A new system for bilayer lipid membrane capacitance measurements: method, apparatus and applications.

A new method and a new apparatus for capacitance measurements on bilayer lipid membranes are described. The membrane is charged and discharged with a constant current during the measurement. The charge-discharge cycle duration, which is proportional to the membrane capacitance, is measured. The measured time period is converted into a binary number by digital systems and then this number is either further converted into a constant capacity-proportional voltage or read out by the computer. The apparatus makes it possible to measure the capacitances of voltage-polarized membranes. Application of the apparatus to capacitance measurements of bilayer lipid membranes during their potential on the capacitance is presented. The capacitances of membranes stimulated by rectangular voltage pulses and of those stimulated by a linearly varying potential were reported.     

BLM Analyzer: a software tool for experiments on planar lipid bilayers

Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a lack of software utilities for assistance during bilayer formation. We present here a multipurpose software tool, the bilayer lipid membrane (BLM) Analyzer which performs high-resolution measurements of bilayer capacitance and resistance using saw-tooth voltage stimulation. Based on the measured values of capacitance and resistance, the BLM Analyzer detects formation, stabilization, and breakage of lipid bilayer, automatically selects appropriate stimulus protocol, compensates for voltage offsets, and issues sound and voice alerts informing about the state of the measurement cycle. The principle of the BLM Analyzer is based on the integration of current responses within four equivalent time segments. It provides capacitance estimates with standard deviation of several femtofarads at temporal resolution of several tens of milliseconds. The functions of the BLM Analyzer were tested experimentally by monitoring formation and thinning of planar lipid bilayer.     

Interaction of influenza virus proteins with planar bilayer lipid membranes. I. Characterization of their adsorption and incorporation into lipid bilayers

Alterations in the surface potential difference (delta U) of asolectin planar bilayer lipid membranes were measured following the adsorption of isolated matrix protein (M-protein) or neuraminidase of influenza virus. The method used was based upon measurement of the bilayer lipid membrane capacitance current second harmonic. The delta U dependence on the M-protein and neuraminidase concentration indicates different mechanisms of adsorption of these viral proteins by the lipid bilayer. The conductance (G0) dependence of the bilayer lipid membrane with different compositions on the concentration of isolated surface glycoproteins, hemagglutinin and neuraminidase, M-protein or neuraminidase was investigated. The change in G0 for M-protein was observed only after adsorption saturation had been achieved. Neuraminidase alone does not affect the membrane conductivity. The surface charge and lipid composition of the lipid bilayer influences the adsorption and incorporation of influenza virus M-protein and surface glycoproteins. The reversibility of protein incorporation into the bilayers was investigated by a perfusion technique. The results show reversibility of surface glycoprotein incorporation while M-protein binding appears to be irreversible.     

Chronopotentiometric Technique as a Method for Electrical Characterization of Bilayer Lipid Membranes

The basic electrical parameters of bilayer lipid membranes are capacitance and resistance. This article describes the application of chronopotentiometry to the research of lipid bilayers. Membranes were made from egg yolk phosphatidylcholine. The chronopotentiometric characteristic of the membranes depends on the current value. For low current values, no electroporation takes place and the voltage rises exponentially to a constant value. Based on these kinds of chronopotentiometric curves, a method of the membrane capacitance and the membrane resistance calculations are presented.     

System for Measuring Planar Lipid Bilayer Properties

We present a system for measuring planar lipid bilayer properties. The system is composed of a control unit, an output stage, an LCR meter, pumps for filling reservoirs, a bath with temperature regulation and a measurement chamber with four electrodes. The planar lipid bilayer is automatically formed using a folding method on apertures of different sizes. The automatization is assured by two syringes, which are clamped in actuators. Actuators are driven and controlled by a control unit via RS-232 communication. The temperature of the planar lipid bilayer can be regulated between 15 and 55 °C. The regulation is assured by insertion of the measurement chamber into the temperature-regulated bath. Different shapes of voltage- or current-clamp signals can be applied to the planar lipid bilayer. By measuring the response of the planar lipid bilayer to the applied signal, the capacitance and breakdown voltage of the planar lipid bilayer can be determined. The cutoff frequencies of the system output stage for voltage- and current-clamp methods are 11 and 17 kHz, respectively.     

