Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes.

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.     

Application of voltammetric techniques to membrane studies

The application of voltammetric methods to planar bilayer lipid membranes (BLM) studies is described. BLM-compound interaction experiments lead to the measurement of the membrane current underlying transport phenomena. From measurements of current/voltage of BLM in unstirred solutions as a function of scan rates, it is possible to obtain both thermodynamic and kinetic information. In past years, a variety of techniques have been used to study the electrical properties of BLMs, but in terms of versatility, the cyclic voltammetric technique is outstanding. Cyclic voltammetry is the definitive means of characterizing the redox process of electroactive membranes.     

Interaction of phospholipid bilayers with polyamines of different length

The tip-dip patch clamp method was used to study the effect of three polyamines, putrescine, spermidine and spermine, on bilayer lipid membrane (BLM) stability. Two kinds of mixed lipid-polyamine membranes were investigated. The poration voltage (V _p), and the closed (σ_cl) and open (σ_op) state conductances for pure and polyamine-treated lipid membranes were determined by the method of current-voltage surfaces. It was demonstrated that putrescine and spermidine destabilized lipid membranes under all circumstances. BLM stabilization by spermine was observed when it was added to preformed membranes. Received: 19 November 1999 / Revised version: 25 February 2000 / Accepted: 25 February 2000     

Detection of transient capacitance increase associated with channel formation in lipid bilayers.

The insertion of alpha- and beta-latrotoxins and sea anemone (Radianthus macrodactilus) toxin into bilayer lipid membranes (BLMs) was investigated using the method of simultaneous conductance/capacitance measurement. All the toxins investigated induced capacitance changes which preceded toxin-induced conductance increases. The processes that may underlie the observed effect are discussed.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

Activity of toxins produced by Pseudomonas syringae pv. syringae in model and cell membranes

We studied effects of toxins produced by a bacterium Pseudomonas syringae pv. syringae on the conductance of bilayer lipid membranes (BLM). The used toxins were as follows: syringopeptin 22A (SP22A), syringomycin E (SPE), syringostatin A (SSA), syringotoxin B (STB), and methylated syringomycin E (CH3-SRE). All toxins demonstrated channel-forming activity. The threshold sequence for toxin activity was SP22A > SRE approximately equal to SSA > STB > CH3-SRE, and this sequence was independent of lipid membrane composition, and NaCl concentration (pH 6) in the membrane bathing solution (in the range of 0.1-1.0 M). This sequence correlated with relative bioactivities of toxins. In addition, SRE demonstrated a more potent antifungal activity than CH3-SRE. These findings suggest that ion channel formation may underlie the bioactivities of the above toxins. The properties of single ion channels formed by the toxins in BLMs were found to be similar, which points to the similarity in the channel structures. In negatively charged membranes, bathed with diluted electrolyte solutions (0.1 M NaCl), the channels were seen to open with positive transmembrane potentials (V) (from the side of toxin addition), and close with negative potentials. In uncharged membranes the opposite response to a voltage sign was observed. Increasing the NaCl concentration up to 1 M unified the voltage sensitivity of channels in charged and uncharged membranes: channels opened with negative V, and closed with positive V. With all systems, the voltage current curves of single channels were similarly superlinear in the applied voltage and asymmetric in its sign. It was found that the single channel conductance of STB and SSA was higher than that of other toxin channels. All the toxins formed at least two types of ion channels that were multiple by a factor of either 6 or 4 in their conductance. The results are discussed in terms of the structural features of toxin molecules.     

