Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.     

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.     

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.     

Alzheimer's beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism in a hypothalamic GnRH neuronal cell line

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.     

Fresh and globular amyloid beta protein (1-42) induces rapid cellular degeneration: evidence for AbetaP channel-mediated cellular toxicity.

Amyloid beta peptides (AbetaP) deposit as plaques in vascular and parenchymal areas of Alzheimer's disease (AD) tissues and Down's syndrome patients. Although neuronal toxicity is a feature of late stages of AD, vascular pathology appears to be a feature of all stages of AD. Globular and nonfibrillar AbetaPs are continuously released during normal cellular metabolism, form calcium-permeable channels, and alter cellular calcium level. We used atomic force microscopy, laser confocal microscopy, and calcium imaging to examine the real-time and acute effects of fresh and globular AbetaP(1-42), AbetaP(1-40), and AbetaP(25-35) on cultured endothelial cells. AbetaPs induced morphological changes that were observed within minutes after AbetaP treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to AbetaP(1-42). AbetaP(1-42)-induced morphological changes were observed at nanomolar concentrations and were accompanied by an elevated cellular calcium level. Morphological changes were prevented by anti-AbetaP antibody, AbetaP-channel antagonist zinc, and the removal of extracellular calcium, but not by tachykinin neuropeptide, voltage-sensitive calcium channel blocker cadmium, or antioxidants DTT and Trolox. Thus, nanomolar fresh and globular AbetaP(1-42) induces rapid cellular degeneration by elevating intracellular calcium, most likely via calcium-permeable AbetaP channels and not by its interaction with membrane receptors or by activating oxidative pathways. Such rapid degeneration also suggests that the plaques, and especially fibrillar AbetaPs, may not have a direct causative role in AD pathogenic cascades.     

Proapoptotic triterpene electrophiles (avicins) form channels in membranes: cholesterol dependence

Avicins, a family of triterpenoid saponins from Acacia victoriae, can regulate the innate stress response in human cells. Their ability to induce apoptosis in transformed cells makes them potential anticancer agents. We report that avicins can form channels in membranes. The conductance reached a steady state after each addition, indicating a dynamic equilibrium between avicin in solution and in the membrane. The high power dependence (up to 10) of the membrane conductance on the avicin concentration indicates the formation of multimeric channels, consistent with the estimated pore radius of 1.1 nm. This radius is too small to allow protein flux across the mitochondrial outer membrane, a process known to initiate apoptosis. Channel formation is lost when avicin's amphipathic side chain is removed, implicating this as the channel-forming region. A small difference in this side chain results in strong cholesterol dependence of channel formation in avicin G that is not found in avicin D. In neutral membranes, avicin channels are nonselective, but negatively-charged lipids confer cation selectivity (5:1, K(+):Cl(-)), indicating that phospholipids form part of the permeation pathway. Avicin channels in the mitochondrial outer membrane may favor apoptosis by altering the potential across this membrane and the intermembrane space pH.     

Amyloid beta peptides mediate hypoxic augmentation of Ca(2+) channels

Clinical studies indicate that neurodegeneration caused by Alzheimer's amyloid beta peptide (AbetaP) formation can be triggered or induced by prolonged (chronic) hypoxia. Here, we demonstrate that 24-h culture of PC12 cells in 10% O(2) leads to induction of a Cd(2+)-resistant Ca(2+) influx pathway and selective potentiation of L-type Ca(2+) current. Both effects were suppressed or prevented by a monoclonal antibody raised against the N'-terminus of AbetaP, and were fully mimicked by AbetaP(1-40 and) AbetaP(1-42), but not by AbetaP(40-1). Potentiation of L-type currents was also induced by exposure to AbetaP(25-35). Our results indicate that hypoxia induces enhancement of Ca(2+) channels, which is mediated by increased AbetaP formation.     

