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.     

Electrochemical elucidation on the mechanism of uncoupling caused by hydrophobic weak acids

Uncoupler-mediated cation transport has been investigated by cyclic voltammetry for the ion transfer from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) in the presence of typical uncouplers, 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) and 2,4-dinitrophenol (DNP). The voltammograms for the ion transfer were in a steady state and exhibited a rotated sigmoidal shape that was almost symmetrical about the origin (0 V, 0 A). The plot of the ion transfer current against pH was a bell-type curve centered on pH approximately = pK(a) + 1, K(a) being the dissociation constant of the uncouplers in the aqueous phase. Taking into account the ion transfer reactions at the W1|BLM and the BLM|W2 interfaces, these properties were well explained by our proposed model which considers that the ion transfer current is attributable to the facilitated transfers of H(+) and Na(+). The buffer action in the aqueous phase was found to play an important role in the facilitated H(+)-transfer across the BLM. The nature of the pH-dependence of the ion transfer current was reasonably explained from an electrochemical viewpoint based on the distribution coefficient of the anionic and neutral forms of SF6847, as estimated from its absorption spectra in liposomal membrane. The proposed model is also valuable for understanding the pH-dependence of uncoupling activity in mitochondria in the literature.     

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.     

Electrical potential distribution over the bilayer lipid membrane due to amphiphilic ion adsorption

Potential drops at the boundary of the bilayer lipid membrane (BLM) due to amphiphilic anion (dodecylsulfate) adsorption have been investigated. The magnitude of these drops was determined by different experimental methods: inner field compensation (IFC), electrophoretic mobility and current relaxation (tetraphenylboron and dipicrylamine were taken as probe anions).The boundary potential (BP) drops (IFC method) do not depend on the electrolyte concentration for neutral membranes. The ζ-potential values in the same conditions are considerably smaller than the BP drops measured by the IFC method. The potential drops, determined with the help of the initial BLM conductivity changes (current relaxation method) coincide with the BP drops (IFC method). The adsorption of amphiphilic ions leads to a decrease in the rate constant of the movement of hydrophobic ions through the BLM (current relaxation method).To explain the results obtained, it is suggested that a potential drop due to amphiphilic ion adsorption is located not only in the diffuse double layer, but also in a layer inside the membrane. The latter is not screened by electrolyte solution ions and could not be registered by the electrophoretic method.     

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.     

Voltammetric study of levomepromazine induced ionic permeability in a model lipid membrane system

The interaction of solid supported liquid films of lipids with levomepromazine is investigated in the present work by voltammetry. It is shown that the levomepromazine penetrates into the films, which results in a readily detectable change of the film permeability to electroactive species. In this respect, possibilities for the employment of supported lipid films as model systems in the research of biomembrane active compounds are briefly discussed.     

A potentiostatic study of hydrophobic ion transfer across lipid bilayer: Part I. Case of zero ion fluxes in adsorption and desorption processes

The transport of tetraphenylborate (TPB^?) and dipicrylamine (DPA^?) ions across glyceryl monoleate decane bilayers is studied under potentiostatic conditions. The transfer mechanism involving three steps (adsorption-translocation-desorption) is retained with the modification that the adsorbed charge, due to the transfer of ions, is taken into consideration. This threecapacitor B.L.M. model enables one to prodict, as is experimentally established, that the electrical driving force of the translocation process is only a fraction of the total applied voltage. In the particular case where the ion fluxes in the adsorption and desorption steps are negligible with respect to the capacitive current, the values of the translocation rate constant, the number of adsorbed ions and the adsorption and desorption capacitance are estimated from the current-time response curves.     

Voltammetric study of the transfer of fluoride ion at the nitrobenzene / water interface assisted by tetraphenylantimony.

