Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.     

Transport of protons and hydrochloric acid through lipid bilayer membranes

Transport of protons and hydrochloric acid through lipid bilayer membranes was studied by a combination of electrical conductance and pH electrode techniques. In the presence of large pH gradients, proton transport occurs primarily by diffusion of molecular HCl. The permeability of egg phosphatidylcholine/decane bilayers to HCl is about 3 cm . s-1, seven to nine order of magnitude higher than the permeability to H+, OH- or Cl-. The HCl permeability of phosphatidylserine or egg phosphatidylcholine/cholesterol (1 : 1) bilayers is about 50% lower than the permeability of egg phosphatidylcholine bilayers. Diffusion of molecular HCl may be an important process in tissues exposed to high HCl concentrations, e.g., gastric mucosa. However, at neutral pH the diffusion of molecular HCl is too slow to contribute significantly to net movements of H+ or Cl-.     

Faster-than-anticipated Na/Cl diffusion across lipid bilayers in vesicles

Maintenance of electrochemical potential gradients across lipid membranes is critical for signal transduction and energy generation in biological systems. However, because ions with widely varying membrane permeabilities all contribute to the electrostatic potential, it can be difficult to measure the influence of diffusion of a single ion type across the bilayer. To understand the electrodiffusion of H⁺ across lipid bilayers, we used a pH-sensitive fluorophore to monitor the lumenal pH in vesicles after a stepwise change in the bulk pH. In vesicles containing the ion channel gramicidin, the lumenal pH rapidly approached the external pH. In contrast, the lumen of intact vesicles showed a two stage pH response: an initial rapid change occurred over ~ 1 min, followed by a much slower change over ~ 24 h. We provide a quantitative interpretation of these results based on the Goldman–Hodgkin–Katz ion fluxes discharging the electrical capacitance of the bilayer membrane. This interpretation provides an estimate of the permeability of the membranes to Na⁺ and Cl⁻ ions of ~ 10^− 8 cm/s, which is ~ 3 orders of magnitude faster than previous reports. We discuss possible mechanisms to account for this considerably higher permeability in vesicle membranes.     

Comparison of double layer potentials in lipid monolayers and lipid bilayer membranes

Summary It is shown that the Gouy-Chapman double layer analysis adequately describes the variation of the surface potential of monolayers of acidic natural lipids over a wide range of surface charge density and salt concentration. It is also shown that the potential which initially appears when an electrolyte gradient is rapidly imposed across a bilayer membrane is due to a difference in the double layer potentials on the two sides of the membrane. This conclusion follows from the fact that the observed bilayer potentials arise much more rapidly than can be accounted for by charge migration across the membrane and from the observation that the bilayer membrane concentration potentials, when measured immediately after establishment of a gradient, are equal to the surface potential change observed when the subphase concentration of a monolayer of the same lipid is changed by an amount equal to the gradient across the bilayer. The bilayer potential and monolayer potential changes, so measured, agree in a number of different electrolyte solutions over a wide range of electrolyte concentrations and surface charge densities. Because of this agreement and the applicability of the Gouy theory to monolayers, initial bilayer potentials may be calculated if the composition of the mixture used to form the membrane is known, provided that the pK's and areas of such components are available. In the absence of this information, membrane potentials may be calculated from electrophoretic data on the membrane lipid mixture; the conditions under which the latter approach is possible have been determined. The experimental results indicate that the composition of monolyers and bilayers spread from the same lipid mixture in decane are very similar, that the composition of the two types of film closely resembles the composition of the solution used to generate them, and that bilayer membranes are close-packed. The evidence further indicates that if any hydrocarbon solvent remains in these bilayers, it must be so situated that it contributes little, if anything, to the surface area. The steady state potential in the bilayer membrane system is frequently not identical with the initial potential which supports the hypothesis that in many cases only a fraction of the electrical conductance of unmodified membranes is caused by the ions which constitute the bulk electrolyte. An expression for the relationship between diffusion and double layer potentials has been derived which shows that, in the absence of any intrinsic selectivity of the hydrocarbon region of the membrane for hydrogen, hydroxyl, or impurity, the two potentials should be identical.     

