Planar Bilayer Membranes Research Articles

35] Use of planar lipid bilayer membranes for rapid screening of membrane active compounds

Planar lipid bilayer membranes (BLMs) have been in use for more than three decades for the study of membrane active compounds) BLMs provide a unique environment that allows the assay of the functional activity of carriers and channels translocating ions across membrane. Although BLMs are in vitro systems that have been used extensively for the measurement of biophysical properties of carriers and channels, their relevance to in vivo phenomena is discussed repeatedly through the more recent technology of patch clamping that has shown that channel properties measured in BLMs correspond qualitatively and often quantitatively to those observed in whole cells. This chapter describes a new BLM perfusion chamber that allows rapid, reversible, and quantitative assay of compounds on the bilayers.

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.

Artificial ion channels of amphiphilic ion pairs composed of oligoether-ammonium and hydrophobic carboxylate or phosphates

5,10,15,20-Tetraoxa-tetracosane-1-trimethylammonium cation was paired with anions from stearic acid, dioctadecyl phosphate, l-α-phosphatidic acid and cardiolipin to afford membrane-insertable amphiphilic ion pairs. These ion pairs were incorporated into planar lipid bilayer membranes to observe single ion channel currents. The ion pair with l-α-phosphatidic acid gave the most stable single ion channel currents with cation selectivity. An ion pair with dioctadecyl phosphate also gave a cation selective supramolecular ion channel. Even the stearate pair afforded single ion channel currents, although the cardiolipin did not give any sign of ion channel formation. These examples provide additional support for supramolecular ion channel formation from simple amphiphilic ion pairs. Among these supramolecular ion channels, only the pair with phosphatidic acid afforded a voltage dependent supramolecular ion channel. The combined evidence strongly suggests that the voltage dependence results simply from the incorporation of charges into the membrane phase.

Unmyristoylated MARCKS-related protein (MRP) binds to supported planar phosphatidylcholine membranes

We have recently shown that unmyristoylated MARCKS-related protein (MRP) does not bind to neutral phospholipid vesicles, unless negatively charged phospholipids are present. Similar behaviour has also been reported for MARCKS itself. Here we have compared the binding of MRP to neutral and negatively charged supported planar lipid bilayer membranes (SPLM) using two-mode waveguide spectroscopy. We find appreciable binding of unmyristoylated MRP to neutral SPLM. We propose that hydrophobic residues in the effector domain constitute an additional factor capable of mediating MRP-membrane interaction.

Effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores.

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a "flat" structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.

Effects of Hydroxyl Radical and Sulfhydryl Reagents on the Open Probability of the Purified Cardiac Ryanodine Receptor Channel Incorporated into Planar Lipid Bilayers

We examined the effects of hydroxyl radical on the ion permeability of the ryanodine receptor, a calcium releasing channel of sarcoplasmic reticulum. The cardiac ryanodine receptor, purified from pig heart, was reconstituted to proteoliposomes and then incorporated into a planar bilayer membrane. A single channel activity with a conductance of 724 pS in 900/200 mM (cis/trans) KCl and an ion selectivity of PK:PCl= 1:0.08 was observed. These characteristics are similar to those observed by the incorporation of the channel through sarcoplasmic reticulum vesicles. Hydroxyl radicals chemically generated by the reaction of H2O2and Cu(ethylenediamine)2at theciscompartment increased the open probability of the channel. Treatment with SH oxidizing reagents from theciscompartment also increased the open probability, and dithiothreitol, a SH reducing agent, reversed the effect. These findings suggest that hydroxyl radicals react with some SH groups of the ryanodine receptor and increase the Ca2+release from sarcoplasmic reticulum through the ryanodine receptor.

Ethanol potentiation of calcium-activated potassium channels reconstituted into planar lipid bilayers.