The transfer of F- ion assisted by an organometallic complex cation tetraphenylantimony (TPhSb+) across the polarized nitrobenzene / water (NB / W) interface has been studied by means of ion-transfer voltammetry. A well-defined voltammetric wave was observed within the potential window at the NB / W interface when tetraphenylantimony tetrakis(4-chlorophenyl) borate and F- ion were present in NB and W, respectively. The voltammogram can be interpreted as being due to the reversible transfer of F- ion assisted by the formation of the TPhSbF complex through the coordination of F- to Sb atom in NB. The formal formation constant of TPhSbF in NB has been determined to be 10(1.95 +/- 0.2 M(-1). No voltammetric wave due to the TPhSb(+)-assisted transfer of other anions such as Cl-, Br, I-, NO3-, CH3COO- and H2PO4(-) ions has been observed within the potential window.     

Determination of trace anions in concentrated weak acids by ion chromatography

Ion-exclusion chromatography (ICE) followed by ion chromatography (IC) was used for the determination of trace anionic contaminants in concentrated weak acids. The ICE separation was used as a pretreatment step to isolate the contaminant anions of strong acids from the excess of matrix ions. Then a fraction containing the analyte ions was separated using IC with suppressed conductivity detection. Microbore–ion-exchange columns were chosen to address the increased purity requirements for use of these concentrated acids in semiconductor applications. The chromatographic conditions were optimized for determining trace chloride, sulfate, phosphate, and nitrate in concentrated 24.5% (v/v) hydrofluoric acid; trace chloride, sulfate, and nitrate in concentrated 85% (w/w) phosphoric acid and trace chloride and sulfate in concentrated 0.7% (v/v) glycolic acid. Method detection limits for the anions of interest were below 100 μg/l.     

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.     

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.     

Photoinduced electron transfer of chlorophyll in lipid bilayer system

Photoinduced electron transfer from chlorophyll-a throughtheinterface of dipalmitoylphosphatidylcholine (DPPC) headgroup of the lipid bilayers was studied with electron magnetic resonance (EMR). The photoproduced radicals were identified with electron spin resonance (ESR) and radical yields of chlorophyll-a were determined by double integration ESR spectra. The formation of vesicles was identified by changes in measured λ_max values from diethyl ether solutions to vesicles solutions indirectly, and observed directly with SEM and TEM images. The efficiency of photosynthesis in model system was determined by measuring the amount of chlorophyll-a radical yields which were obtained from integration of ESRspectra.     

Supported bilayer lipid membrane arrays on photopatterned self-assembled monolayers

This work demonstrates the use of photocleavable cholesterol derivatives to create supported bilayer lipid membrane arrays on silica. The photocleavable cholesteryl tether is attached to the surface by using the reaction of an amine-functionalized self-assembled monolayer (SAM) and the N-hydroxysuccinimide-based reagent 9. The resultant SAM contains an ortho-nitrobenzyl residue that can be cleaved by photolysis by using soft (365 nm) UV light regenerating the original amine surface, and which can be patterned using a mask. The photoreaction yield was approximately 75 % which was significantly higher than previously found for related ortho-nitrobenzyl photochemistry on gold substrates. The SAMs were characterized by means of contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Patterned surfaces were characterized with SEM and AFM. After immersing the patterned surface into a solution containing small unilamellar vesicles of egg phosphatidylcholine (PC), supported lipid membranes were formed comprised of lipid bilayer over the amine functionalized "hydrophilic" regions and lipid monolayer over the cholesteryl "hydrophobic" regions. This was confirmed by fluorescence microscopy and AFM. FRAP studies yielded a lateral diffusion coefficient for the probe molecule of 0.14+/-0.05 microm(2) s(-1) in the bilayer regions and approximately 0.01 microm(2) s(-1) in the monolayer regions. This order of magnitude difference in diffusion coefficients effectively serves to isolate the bilayer regions from one another, thus creating a bilayer array.     

An electrochemical impedance study of the effect of pathogenic bacterial toxins on tethered bilayer lipid membrane

Pathogenic bacteria secrete various virulence factors that can directly interact with the outer lipid bilayer membrane of eukaryotic cells, inducing cell death by apoptosis or necrosis. Such virulence factors account for much of the toxic action associated with bacterial infection; therefore the detection of such proteins could provide a methodology for sensing/detection of pathogenic bacteria in, for example, food or human tissue. Detection and identification of pathogenic bacteria by conventional methods such as plating and counting in laboratory is expensive and time consuming. With growing concerns over emergence and re-emergence of pathogenic bacteria with high resistant to current antibiotics, there is a potential need for effective detection of pathogenic toxins in-vitro. This paper presents the application of tethered bilayer lipid membrane (TBLM) as a sensing platform for the detection of the clinically relevant pathogenic bacterial, Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1 via their secreted virulence factors, using electrochemical impedance spectroscopy (EIS). A non-pathogenic strain of bacteria, E. coli DH5α was used as a control. A clear difference in the impedance of the TBLM for the pathogenic vs. non-pathogenic species was observed.     

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.     

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.     

Dynamics and instabilities of lipid bilayer membrane shapes

Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a ‘slipping mode’ in addition to the conventional ‘bending mode’ for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions.     

Cation selective ion channels formed by macrodiolide antibiotic elaiophylin in lipid bilayer membranes

The macrodiolide antibiotic elaiophylin (1) forms stable, long-lasting cation selective ion channels in planar lipid bilayer membranes prepared from soybean phosphatidylcholine. Current of the single ion channel displayed two sublevels corresponding to the two substates of the channel conductance: a slow substate, with about 5 s of mean dwell time in the open state at 40% level of the total amplitude conductance, and a fast substate of higher conductance with dwell times in the open and closed state of about 0.1 s. Amplitude conductances of the single ion channels in 200 mM of LiCl, NaCl, KCl, RbCl and CsCl were 75, 140, 220, 240 and 226 pS, and the conductance was linear function of the electrolyte concentration. Ratios of cation to anion permeabilities of the channel for NaCl and KCl were 8+/-2 and >24, respectively. A molecular model of the channel structure is suggested.     

Kinetics of weak distance-dependent hole transfer in DNA by adenine-hopping mechanism

The kinetics of hole transfer in DNA by adenine-hopping mechanism was investigated by the combined pulse radiolysis-laser flash photolysis method. The hole transfer from Ptz*+* to oxG across the (A)n-bridge preceded by the A-hopping mechanism and the weak distance-dependent hole transfer with the rates faster than 108 s-1 over the distance range of 7-22 A was demonstrated. In contrast, hole transfer from oxG*+ to Ptz followed the single-step super exchange mechanism. Thus, two different processes for the hole transfer across the identical (A)n-bridge in DNA have been demonstrated. The results clearly show that the mechanism of hole transfer in DNA strongly depends on the redox nature of the oxidant, whether it produces only G*+ or both A*+ and G*+.     

Impact on lipid membrane organization by free branched-chain fatty acids

Here, we exploit the non-invasive techniques of solid-state NMR (nuclear magnetic resonance) and differential scanning calorimetry (DSC) to study the effect of free iso and ante-iso branched chain fatty acids (BCFAs) on the physicochemical properties of lipid membranes. Free fatty acids are present in biological membranes at low abundance, but can influence the cellular function by modulating the membrane organization. Solid state NMR spectra of dimyristoylphosphatidylcholine (DMPC) lipid membranes containing either free 12-methyltetradecanoic acid (a15:0) or free 13-methyltetradecanoic acid (i15:0), show significant differences in their impact on the lipid bilayer. Chain order profiles obtained by deuterium NMR on fully deuterated DMPC-d(67) bilayers revealed an ordering effect induced by both fatty acids on the hydrophobic membrane core. This behavior was also visible in the corresponding DSC thermograms where the main phase transition of DMPC bilayers-indicative of the hydrophobic membrane region-was shifted to higher temperatures, with the iso isomer triggering more pronounced changes as compared to the ante-iso isomer. This is probably due to a higher packing density in the core of the lipid bilayer, which causes reduced diffusion across membranes. By utilizing the naturally occurring spin reporters nitrogen-14 and phosphorus-31 present in the hydrophilic DMPC headgroup region, even fatty acid induced changes at the membrane interface could be detected, an observation reflecting changes in the lipid headgroup dynamics.