series of experiments that show transfer of these compounds across the lipid membrane using the stopped‐flow technique. The results obtained demonstrate that DPhT and TPhT first adsorb onto the lipid bilayer surface, in a diffusion‐controlled manner and within a very short time (0.05 s), whereas the membrane crossing was observed to be on the order of a minute. The adsorption process was easily fitted with a single exponential for both the compounds studied, indicating a single process phenomenon. The longer time kinetics (characteristic of membrane crossing) showed a complex dependence on compound concentration and the presence of cholesterol in the membrane. On passing from the outer to the inner surface of the bilayer, organotins undergo desorption and enter the liposome interior, which has been shown in lipid monolayer desorption studies. In conclusion, it can be stated that amphiphilic DPhT and TPhT permeate the liposome membrane. Copyright © 2005 John Wiley & Sons, Ltd.     

A needle-and-thread approach to bilayer transport: permeation of a molecular umbrella-oligonucleotide conjugate across a phospholipid membrane

A di-walled molecular umbrella, composed of two choloyl groups, one spermidine moiety, and a 5-thiol(2-nitrobenzoyl) "handle", was covalently attached to a 16-mer oligonucleotide (S-dT16) through a disulfide bond. Incubation of this conjugate (1) with vesicles made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (95/5, mol/mol) (200 nm diameter, extrusion) that contained entrapped glutathione (GSH) at 37 degrees C resulted in the liberation of the oligonucleotide and the umbrella-bound 5-mercapto(2-nitrobenzoyl) moiety (USH) via thiolate-disulfide interchange. The appearance of USH, together with the absence of leakage of entrapped GSH and a vesicular capture of the oligonucleotide that matches the extent of USH formation, provides compelling evidence for delivery of S-dT16 into the aqueous compartment of these vesicles. In a sense, the molecular umbrella functions like a "needle" in providing a pathway for the oligonucleotide (the "thread") to cross the membrane.     

Characterization and control of lipid layer fluidity in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the distal lipid layer in hybrid bilayer membranes (HBMs) under water was investigated using vibrational sum frequency spectroscopy (VSFS). VSFS has unique sensitivity to order/disorder transitions in the lipid acyl chains and can determine T(m) for the lipid monolayers in HBMs. We recently reported the observation that T(m) is raised and the transition width is broadened for the overlying phospholipid monolayer in HBM systems formed on densely packed crystalline self-assembled monolayers (SAMs) as compared to that of vesicles in solution. In this report, we establish that T(m) for the lipid layer of HBMs can be controlled by proper choice of the SAM underlayer. The SAM underlayer of the HBM was systematically altered by using an alkane thiol, a saturated thiolipid, a mixed SAM of a saturated lipid-pyridine disulfide, and finally a mixed SAM of an unsaturated lipid-pyridine disulfide. T(m) was measured for two different chain length saturated phosphatidylcholine lipid overlayers on these SAMs. The values obtained show that Tm of the lipid layer of HBMs is sensitive to the composition and/or packing density of molecules in the underlying SAM.     

Interfacial constraints on water and proton transport across nafion membranes

Water and proton transport across a Nafion membrane are measured as functions of water activity and applied electric potential with a polymer electrolyte hydrogen pump. Water and proton transport across the membrane must match water and proton transport entering and leaving the electrode/membrane/vapor three phase interfaces at the anode and cathode. At low applied electric potential proton and water fluxes are correlated. At moderate to high applied electric potential the proton current is constant, independent of applied electric potential, while the water transport increases with increasing electric potential. At high applied electric potential water and proton transport become uncoupled at the membrane interfaces; water is transported across the membrane/vapor interface and protons are transported across the membrane/electrode interface. The applied electric potential drives electro‐osmosis to redistribute the water in the membrane. Water redistribution is limited by the interfacial transport of water across the membrane/vapor interface. © 2015 Wiley Periodicals, Inc. J. Polym. Sci. Part B: Polym. Phys. 2015 , 53, 1580–1589     

Triggered release of molecules across droplet interface bilayer lipid membranes using photopolymerizable lipids

A combination of nonpolymerizable phospholipids (DPPC or DPhPC) and a smaller amount of cross-linking photopolymerizable phospholipids (23:2 DiynePC) is incorporated in an unsupported artificial lipid bilayer formed using the droplet interface bilayer (DIB) approach. The DIB is formed by contacting lipid monolayer-coated aqueous droplets against each other in a dodecane-lipid medium. Cross-linking of the photopolymerizable lipids incorporated in the DIB was obtained by exposure to UV-C radiation (254 nm), resulting in pore formation. The effect of cross-linking on the DIB properties was characterized optically by measuring the diffusion of selectively encapsulated dye molecules (calcein) from one droplet of the DIB to the other droplet. Changes in DIB conductivity due to UV-C exposure were investigated using current-voltage (I-V) measurements. The leakage of dye molecules across the DIB and the increase in DIB conductivity after UV-C exposure indicates the formation of membrane pores. The results indicate that the DIB approach offers a simple and flexible platform for studying phototriggered drug delivery systems in vitro.     