We examined the actions of ethanol on the single channel properties of large conductance Ca2+-activated K+ (BK) channels isolated from skeletal muscle T-tubule membranes and incorporated into planar lipid bilayer membranes. We have taken advantage of this preparation, because it lacks most elements of cellular complexity, including cytoplasmic constituents and complex membrane lipid composition and architecture, to examine the minimum requirements for the effects of alcohol. Clinically relevant concentrations (25-200 mM) of ethanol increased the activity of BK channels incorporated into bilayers composed of phosphatidylethanolamine (PE) alone or PE and phosphatidylserine. The potentiation of channel activity by ethanol was attributable predominantly to a decrease in the average amount of time spent in closed states. Ethanol did not significantly affect the current amplitude-voltage relationship for BK channels, indicating that channel conductance for K+ was unaffected by the drug. Although base-line characteristics of BK channels incorporated into bilayers composed only of PE differed from those of channels in PE/ phosphatidylserine in a manner expected from the change in bilayer charges, the actions of ethanol on channel activity were qualitatively similar in the different lipid environments. The effects of ethanol on single channel properties of BK channels in the planar bilayer are very similar to those reported for the action of ethanol on neurohypophysial BK channels studied in native membrane, and for cloned BK channels expressed in Xenopus laevis oocytes, which suggests that ethanol's site and mechanism of action are preserved in this greatly simplified preparation.

Theory of skin electroporation: implications of straight-through aqueous pathway segments that connect adjacent corneocytes.

Previous in vitro experiments have shown that transdermal high-voltage pulses (Uskin approximately 100 V; duration approximately 1 ms) create local transport regions (LTR) away from appendages in human skin. Quantitative interpretation of the associated ionic and molecular transport led to the view that a large number of aqueous pathways were created, and these connect the corneocytes within an LTR. Here we use the "brick wall" model of the stratum corneum, modified so that morphology important to understanding electrical behavior is emphasized. In this model a minimum-size LTR is regarded as an idealized stack of corneocytes in which the 5-6 multilamellar lipid bilayer membranes between adjacent corneocytes are electroporated. As in artificial planar bilayer and cell membrane electroporation, a distribution of pathway sizes is expected during pulsing, and during recovery after pulsing individual pathway segments are expected to shrink and close randomly, with a time constant tau(seg) that depends on temperature and on lipid composition. Numerical simulations based on stochastic closure of individual segments were used to predict the electrical conductance G(LTR)(t) of a minimum-size LTR after pulsing stops. These theoretical results show that simple exponential decay, G(LTR)(t) = G(LTR)(0)exp(-t/tau(seg)), occurs with minimal fluctuations if the number of pathways is large (np > 10(2)), but for much smaller values the conduction decreases erratically. A "stochastic bottleneck" leading to complete closure is reached only at about np < 3. Thus, for the same number of electrically created pathways, the stratum corneum will remain "open" longer if the pathways are located within an LTR than if the same number of pathways are distributed sparsely over the skin. These predictions are relevant to postpulse transport, including the trapping of linear macromolecules that can hold pathway segments open for prolonged intervals.

Cluster Organization of Ion Channels Formed by the Antibiotic Syringomycin E in Bilayer Lipid Membranes

The cyclic lipodepsipeptide, syringomycin E, when incorporated into planar lipid bilayer membranes, forms two types of channels (small and large) that are different in conductance by a factor of sixfold. To discriminate between a cluster organization-type channel structure and other possible different structures for the two channel types, their ionic selectivity and pore size were determined. Pore size was assessed using water-soluble polymers. Ion selectivity was found to be essentially the same for both the small and large channels. Their reversal (zero current) potentials with the sign corresponding to anionic selectivity did not differ by more than 3 mV at a twofold electrolyte gradient across the bilayer. Reduction in the single-channel conductance induced by poly(ethylene glycol)s of different molecular weights demonstrated that the aqueous pore sizes of the small and large channels did not differ by more than 2% and were close to 1 nm. Based on their virtually identical selectivity and size, we conclude that large syringomycin E channels are clusters of small ones exhibiting synchronous opening and closing.