On measurements of the higher harmonics of transmembrane current

The higher harmonics of the current caused by an alternating voltage applied to bilayer lipid membranes made of diphytanoyl phosphatidylcholine (DPhPC) in decane and tetradecane were measured. A universal relation between the amplitudes of harmonics was proposed and experimentally verified. This allowed the coefficients of expansion of the capacitance in even powers of voltage to be calculated for the DPhPC membrane in tetradecane; it also permitted comparison of the inhomogeneity in the thickness of the DPhPC membranes in decane and tetradecane.     

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.     

Electrical noise from lipid bilayer membranes in the presence of hydrophobic ions

In the presence of the hydrophobic ion dipicrylamine, lipid bilayer membranes exhibit a characteristic type of noise spectrum which is different from other forms of noise described so far. The spectral density of current noise measured in zero voltage increases in proportion to the square of frequency at low frequencies and becomes constant at high frequencies. The observed form of the noise spectrum can be interpreted on the basis of a transport model for hydrophobic ions in which it is assumed that the ions are adsorbed in potential-energy minima at either membrane surface and are able to cross the central energy barrier by thermal activation. Accordingly, current-noise results from random fluctuations in the number of ions jumping over the barrier from right to left and from left to right. On the basis of this model the rate constant ki for the translocation of the hydrophobic ion across the barrier, as well as the mean surface concentration Nt of adsorbed ions may be calculated from the observed spectral intensity of current noise. The values of ki obtained in this way closely agree with the results of previous relaxation experiments. A similar, although less quantitative, agreement is also found for the surface concentration Nt.     

Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier.

The voltage dependence of capacitance was measured by using the setup which was almost the same as that for the study of ion channels. The coefficient which represents the voltage dependence of capacitance itself also changes as a function of the duration of voltage application if hexadecane is contained in bilayer lipid membrane (BLM). The method of Alvarez, O., and R. Latorre (1978. Biophys. J. 21:1-17) was extended to treat BLM with hexadecane.     

Optimizing planar lipid bilayer single-channel recordings for high resolution with rapid voltage steps.

We describe two enhancements of the planar bilayer recording method which enable low-noise recordings of single-channel currents activated by voltage steps in planar bilayers formed on apertures in partitions separating two open chambers. First, we have refined a simple and effective procedure for making small bilayer apertures (25-80 micrograms diam) in plastic cups. These apertures combine the favorable properties of very thin edges, good mechanical strength, and low stray capacitance. In addition to enabling formation of small, low-capacitance bilayers, this aperture design also minimizes the access resistance to the bilayer, thereby improving the low-noise performance. Second, we have used a patch-clamp headstage modified to provide logic-controlled switching between a high-gain (50 G omega) feedback resistor for high-resolution recording and a low-gain (50 M omega) feedback resistor for rapid charging of the bilayer capacitance. The gain is switched from high to low before a voltage step and then back to high gain 25 microseconds after the step. With digital subtraction of the residual currents produced by the gain switching and electrostrictive changes in bilayer capacitance, we can achieve a steady current baseline within 1 ms after the voltage step. These enhancements broaden the range of experimental applications for the planar bilayer method by combining the high resolution previously attained only with small bilayers formed on pipette tips with the flexibility of experimental design possible with planar bilayers in open chambers. We illustrate application of these methods with recordings of the voltage-step activation of a voltage-gated potassium channel.     

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.     

Changes in the electrical and visco-elastic properties of bilayer lipid membranes during interaction with proteins and lipoproteins

A method for simultaneous registration of planar bilayer lipid membrane (BLM) DC conductance G, capacitance C, surface potential difference delta phi and transversal elasticity module E is developed. C, delta phi and E are proportional to the amplitude of the first, second and third harmonics of capacitance current respectively. A comparative study of the interaction of BLM with very low density lipoproteins (VLDL), influenza virus matrix protein (M-protein) and yeast invertase was carried out. The kinetics of delta phi, E and G changes at different concentrations of VLDL, and dependence of delta phi and G on M-protein and invertase concentration was investigated. It is shown for VLDL invertase and M-protein that the changes in delta phi and E occur before the change in G. The method used permits to study peculiarities of individual stages of interaction between charge particles, supramolecular structures and lipid membranes.     