Migration of poly-L-lysine through a lipid bilayer

When a giant vesicle, composed of neutral and anionic lipid (90:10 mol %), comes into contact with various poly-l-lysines (MW 500-29 300), ropelike structures form within the vesicle interior. By using fluorescence lipids and epi-fluorescence microscopy, we have shown that both neutral and anionic lipids are constituents of the ropes. Evidence that the ropes are also comprised of poly-l-lysine comes from two experiments: (a) direct microinjection of poly(acrylic acid) into rope-containing vesicles causes the ropes to contract into small particles, an observation consistent with a polycation/polyanion interaction; and (b) direct microinjection of fluorescein isothiocyanate (a compound that covalently labels poly-l-lysine with a fluorescent moiety) into rope-containing vesicles leads to fluorescent ropes. The results may be explained by a model in which poly-l-lysine binds to the vesicle exterior, forms a domain, and enters the vesicle through defects or at the domain boundary. The model helps explain the ability of poly-l-lysine to mediate the permeation of a cancer drug, doxorubicine, into the vesicle interior.     

Characterization of solvation properties of lipid bilayer membranes in liposome electrokinetic chromatography

The nature of solute interactions with biomembrane-like liposomes, made of naturally occurring phospholipids and cholesterol, was characterized using electrokinetic chromatography (EKC). Liposomes were used as a pseudo-stationary phase in EKC that provided sites of interactions for uncharged solutes. The retention factors of uncharged solutes in liposome EKC are directly proportional to their liposome-water partition coefficients. Linear solvation energy relationship (LSER) models were developed to unravel the contributions from various types of interactions for solute partitioning into liposomes. Size and hydrogen bond acceptor strength of solutes are the main factors that determine partitioning into lipid bilayers. This falls within the general behavior of solute partitioning from an aqueous into organic phases such as octanol and micelles. However, there exist subtle differences in the solvation properties of liposomes as compared to those of octanol and various micellar pseudo-phases such as aggregates of sodium dodecyl sulfate (SDS), sodium cholate (SC), and tetradecylammonium bromide (TTAB). Among these phases, the SDS micelles are the least similar to the liposomes, while octanol, SC, and TTAB micelles exhibit closer solvation properties. Subsequently, higher correlations are observed between partitioning into liposomes and the latter three phases than that into SDS.     

Adsorption of gentamicin onto a bilayer lipid membrane

Addition of the aminoglycoside antibiotic, gentamicin (GM), to one side of a bilayer lipid membrane (BLM) results in a potential difference across the membrane. Evidence is presented that the membrane potential is caused by the adsorption of GM, bearing four positive charges, on the BLM surface. The experimental results are subjected to a quantitative analysis based on the double-layer theory and the Langmuir adsorption isotherm. The adsorption is saturated (i.e., the BLM is fully covered) at the bulk GM concentration of about 80 μmol/1. At this point, the calculated GM-induced increase in the BLM surface charge density is σ = 0.0054 C m⁻², which is equivalent to one positive charge per 50 lipids or one molecule of GM per 200 lipids.     

Planar bilayer lipid memebranes

The planar bilayer lipid membrane, also known as lipid bilayer membrane, black lipid membrane or simply BLM(s), for short, has been investigated since its inception in 1960, the details of which have been described in a monograph published in 1974. This review is a report on the advances in the BLM research since that time.After a brief introduction, the first five sections consider various aspects of experimental methods, optical properties, thermodynamics of lipid bilayers, permeability, and electrical properties of BLMs. Section 7 deals with the use of BLM as energy transducer, particularly the transduction of light into electrical energy. Section 8, the longest portion of the paper, is devoted to modelling of biomembranes, such as the plasma membrane of cells, the thylakoid membrane of chloroplasts, the cristae membrane of mitochondria, the visual receptor membrane of the eye, and the nerve membrane. The concluding section points out that studies of BLMs facilitate the initial testing of working hypothses and may lead to a better choice of appropriate $\text{in vivo}$ and reconstituted membrane experiments.