Kinetic characteristics of the excitability-inducing material channel in oxidized cholesterol and brain lipid bilayer membranes

The kinetic characteristics of the opening and closing of the excitability-inducing material (EIM) channel in oxidized cholesterol and in brain lipid bilayers are compared. The kinetics of the opening and closing of individual ion-conducting channels in bilayers doped with small amounts of EIM are determined from discrete fluctuations in ionic current. The kinetics for approach to steady-state conductance are determined for lipid bilayers containing many channels. Steady-state and kinetic characteristics for the EIM channel incorporated in brain lipid bilayers can be accounted for by the model developed for the EIM channel incorporated in oxidized cholesterol membranes. Relaxation time, calculated from rate constants of single-channel membranes or directly measured in many-channel membranes is strongly temperature dependent, and is always shorter in brain lipid membranes. Changes in temperature do not affect the interaction of the electric field and the open channel, but the open configuration of the EIM channel in brain lipid bilayers is stablized with increasing temperature. The configurational energy difference between the open and closed channel, calculated from temperature studies, is larger in brain lipid bilayers. The energy barrier which separates the two configurations of the channel is larger in oxidized cholesterol bilayers.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

β—淀粉样蛋白31—35片段对海马神经元分离膜片Ca^2＋激活大电导钾通道的抑制

为了确定β－淀粉样蛋白（AβP）在影响神经元电生理特性并导致神经毒作用时的最短活性序列,实验采用片钳技术,在急性分离的大鼠海马CA1区锥体细胞的“内面向外”式膜片上,观察了AβP的31－35和25－35片段对C^2 a激活大电导钾（BK）通道活动的影响,结果显示,浴液中给预5umol/L的AβP31－35后,BK通道的平均开放概率（P0）和开放频率在1－3min内分别减少了85．8％（P＜0．01）和72．1％（P＜0．01）,平均开放时间减少了41．1％（P＜0．01）,平均电流幅度则无明显改变（P＞0．05）,给予同样摩尔浓度的AβP25－35后,BK通道平均P0减少了85．5％（P＜0．01）,平均开放时间减少51．4％,（P＜0．05）,结果提示：两种AβP片段对海马神经元BK通道具有抑制作用,。这可能与AβP的神经性作用有关,AβP－31－35片段可能是AβP分子中影响细胞电生理特性的最小活性序列。     

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.     

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.     

Bilayer-gel membranes. Formation and some properties.

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields "bilayer-gel" membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-L-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

Bilayer-gel membranes. Formation and some properties

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields ‘bilayer-gel’ membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-l-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

Mechanism of ion transport through the anion-selective channel of the Pseudomonas aeruginosa outer membrane

Protein P trimers isolated and purified from Pseudomonas aeruginosa outer membrane were reconstituted in planar lipid bilayer membranes from diphytanoyl phosphatidylcholine. The protein trimers formed highly anion-specific channels with an average single channel conductance of 160 pS in 0.1 M Cl solution. A variety of different nonvalent anions were found to be permeable through the channel, which suggests a channel diameter between 0.5 and 0.7 nm. The selectivity for the halides followed the Eisenman sequence AVI (without At-). The ion transport through the protein P channel could be explained reasonably well by a one-site, two-barrier model. The stability constant of the binding of Cl- to the site was 20 M-1 at neutral pH. The binding of anions to the site was pH dependent, which suggested that several charges are involved in the closely spaced selectivity filter. Permeability ratios for different anions as calculated from bi-ionic potentials showed agreement with corresponding ratios of single channel conductances. The protein P channels were not voltage-gated and had lifetimes of the order of several minutes. The current-voltage curves were linear for membrane potentials up to 150 mV, which suggested that Nernst-Planck-type barriers rather than Eyring barriers were involved in the movement of anions through the protein P channel.     

Potential-dependent formation of single conducting ion channels in lipid bilayers induced by the polyene antibiotic levorin A2

The aromatic polyene antibiotic levorin A₂ forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3–0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A₂ channels is observed in lipid membranes. The system responsible for the ion-channel selectivity is localized on the hydrophilic side of the lactone ring of the polyene molecule.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.     

Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel

Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.     

SR-BI is required for microvillar channel formation and the localization of HDL particles to the surface of adrenocortical cells in vivo

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl ester into liver and steroidogenic tissues. In steroidogenic cells, juxtaposed microvilli, or microvilli snuggled against the plasma membrane create microvillar channels that fill with HDL. Microvillar membranes contain SR-BI and are believed to be the site of HDL cholesteryl ester uptake. A recent study showed that SR-BI expression in insect cells elicits membrane structures that contain SR-BI, bind HDL, and closely resemble the ultrastructure of microvillar channels. In the present study we compared the ultrastructure of adrenal gland microvillar membranes in Srb1+/+ and Srb1-/- mice to test whether SR-BI is required for the formation of microvillar channels. The results show that SR-BI is absolutely required for microvillar channel formation and that the microvillar membranes of Srb1-/- mice are 17% thinner than in Srb1+/+ mice.We conclude that SR-BI has a major influence on plasma membrane ultrastructure and organization in vivo.