Identification of the cable parameters in the somatic shunt model

 We show that the first five moments of the soma potential and soma current uniquely and stably determine the soma conductance and capacitance and the dendritic electrotonic length, conductance, and capacitance in the so-called somatic shunt model of the passive behavior of a neuron. We test our resulting input admittance algorithm on synthetic data and demonstrate the regularizing effect of knowledge of the ratio of soma to dendrite surface areas. Received: 9 June 1999 / Accepted in revised form: 24 January 2000     

Pore Formation in Lipid Bilayer Membranes made of Phosphatidylcholine and Cholesterol Followed by Means of Constant Current

This paper describes the application of chronopotentiometry to lipid bilayer research. The experiments were performed on bilayer lipid membranes composed of phosphatidylcholine and cholesterol and formed using the painting technique. Chronopotentiometric (U = f(t)) measurements were used to determine the membrane capacitance, resistance, and breakdown voltage as well as pore conductance and diameter.     

The nicotinic acetylcholine receptor: from molecular model to single-channel conductance

The nicotinic acetylcholine receptor (nAChR) is the archetypal ligand-gated ion channel. A model of the α7 homopentameric nAChR is described in which the pore-lining M2 helix bundle is treated atomistically and the remainder of the molecule is treated as a “low resolution” cylinder. The surface charge on the cylinder is derived from the distribution of charged amino acids in the amino acid sequence (excluding the M2 segments). This model is explored in terms of its predicted single-channel properties. Based on electrostatic potential profiles derived from the model, the one-dimensional Poisson-Nernst-Planck equation is used to calculate single-channel current/voltage curves. The predicted single-channel conductance is three times higher (ca. 150 pS) than that measured experimentally, and the predicted ion selectivity agrees with the observed cation selectivity of nAChR. Molecular dynamics (MD) simulations are used to estimate the self-diffusion coefficients (D) of water molecules within the channel. In the narrowest region of the pore, D is reduced ca. threefold relative to that of bulk water. Assuming that the diffusion of ions scales with that of water, this yields a revised prediction of the single-channel conductance (ca. 50 pS) in good agreement with the experimental value. We conclude that combining atomistic (MD) and continuum electrostatics calculations is a promising approach to bridging the gap between structure and physiology of ion channels. Received: 2 August 1999 / Revised version: 5 November 1999 / Accepted: 9 November 1999     

Studies of Electric Capacitance of Membranes: I. A Model Membrane Composed of a Filter Paper and a Lipid Analogue

A hydrophobic filter paper of a given pore size containing a synthetic lipid, i.e. dioleyl phosphate, was interposed between aqueous electrolyte solutions having the same chemical composition and temperature. The electric capacitance and conductance of the membrane immersed in various concentrations of KCl were measured in the frequency range from 20 to 3 × 10⁶ cycle/sec. The observed capacitance and conductance were found to be strongly dependent on the applied frequency. A theory is proposed to account for this dispersion of impedance observed in the present membrane-electrolyte system. The dispersion is attributed to the formation of bilayer membranes of the lipid inside the filter paper. The effects of the salt concentration, the adsorbed quantity of the lipid, and the pore size of the filter paper on the capacitance and conductance of the membrane are discussed in terms of the distribution function of bilayers formed within the filter paper.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Millimeter microwave effect on ion transport across lipid bilayer membranes

The effects of millimeter microwaves in the frequency range of 54–76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB_?). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% ± 0.5%. At the same time, membrane current induced by TPhB transport increased by 5% ± 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% ± 0.4%). No “resonance-like” effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 °C. © 1995 Wiley-Liss, Inc.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Effect of ultrasound on a bilayer lipid membrane.

The effects of continuous wave ultrasound at a frequency of 1 MHz in the intensity range of 0-1.4 W/cm2 on an oxidized cholesterol bilayer lipid membrane (BLM) were observed. Ultrasound at 1.5 W/cm2 broke the membrane; in the range from 0.5 to 1.4 W/cm2, it accelerated the draining of the bulk lipid solution from the annulus to the Teflon support. At all intensities it has no effect on the conductance, the capacitance, or the dependence of each on the voltage applied across the membrane. Electrical parameters were measured in the presence of aqueous solutions of NaCl, KCl, and distilled water. The motivation and results of this project are explained in relation to an overall objective of determining the specific effects of ultrasound on biological membranes.     

Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.