The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin.
Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE.
Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open.
A better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and for in vitro assessment, variants of Enterococcus faecalis that are resistant to different doses of the fungal AMP alamethicin (Almr) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Almr variants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Almr variants showed cross-resistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Almr variants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using an in vitro colorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.
Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca2+-permeable cation channel in the regulation of cytosolic Ca2+ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.
Ca2+ channel; two-pore channel 1; intracellular localization; plant cells
Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ~7, the net charge of alamethicin is −1 whereas that of melittin is +5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.
molecular dynamics simulations; antimicrobial peptides; toroidal pore; barrel-stave pore; melittin; alamethicin
Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.
molecular dynamics simulations; antimicrobial peptides; toroidal pore; barrel-stave pore; implicit solvent
The induction of plant defences involves a sequence of steps along a signal transduction pathway, varying in time course. In this study, the effects of induction of an early and a later step in plant defence signal transduction on plant volatile emission and parasitoid attraction are compared. Ion channel-forming peptides represent a class of inducers that induce an early step in signal transduction. Alamethicin (ALA) is an ion channel-forming peptide mixture from the fungus Trichoderma viride that can induce volatile emission and increase endogenous levels of jasmonic acid (JA) and salicylic acid in plants. ALA was used to induce an early step in the defence response in Brussels sprouts plants, Brassica oleracea var. gemmifera, and to study the effect on volatile emission and on the behavioural response of parasitoids to volatile emission. The parasitoid Cotesia glomerata was attracted to ALA-treated plants in a dose-dependent manner. JA, produced through the octadecanoid pathway, activates a later step in induced plant defence signal transduction, and JA also induces volatiles that are attractive to parasitoids. Treatment with ALA and JA resulted in distinct volatile blends, and both blends differed from the volatile blends emitted by control plants. Even though JA treatment of Brussels sprouts plants resulted in higher levels of volatile emission, ALA-treated plants were as attractive to C. glomerata as JA-treated plants. This demonstrates that on a molar basis, ALA is a 20 times more potent inducer of indirect plant defence than JA, although this hormone has more commonly been used as a chemical inducer of plant defence.
Alamethicin; Brussels sprouts; Cotesia glomerata; jasmonate; parasitoid host-location behaviour; peptaibol; octadecanoid pathway; salicylate; volatile emission
Structures of membrane associated peptides and molecular interactions between peptides and cell membrane bilayers govern biological functions of these peptides. Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study such structures and interactions at the molecular level. In this research, SFG has been applied, supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), to characterize interactions between alamethicin (a model for larger channel proteins) and different lipid bilayers in the absence of membrane potential. The orientation of alamethicin in lipid bilayers has been determined using SFG amide I spectra detected with different polarization combinations. It was found that alamethicin adopts a mixed α-helical and 310- helical structure in fluid-phase lipid bilayers. The helix (mainly α-helix) at the N-terminus tilts at about 63° versus the surface normal in a fluid-phase 1,2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine-1,1,2,2-D4-N,N,N-trimethyl-D9 (d-DMPC)/1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bilayer. The 310 helix at the C-terminus (beyond the Pro14 residue) tilts at about 43° versus the surface normal. This is the first time to apply SFG to study a 310 helix experimentally. When interacting with a gel-phase lipid bilayer, alamethicin lies down on the gel-phase bilayer surface and/or aggregates, which does not have significant insertion into the lipid bilayer.
Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analogue MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136 ns to 216 ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for the toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melitin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.
molecular dynamics simulations; antimicrobial peptides; charge distribution; imperfect amphipathicity; melittin; magainin MG-H2
There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP’s interaction with lipid membranes. In this study we show that oriented-sample 31P solid-state NMR spectroscopy can be used to probe the membrane perturbations and -disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.
Alamethicin is a well-studied channel-forming peptide that has a prototypical amphipathic helix structure. It permeabilizes both microbial and mammalian cell membranes, causing loss of membrane polarization, and leakage of endogenous contents. Antimicrobial peptide-lipid systems have been studied quite extensively and have led to significant advancements in membrane biophysics. These studies have been performed on lipid bilayers that are generally charged or zwitterionic and restricted to a thickness range of 3 – 5 nm. Bilayers of amphiphilic diblock copolymers are a relatively new class of membranes that can have significantly different material properties compared with those of lipid membranes. In particular, they can be made un-charged, non-zwitterionic, and much thicker than their lipid counterparts. In an effort to extend studies of membrane-protein interactions to these synthetic membranes, we have characterized the interactions of alamethicin and several other membrane-active peptides with diblock copolymer bilayers. We find that although alamethicin is too small to span the bilayer, the peptide interacts with, and ruptures thick polymer membranes.
Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e., from leaf to leaf, the “system potential” (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in substomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.
alamethicin; long distance electrical signal; depolarization; non-invasive recording
Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 μM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 μM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 μM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 μM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.
Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split alpha/310 helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer, and measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH=6.7 the α-helix at the N-terminus and the 310 helix at the C-terminus tilt at about 72° (θ1) and 50° (θ2) versus the surface normal, respectively. When pH increases to 11.9, θ1 and θ2 decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ1 for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.
Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO3−) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO3− efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled us to record the activation of slow-type deactivating anion channel currents. Cryptogein-induced plasma membrane depolarization and Ca2+ influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO3− efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO3− efflux in mediating proteases activation was further assessed. It is shown that cryptogein induced the activation of three proteases with apparent molecular masses of 95, 190 and 240 kDa. Their activation occurred independently on the anion efflux and, together with cell death, was strongly reduced by cycloheximide and the protease inhibitor PMSF. In contrast, the NO3− efflux was shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during the HR. Collectively, our data indicate that anion efflux is an early prerequisite to morphological and biochemical events participating to cell death.
anion channels; calcium; cell death; cryptogein; plasma membrane depolarization; proteases
Alamethicin has been extensively studied as an antimicrobial peptide and is widely used as a simple model for ion channel proteins. It has been shown that the antimicrobial activity of peptides is related to their membrane orientation. In this study, we determined the relationship between the solution concentration of alamethicin and its membrane orientation in lipid bilayers using sum frequency generation (SFG) vibrational spectroscopy. Our SFG results indicated that the alamethicin molecules more or less lay down on the surface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers at a low peptide concentration of 0.84 μM; the α-helix segment tilts at about 88°, and 310-helix segment tilts at about 58° versus the surface normal. However, when the peptide concentration was increased to 15.6 μM, we observed that alamethicin molecules further inserted into the lipid bilayers: the α-helical component changes its orientation to make a 37° tilt from the lipid bilayer normal, and the 310-helical component tilts at about 50° versus the surface normal. This is in agreement with the barrel-stave mode for the alamethicin–cell membrane interaction as reported previously. Additionally, we have also studied membrane orientation of alamethicin as a function of peptide concentration with SFG. Our results showed that the membrane orientation of the alamethicin α-helical component changed substantially with the increase of the alamethicin concentration, while the membrane orientation of the 310-helical component remained more or less the same.
Sum frequency generation vibrational spectroscopy; antimicrobial peptide; supported lipid bilayer; α-helix; 310-helix
To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.
While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin - a model voltage-gated ion channel - was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.
The use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.
This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW ~30 kD) to the ion channel-forming peptides (MW ~2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore, or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor – 4-carboxybenzenesulfonamide – to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of ~2 µM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.
The photodynamic effects of sulphonated aluminium phthalocyanine (SALPC) have been compared on cultured AR4-2J cells of a pancreatic carcinoma cell line and on exocrine cells of the normal phenotype freshly isolated from the rat pancreas; a multi-channel perifusion system was used for this kinetic study in vitro. Whereas light alone or SALPC alone was without effect on either cell type, photon activation of cellularly-bound SALPC with light greater than 570 nm permeabilised the cells and caused an increase in amylase secretion from normal acinar cells but a dose-dependent inhibition (10(-7) to 10(-5) M) of amylase release from AR4-2J cells. In contrast, direct permeabilisation of the plasma membrane with digitonin, 10 micrograms ml-1, evoked a marked release of amylase from both types of cell. Elevation of [Ca2+]i by the ionophore A23187, 10(-6) M, elicited secretion of amylase from normal cells but had little effect on AR4-2J cells. Finally, it was established that the differential photodynamic effects of SALPC on amylase release were not attributable to any topographical differences in the microanatomical organisation of normal or tumour-derived cells; furthermore, the structural integrity of normal and AR4-2J cells was maintained after the photodynamic action of SALPC. It is concluded that the generation of singlet oxygen is responsible for permeabilisation of both types of cell and that photon-activated SALPC has functionally distinct effects on the constitutive secretion of amylase of tumour cells and the regulated secretory pathway of normal cells. These observations may be important in the development of drugs with a selective photodynamic action on pancreatic tumour cells.
Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10–15 mM nitrate.
Mycorrhiza; Nitric oxide; Nitrite:NO reductase; Palmitvaccenic acid; Plasma membrane; Root
Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.
Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.
electric conductivity; lipid bilayers
X-ray scattering features induced by aggregates of alamethicin (Alm) were obtained in oriented stacks of model membranes of DOPC(diC18:1PC) and diC22:1PC. The first feature obtained near full hydration was Bragg rod in-plane scattering near 0.11 Å-1 in DOPC and near 0.08 Å-1 in diC22:1PC at 1:10 Alm:lipid ratio. This feature is interpreted as bundles consisting of N Alm monomers in a barrel-stave configuration surrounding a water pore. Fitting the scattering data to previously published MD simulations indicates that the number N of peptides per bundle is N=6 in DOPC and N≥9 in diC22:1PC. The larger bundle size in diC22:1PC is explained by hydrophobic mismatch of Alm with the thicker bilayer. A second diffuse scattering peak located at qr≈0.7 Å-1 is obtained for both DOPC and diC22:1PC at several peptide concentrations. Theoretical calculations indicate that this peak can not be caused by the Alm bundle structure. Instead, we interpret it as due to two-dimensional hexagonally packed clusters in equilibrium with Alm bundles. As the relative humidity was reduced, interactions between Alm in neighboring bilayers produced more peaks with three dimensional crystallographic character that do not index with the conventional hexagonal space groups.
alamethicin; aggregation; hydrophobic mismatch; water pore; helix bundle; ion channel
According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, β-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name.
Membrane-active peptides; Lipid-mixing; Membrane fusion; Circular dichroism; Conformational changes; Designer-made sequences; Peptide-lipid interactions