Evidence for voltage-sensitive reaction steps in the mechanism of the red beet plasma membrane H +-ATPase

The effects of an imposed voltage on the ATP hydrolytic and transport activities of the red beet ( Beta vulgaris L.) plasma membrane H +-ATPase were examined following its purification and reconstitution into proteoliposomes or a planar bilayer membrane. When a large opposing (interior-positive) membrane voltage was established in proteoliposomes, ATP hydrolytic activity of the plasma membrane H +-ATPase decreased, but its sensitivity to vanadate was also reduced. Although an opposing voltage decreased ATP-dependent electrical current generation by the H +-ATPase, little change in the vanadate sensitivity of this activity was observed. These results were interpreted in terms of a voltage-sensitive reaction occurring at the E 1P → E 2P transition step in the enzyme reaction mechanism. The close fit of current-voltage (I/V) data to a two-state reaction-kinetic model for the red beet plasma membrane H +-ATPase, in a planar bilayer or proteoliposomes, was also consistent with the presence of a voltage-sensitive step in the mechanism being involved in charge translocation. Moreover, direct measurement of rate constants for the E 1P → E 2P reaction based upon dephosphorylation kinetics confirmed voltage-sensitivity for this step. The results of this study are discussed in terms of a role for the E 1P → E 2P reaction step in H + translocation and the possibility of the plant plasma membrane H +-ATPase displaying “slip” in the presence of an opposing membrane voltage.

Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin.

The transport of genetic material across biomembranes is a process of great relevance for several fields of study. However, much remains to be learned about the mechanisms underlying transport, one of which implies the involvement of proteic DNA-conducting pores. Entry of genetic material into mitochondria has been observed under both physiological and pathological conditions. We report here that double-stranded DNA can move through a planar bilayer membrane containing isolated mitochondrial porin (voltage-dependent anion channel). The transport is driven by the applied electrical field, and the presence of DNA is associated with a decrease of current conduction by the pores. The passage of DNA does not take place if the bilayer has not been doped with any protein or in the presence of both reconstituted porin and anti-porin antibody. Translocation does not occur if the bilayer contains Shigella sonnei maltoporin, gramicidin A channels, or a 30 pS anion-selective channel plus other proteins. These results show that mitochondrial porin is capable of mediating the transport of genetic material, revealing a new property of this molecule and further confirming the idea that DNA can move through proteic pores.

Molecular mechanisms of polymyxin B-membrane interactions: direct correlation between surface charge density and self-promoted transport.

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoyl-phosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d = 2.4 nm +/- 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction.

Signal transduction across alamethicin ion channels in the presence of noise

We have studied voltage-dependent ion channels of alamethicin reconstituted into an artificial planar lipid bilayer membrane from the point of view of electric signal transduction. Signal transduction properties of these channels are highly sensitive to the external electric noise. Specifically, addition of bandwidth-restricted "white" noise of 10-20 mV (r.m.s.) to a small sine wave input signal increases the output signal by approximately 20-40 dB conserving, and even slightly increasing, the signal-to-noise ratio at the system output. We have developed a small-signal adiabatic theory of stochastic resonance for a threshold-free system of voltage-dependent ion channels. This theory describes our main experimental findings giving good qualitative understanding of the underlying mechanism. It predicts the right value of the output signal-to-noise ratio and provides a reliable estimate for the noise intensity corresponding to its maximum. Our results suggest that the alamethicin channel in a lipid bilayer is a good model system for studies of mechanisms of primary electrical signal processing in biology showing an important feature of signal transduction improvement by a fluctuating environment.

DNA Translocation Across Planar Bilayers Containing Bacillus subtilis Ion Channels

The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood. We have developed a new methodology to study the translocation of genetic material via pores in a reconstituted system, using techniques from electrophysiology and molecular biology. We report here that planar bilayer membranes become permeable to double-stranded DNA (kilobase range) if Bacillus subtilis membrane vesicles containing high conductance channels have been fused into them. The translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a polyanion, is applied. It is not an aspecific permeation through the phospholipid bilayer, since it does not take place if no proteins have been incorporated into the membrane. The transport is also not due simply to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multi-protein vesicle fraction exhibiting 30-picosiemens anion-selective channel activity. The presence of DNA alters the behavior of the bacterial channels, indicating that it interacts with the pores and may travel through their lumen. These results support the idea that DNA translocation may take place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological experiments may be constituents of the DNA translocating machinery in these organisms.

Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor

The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.