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.     

Voltage-dependent capacitance in lipid bilayers made from monolayers.

Electrocompression has been measured in lipid bilayers made by apposition of two monolayers. The capacitance C(V), as a function of membrane potential, V, was found to be well described by C(V) = C(O) [1 + alpha(V + delta psi)2] where C(O) is the capacitance at V = O, alpha is the fractional increase in capacitance per square volt, and delta psi is the surface potential difference. In lipid bilayers made from monolayers alpha has a value of 0.02 V-2, which is ca. 500-fold smaller than the value found in solvent containing membranes. In asymmetric bilayers made of one neutral and one negatively charged monolayer, delta psi values were found to be those expected from independent measurements of surface charge density. If the fractional increase in capacitance found here is a good approximation to that of biological membranes, nonlinear capacitative charge displacement derived from electrostriction is expected to be less than 1% of the total gating charge displacement found in squid axons.     

Potential generation in bilayer lipid membranes in the NADH-flavin mononucleotide-ubiquinone-6-O2 system

Membrane conductance and generation of transmembrane potential by the NADH oxidation reactions in the NADH-flavin mononucleotide-ubiquinone-6-O₂ system have been studied. It has been shown that in solutions of a relatively low buffer capacity at pH 5.8 in the presence of a proton carrier, a potential is generated, the value of which depends on the concentration of the reducer and amounts to 40–60 mV. In the absence of a proton carrier at pH 8, a potential arises, which suggests a transmembrane negative charge transfer. Bilayer lipid membranes have been shown to possess proton selectivity if the reaction is run at pH 3.7. At a pH higher than 5.8 the proton selectivity disappears. Schemes of potential generation in lipid bilayers in different conditions are suggested and discussed.     

Tributyltin-mediated exchange diffusion of halides in lipid bilayers

This paper describes the effect of tributyltin (TBT) on the inorganic anion permeability of lipid bilayers. When this compound is added in micromolar concentrations to one or both sides of a phosphatidyl ethanolamine (PE) membrane formed in 0.1 M NaCl or KCl (pH 7), there is no change in the electrical conductance. Under these circumstances, the Cl self-exchange flux measured with 36Cl (MCl) increases from a value of approximately 10(-12) mol.cm-2.s-1, to approximately 10(-8) mol.cm-2.s-1. It was further found that the relation between chloride flux and [TBT] and [Cl] can be described as: MCl = B[TBT] [Cl]. When chloride was replaced by an equimolar concentration of different univalent anions in the trans compartment, the heteroexchange flux of chloride followed the sequence: I greater than Br greater than Cl greater than F greater than NO3. Under all experimental conditions tested, the chloride flux was always more than 10(3) times the maximum flux predicted from the value of the membrane conductance, and at least 100 times higher than the expected fluxes of ion pairs (TBT-Cl) diffusing across the unstirred layers. Thus, the mechanism by which tributyltin increases anion permeability in bilayers seems to be that of an obligatory exchange diffusion, with the reaction between tributyltin and the halides occurring at the membrane surface. Measurements of interfacial potentials indicate that tributyltin chloride lowers the positive intrinsic dipole potential of PE membranes by approximately 70 mV (at a TBT concentration of 30 microM) without substantial alteration of other parameters of the bilayer. The estimated adsorption coefficient of TBT-Cl was found to be 3 x 10(-4) cm.     