Theory of cation-phospholipid-induced shape changes in a lipid bilayer couple

A quantitative model of ion binding and molecular interactions in the lipid bilayer membrane is proposed and found to be useful in examining the factors underlying such membrane characteristics as shape, sidedness, stability and vesicle size at various cation concentrations. The lipid membrane behaves as a bilayer couple whose preferential radius of curvature depends on the expansion or contraction of one monolayer relative to the other. It is proposed that molecular packing may be altered by electrostatic repulsion of adjacent like-charged phospholipid headgroups, or by bringing two headgroups closer together by divalent cation crossbridging. The surface concentrations of each type of cation-phospholipid complex can be described by simple binding equilibria and the Gouy-Chapman-Stern formulation for the surface potential in a diffuse double layer. The asymmetric distribution of acidic phospholipids in most biological membranes can account for the differential effects of identical ionic environments on either side of the bilayer. The fraction of vesicle material which tends to have a right-side-out orientation may be approximated by a normal distribution about the mean curvature. The theory generates vesicle sidedness distributions that, when fitted to experimental results from human erythrocyte membranes, provide an alternative method of estimating intrinsic cationphospholipid dissociation constants and other molecular parameters of the bilayer. The results also corroborate earlier suggestions that the Gouy-Chapman theory tends to overestimate free counter-ion concentrations at the surface under large surface potentials.     

Formation of "solvent-free" black lipid bilayer membranes from glyceryl monooleate dispersed in squalene.

A simple technique for forming "black" lipid bilayer membranes containing negligible amounts of alkyl solvent is described. The membranes are formed by the method of Mueller et al (Circulation. 1962. 26:1167.) from glyceryl monooleate (GMO) dispersed in squalene. The squalene forms an annulus to satisfy the boundary conditions of the planar bilayer but does not appear to dissolve noticeably in the bilayer itself. The specific geometric capacitance (Cg) of the membranes at 20 degrees C formed by this technique is 0.7771 +/- 0.0048 muF/cm2. Theoretical estimates of Cg for solvent-free bilayers range from 0.75 to 0.81 muF/cm2. Alkane-free GMO bilayers formed from n-octadecane by the solvent freeze-out method of White (Biochim. Biophys. Acta. 1974. 356:8) have values of Cg = 0.7903 +/- 0.0013 muF/cm2 at 20.5 degrees C. The agreement between the various values of Cg strongly suggests that the bilayers are free of squalene. DC potentials applied to the bilayers have no detectable effect on the value of Cg, as expected for solvent-free films. The ability to form bilayers essentially free of the solvent used in the forming solution makes it possible to determine the area per molecule of the surface active lipid in the bilayer. The area per molecule of GMO at 20 degrees C is estimated to be 37.9 +/- 0.2 A2.     

Electrical Response to Vibration of a Lipid Bilayer Membrane

The discovery and characterization of a vibration response in a black lipid bilayer membrane is the topic of this paper. An electrical vibration response is obtained when the membrane is under voltage clamp and a weaker, but significant, response is obtained under current clamp. The effect arises from an induced variation in the membrane capacitance. It is further shown that the capacitance variation arises from a change in the membrane area as the membrane undergoes drumhead vibration. Possible physiological significance in mechanoreception is discussed.     

Interaction of influenza virus proteins with planar bilayer lipid membranes. II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (delta U), transversal elasticity module (E1) and membrane conductivity (G0) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 X 10(5) M-1 and 1.3 X 10(4) M-1 for rimantadine and amantadine, respectively. The changes in G0 took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 micrograms/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Surface potential determination in planar lipid bilayers: a simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba(2+)) to the bathing solution. Ba(2+) ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density. Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667-687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

A study of inhomogeneity of bilayer lipid membranes by measuring the higher harmonics of the transmembrane current

The higher harmonics of the alternating current in bilayer lipid membranes induced by a sinusoidal voltage applied to the membrane were measured. The bilayer lipid membranes were prepared from diphytanoylphosphatidylcholine in decane and tetradecane; a 16-bit analog-to-digital converter was used for the measurements. A sinusoidal voltage was formed with a 16-bit digital-to-analog converter. The dynamic measurement range reached 90 dB. The coefficients α and β of the expansion of capacitance C in terms of the membrane voltage U—C = C ₀(1 + αU ² + βU ⁴)—were determined from the measurement results. It was shown within the framework of the electrostriction model that a certain ratio of the coefficients α and β characterizes the inhomogeneity of the membrane with respect to its thickness and Young’s modulus of elasticity.     

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side.