The Role of the Cytoplasmic Tail Region of Influenza Virus Hemagglutinin in Formation and Growth of Fusion Pores

The effect of the cytoplasmic tail of influenza hemagglutinin (HA) (H3 subtype) on fusion kinetics and pore growth was examined. An SV40 recombinant virus was used to express wild-type (WT) HA and HA mutants containing changes in the HA cytoplasmic tail. HA and its mutants were expressed in CV-1 cells and the ability of these cells to fuse to either red blood cells (RBCs) or planar bilayer membranes was determined quantitatively. The percentage of cells expressing HA and the levels of expression were the same for WT HA or HA lacking its cytoplasmic tail (CT−), and for a mutant, MAY, in which the three HA C-terminal cysteine residues were replaced to block the addition of palmitate. When RBCs were colabeled with large and small aqueous dyes and fused to CV-1 cells expressing WT HA, transfer of the large dye was significantly slower and extent of transfer was lower than that of the small dye, indicating that pores did not expand quickly to large diameters. An absence of the HA cytoplasmic tail did not alter the time course of spread for either dye. When CV-1 cells expressing WT HA were fused to planar membranes, small pores tended to open and close repetitively (“flicker”) before a pore would continue to either grow irreversibly to large conductances or grow to intermediate sizes and then contract. For HA mutants CT−and MAY, flickering was less likely to occur, but these pores did evolve in a manner identical to WT HA postflicker pores. We conclude that palmitate covalently linked to cysteine residues of the HA cytoplasmic tail is required for pore flickering, but that the tail does not play an important role in subsequent pore enlargement.

Mechanisms of action of bactericidal/permeability-increasing protein BPI on reconstituted outer membranes of gram-negative bacteria.

The mechanisms of interaction of the recombinant N-terminal portion of bactericidal/permeability-increasing protein, rBPI21, with various planar asymmetric and symmetric bilayer membranes, including the lipid matrix of the outer membrane of Gram-negative bacteria, were investigated via electrical measurements. For the lipopolysaccharide (LPS) leaflet of the outer membrane, isolated deep rough mutant LPS of Escherichia coli strain F515 (F515 LPS) and Proteus mirabilis strain R45 (R45 LPS) were used. The addition of rBPI21 to the LPS side of asymmetric LPS/phospholipid membranes, as well as to black lipid membranes made from dioleoylphosphatidylglycerol (DOPG), led to membrane rupture. The innermembrane potential difference resulted in a slight increase from 0 to 5 mV for symmetric DOPG membranes but changed for asymmetric F515 LPS/PL membranes from -36 to +8 mV and for R45 LPS/PL membranes from -37 to -5 mV following the addition of rBPI21. In all cases, the addition of rBPI21 led to an increase in membrane current. The effect of rBPI21 on the innermembrane potential difference of LPS/PL membranes was significantly reduced in the presence of 40 mM MgCl2 (shift from -36 to -31 mV for F515 LPS). On the basis of these results and from our studies on the interaction of rBPI21 with lipid monolayers and aggregates [Wiese, A., et al. (1997) Biochemistry 36, 10301-10310], a model is discussed explaining how the observed membrane rupture, increase of membrane current, and change of transmembrane potential as induced by rBPI21 may contribute to bacterial dysfunction.

Pore-forming properties of proteolytically nicked staphylococcal alpha-toxin: the ion channel in planar lipid bilayer membranes.

Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.

The effect of blue light o electron transport in photosystem II reconstituted in planar bilayer lipid membrane

Photosystem II particles (PS II) isolated from bean leaves were reconstituted to planar lipid bilayer membranes (BLM). The light-driven electron transport across PS II-BLM has been observed. The direction of the electric current was consistent with the vectorial electron transport within PS II, i.e. between water and exogenous electron acceptor ferricyanide which was present only at one side of BLM. The photocurrent induced by blue light absorbed by both carotenoid and chlorophyll pigments was found to be much higher than that induced by red light absorbed exclusively by chlorophyll despite higher intensity of the red light applied, in agreement with the rate of photosynthetic oxygen evolution induced by blue and red light (Gruszecki et al., z. Naturforsch., 50c (1995) 61–68).

Transmembrane insertion of the colicin Ia hydrophobic hairpin.

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an alpha-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia.