Formation of cation channels in planar lipid bilayers by brefeldin A.

Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms.     

The mode of action of Vibrio cholerae cytolysin. The influences on both erythrocytes and planar lipid bilayers.

The interaction with erythrocytes of cholera cytolysin (CC) obtained from a non-01 Vibrio cholerae strain results in the osmotic rupture of target cells upon formation by CC of the waterfilled pores in their membranes. The aggregation of several toxin monomers is required for the formation of one CC channel with a radius of 0.9-1.0 nm. The investigations using planar bilayer lipid membranes suggest that the CC-induced pore is an interprotein anion selective channel carrying a fixed positive charge. The role of the charge was supported by the influence of pH on the selectivity, single conductance and voltage gating of the CC channels. The ability of the CC to modify both model and natural membranes has a maximum at pH 6.0-7.0. It was found that CC channels insert into the membrane asymmetrically. The effect of proteolytic treatment of the channel by papain also indicates that the two entrances of the channel protrude from the plane of the membrane into the solution for different distances. It is proposed that the biological effects of the non-01 V. cholera cytolysin are based on its channel-forming activity.     

Aerolysin,a hemolysin fromAeromonas hydrophila,forms voltage-gated channels in planar lipid bilayers

Summary The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between –70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2–3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.     

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.     

Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers.

Single epithelial Na+ channels incorporated into planar lipid bilayers were studied to determine the effects of Na concentration on its own conductance. Amiloride-sensitive Na+ channels were obtained from apical membrane vesicles made from A6 cells, a continuous epithelial cell-line derived from amphibian kidney. Single-channel conductance was found to be a saturable function of Na+ concentration, with a Michaelis constant of approximately 17 or 47 mM, for a Gmax of approximately 4 or 44 pS, respectively.     

Gaegurin 4, a peptide antibiotic of frog skin, forms voltage-dependent channels in planar lipid bilayers.

Gaegurin 4 (GGN4) is a cationic peptide of 37 amino acids (MW 3748) isolated from the skin of Rana rugosa. It has shown a broad spectrum antimicrobial activity in vitro against Gram-negative and -positive bacteria, fungi and protozoa. To understand its mechanism of antimicrobial action, we examined the effect of GGN4 on the membrane conductance and the electrical properties of GGN4-induced pores in planar lipid bilayers under voltage clamp. Natural and synthetic GGN4 (0.01-1 microg/mL) increased the membrane conductance in a concentration-dependent manner, but GGN4 (1-23), an N-terminal fragment of the peptide with little antimicrobial activity, failed to increase the conductance. At symmetrical 100 mM KCI, unitary conductances of about 120 pS were frequently observed. Their current-voltage relations were linear and open state probabilities were close to 1, but longer closing events were seen more frequently at negative voltages. In addition, GGN4-induced pores were selective for cation over anion, the permeability ratio of K+ to Cl- being 6: 1 in neutral and 7: 1 in acidic lipid bilayers. In conclusion, our results indicate that GGN4 forms voltage-dependent and cation-selective pores in planar lipid bilayers. The ionophoric property of GGN4 is likely to contribute to its antimicrobial activity.     

Phospholipase A2 and small, dense low-density lipoprotein

High levels of small, dense LDL in plasma are associated with increased risk for cardiovascular disease. There are some biochemical characteristics that may render small, dense LDL particles more atherogenic than larger, buoyant LDL particles. First, small, dense LDL particles contain less phospholipids and unesterified cholesterol in their surface monolayer than do large, buoyant LDL particles. This difference in lipid content appears to induce changes in the conformation of apolipoprotein B-100, leading to more exposure of proteoglycan-binding regions. This may be one reason for the high-affinity binding of small, dense LDL to arterial proteoglycans. Reduction of the phospholipid content in the surface monolayer LDL by treatment with secretory phospholipase A2 (sPLA2) forms small, dense LDL with an enhanced tendency to interact with proteoglycans. Circulating levels of sPLA2-IIA appears to be an independent risk factor for coronary artery disease and a predictor of cardiovascular events. In addition, in-vivo studies support the hypothesis that sPLA2 proteins contribute to atherogenesis and its clinical consequences. These data suggest that modification of LDL by sPLA2 in the arterial tissue or in plasma may be a mechanism for the generation of atherogenic lipoprotein particles in vivo, with a high tendency to be entrapped in the arterial extracellular matrix.     

An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A

The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A ( J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.     

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.     

A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal delta-endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation-selective channels in the bilayers, permeable to K+ and Na+ but not to N-methylglucamine or Cl-, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid-osmotic lysis.     

An outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of Rhizobium leguminosarum lipid A

The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A). With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A). The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

Like diphtheria toxin and the N-terminal (M_r 23 000) region of fragment B, CB1 (M_r 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (M_r 23 000 and 13 000) share a common segment (M_r 6300) containing the predicted amphipathic, -helical, transverse lipid-associating domain (M_r 2750) of fragment B[J. Cell Biol. (1980) 87, 837–840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172–176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C-terminal region of CB1 is proposed.     

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.