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Electrically silent anion transport through lipid bilayer membranes containing a long-chain secondary amine

The permeability properties of planar lipid bilayers made from egg lecithin, n-decane and a long-chain secondary amine (n-lauryl [trialkylmethyl]amine) are described. Membranes containing the secondary amine show halide selectivity and high conductance at pH less than 6, as estimated by measurements of zero-current potentials generated by NaBr activity gradients. In the absence of halide ions, the membranes show H+ selectivity, although the total membrane conductance is relatively low. In 0.1 M NaBr both the membrane conductance (Gm) and the Br- self-exchange flux (JBr) are proportional to H+ concentration over the pH range of 7 to 4, and both JBr and Gm saturate at pH less than 4. However, JBr is always more than 100 times the flux predicted from Gm and the transference number for Br-. Thus, greater than 99% of the observed (tracer) flux is electrically silent and is not a Br2 or HBrO flux because the reducing agent, S2O3=, has no effect on JBr. At pH 7, JBr is proportional to Br- concentration over the range of 1-340 mM, with no sign of saturation kinetics. Both urea and sulfate tracer permeabilities are low and are unaffected by pH. The results can be explained by a model in which the secondary amine behaves as a monovalent, titratable carrier which exists in three chemical forms (C, CH+, and CHBr). Br- crosses the membrane primarily as the neurtal complex (CHBr). The positively charged carrier (CH+) crosses the membrane slowly compared to CHBr, but CH+ is the principal charge carrier in the membrane. At neurtal pH greater than 99% of the amine is in the nonfunctional form (C), which can be converted to CH+ or CHBr by increasing the H+ or Br- concentrations. The permeability properties of these lipid bilayers resemble in many respects the permeability properties of red cell membranes.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.     

Influence of dolichyl phosphate on permeability and stability of bilayer lipid membranes

The ionic permeability coefficients, ionic transference numbers, activation energy of ion transport and breakdown voltage of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl 12-phosphate have been studied. The electrical measurements showed that dolichyl phosphate in phospholipid bilayers decreases membrane permeability, changes membrane ionic selectivity and increases membrane stability. These results are discussed in light of the aggregation behavior and the intramolecular clustering of a dolichyl phosphate molecule in phospholipid membranes. From our data we suggest that the hydrophilic part of dolichyl phosphate molecules regulates their behavior in membranes.     

Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes

Purple membranes were adsorbed on freestanding lipid bilayers, termed nano-black lipid membranes (nano-BLMs), suspending the pores of porous alumina substrates with average pore diameters of 280 nm. Nano-BLMs were obtained by first coating the upper surface of the highly ordered porous alumina substrates with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and octadecylamine dissolved in n-decane onto the hydrophobic submonolayer. By means of impedance spectroscopy, the quality of the nano-BLMs was verified. The electrical parameters confirm the formation of single lipid bilayers with high membrane resistances covering the porous matrix. Adsorption of purple membranes on the nano-BLMs was followed by recording the photocurrents generated by bacteriorhodopsin upon continuous light illumination. The membrane system exhibits a very high long-term stability with the advantage that not only transient but also stationary currents are recordable. By adding the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone the conductivity of the nano-BLMs increases, resulting in a higher stationary current, which proves that proton conductance occurs across the nano-BLMs.     

Effect of increased lipid packing on the surface charge of micelles and membranes

We have investigated the responsiveness of micelle and bilayer surfaces to changes in bulk pH through titrations, and to changes in lipid packing through the application of high hydrostatic pressure using two fluorescent, pH-sensitive surface probes. In micelles, the surface is very sensitive to bulk pH while in phosphatidylcholine and phosphatidic acid bilayers the surface charge changed little through a large pH region. Application of pressure on micelles causes proton dissociation due to the volume reduction achieved from the contraction of water around the charges (electrostriction). However, in bilayers, the effect of electrostriction is greatly reduced, most likely due to the energy needed to expand and hydrate the surface. The sign and amount of change in dissociation the probe undergoes with pressure depend on the initial degree of probe dissociation, which is in turn dependent on the particular surface rather than the charge of the lipid head groups comprising the bilayer. This finding may limit the use of fluorescent probes to determine the exact surface potential. By assuming the change in delta V for proton dissociation from the probe is constant for a given pH, we can calculate the changes in local pH that occur under pressure relative to a neutral or uncharged system. In doing so, we find that the local pH around the probe in bilayers changes very little (approximately 0.1 pH unit or less) in the first 2000 bars. Also, if pressure data are coupled with titration curves, we can determine the change that the bulk pH must undergo to produce the observed change in dissociation seen under pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm² for DPhyPC bilayers and 0.75 and 0.77 μF/cm² for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm² were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Reversible binding of substance P to artificial lipid membranes studied by capacitance minimization techniques

Interaction of substance P with electrically neutral, planar lipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and with anionic bilayers prepared from mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and brain phosphatidylserine was measured using the capacitance minimization method for monitoring the membrane surface potential caused by the positive charges and electric dipole moment of adsorbed peptide. Substance P bound to the electrically neutral bilayers from 9 mM KCl (buffered to pH 5.5 with 2.0 mM 2-(N-morpholino)ethanesulfonate) with a maximal binding density of about 1 x 10(-2) molecules per nm2 and a dissociation constant of about 2 x 10(-4) M. Measurement of the surface potential at different ionic strengths (shielding of surface charges) allowed distinction between the fixed-charge surface potential and a dipole potential. Ascribing this dipole potential to membrane-bound substance P would imply an effective dipole moment normal to the bilayer surface of about 20 Debye per molecule. Magnitude and polarity are consistent with an alpha-helical domain at the C-terminal end of substance P which is oriented normal to the surface of the membrane, and inserted so as to be inaccessible to the aqueous phase. Consistent measurements were obtained with anionic membranes at low substance P concentrations (10(-7)-10(-6) M; pH 7.2). They indicated electrostatic accumulation of the triply charged peptide on the surface of the membrane followed by hydrophobic interaction with the same parameters as for neutral membranes. The results agree with the membrane structure of substance P determined with infrared attenuated total reflection spectroscopy, circular dichroism measurements, and thermodynamic estimations.     

Monitoring the permeability profile of lipid membranes with spin probes

The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows first-order kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile. The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the half-time of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.     

Modulation of proton transfer in the water wire of dioxolane-linked gramicidin channels by lipid membranes

Proton conductance (g(H)) in single SS stereoisomers of dioxolane-linked gramicidin A (gA) channels were measured in different phospholipid bilayers at different HCl concentrations. In particular, measurements were obtained in bilayers made of 1,2-diphytanoyl 3-phosphocholine (DiPhPC) or its ethylated derivative 1,2-diphytanoyl 3-ethyl-phosphocholine (et-DiPhPC,). The difference between these phospholipids is that in et-DiPhPC one of the phosphate oxygens is covalently linked to an ethyl group and cannot be protonated. In relatively dilute acid solutions, g(H) in DiPhPC is significantly higher than in et-DiPhPC. At high acid concentrations, g(H) is the same in both diphytanoyl bilayers. Such differences in g(H) can be accounted for by surface charge effects at the membrane/solution interfaces. In the linear portion of the log g(H)-log [H] relationship, g(H) values in diphytanoyl bilayers were significantly larger (approximately 10-fold) than in neutral glyceryl monooleate (GMO) membranes. The slopes of the linear log-log relationships between g(H) and [H] in diphytanoyl and GMO bilayers are essentially the same (approximately 0.76). This slope is significantly lower than the slope of the log-log plot of proton conductivity versus proton concentration in aqueous solutions (approximately 1.00). Because the chemical composition of the membrane-channel/solution interface is strikingly different in GMO and diphytanoyl bilayers, the reduced slope in g(H)-[HCl] relationships may be a characteristic of proton transfer in the water wire inside the SS channel. Values of g(H) in diphytanoyl bilayers were also significantly larger than in membranes made of the more common biological phospholipids 1-palmitoyl 2-oleoyl phosphocholine (POPC) or 1-palmitoyl 2-oleoyl phosphoethanolamine (POPE). These differences, however, cannot be accounted for by different surface charge effects or by different internal dipole potentials. On the other hand, maximum g(H) measured in the SS channel does not depend on the composition of the bilayer and is determined essentially by the reduced mobility of protons in concentrated acid solutions. Finally, no experimental evidence was found in support of a lateral proton movement at the phospholipid/solution interface contributing to g(H) in single SS channels. Protein-lipid interactions are likely to modulate g(H) in the SS channel.