Channel-forming Activity Research Articles

The effect of dipole potential of lipid bilayers on the properties of ion channels formed by cyclic lipodepsipeptide syringomycin E

The effect of membrane dipole potential ( ϕ d ) on the properties of ion channels formed in bilayer lipid membranes by syringomycin E (SRE), a toxin produced by Pseudomonas syringae , has been studied. It has been shown that ϕ d affects the conductance and lifetime of elementary SRE channels as well as their cluster organization, in particular, the number of elementary channels synchronously opened in the cluster and the life- time of these clusters. The channel-forming activity of SRE was found to be ϕ d -dependent. The analysis of experimental data has revealed that (i) the mechanisms of the observed effects involve the dipole-dipole and charge-dipole interactions responsible for the cooperative functioning of the elementary SRE channels; (ii) about 95% of membrane dipole potential is shielded in the SRE pore; and (iii) the channel-forming activity of SRE is mainly determined by the gating charge of the SRE channels. At the same time, the partition coefficient for the toxin distribution between the membrane and aqueous phase as well as the chemical component of the channel formation work are also responsible for the ϕ d -dependence of the SRE channel forming activity.

Translocation of an Aib-containing peptide through cell membranes

The biophysical characteristics and channel-forming activity of peptaibols inserted into artificial membranes have been studied over the last 30 years. However, to our knowledge, no studies have addressed directly their behavior in living cells. In this work, a novel strategy has been employed to precisely assess the living cell membrane-penetrating activity of a fluorescein-labeled Aib (α-aminoisobutyric acid)-containing peptide derived from a peptaibol, trichorovin-XIIa (TV-XIIa). We have demonstrated for the first time that the peptide containing an unusual amino acid residue, Aib, is taken up by cells via a non endocytic pathway. The replacement of Aib in the TV-XIIa sequence with Ala inhibits the cellular uptake.

Altering the Activity of Syringomycin E via the Membrane Dipole Potential

The membrane dipole potential is responsible for the modulation of numerous biological processes. It was previously shown (Ostroumova, O. S.; Kaulin, Y. A.; Gurnev, P. A.; Schagina, L. V. Langmuir 2007, 23, 6889-6892) that variations in the dipole potential lead to changes in the channel properties of the antifungal lipodepsipeptide syringomycin E (SRE). Here, data are presented demonstrating the effect of the membrane dipole potential on the channel-forming activity of SRE. A rise in the dipole potential is accompanied by both an increase in the minimum SRE concentration required for the detection of single channels at fixed voltage and a decrease in the steady-state number of open SRE channels at a given SRE concentration and voltage. These alterations are determined by several factors: gating charge, connected with translocations of lipid and SRE dipoles during channel formation, the bilayer-water solution partitioning of SRE, and the chemical work related to conformational changes during channel formation.

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta6.3 single-stranded and beta5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.

Oral secretions from herbivorous lepidopteran larvae exhibit ion channel-forming activities

Feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Various OS-derived molecules must be involved in subsequent processes including the induction of plant defence reactions. Using the planar lipid bilayer membrane technique, isolated OS were analyzed with respect to their membrane activities. Transmembrane ion fluxes were generated by OS of eight different lepidopteran larvae, all of which form comparable ion channels in artificial membranes. Currents were characterized by long lasting open times and conductivities from 250 pS up to 1100 pS. Channels formed by Spodoptera exigua secretions showed a preference for cations over anions. OS also induced a transient increase of the cytosolic calcium concentration in soybean cells, determined by the aequorin technique. Known compounds of the OS, fatty-acid–glutamine conjugates, also interfered with the membrane but were unable to form stable channels. Since ion fluxes and depolarization are early responses upon insect feeding, OS-derived components may be involved in the elicitation process by direct interaction with the plant membranes.

The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori, the bacterium associated to gastroduodenal ulcers and stomach cancers. VacA induces formation of cellular vacuoles that originate from late endosomal compartments. VacA forms an anion-selective channel and its activity has been suggested to increase the osmotic pressure in the lumen of these acidic compartments, driving their swelling to vacuoles. Here, we have tested this proposal on isolated endosomes that allow one to manipulate at will the medium. We have found that VacA enhances the v-ATPase proton pump activity and the acidification of isolated endosomes in a Cl- dependent manner. Other counter-anions such as pyruvate, Br-, I- and SCN- can be transported by VacA with stimulation of the v-ATPase. The VacA action on isolated endosomes is associated with their increase in size. Single amino acid substituted VacA with no channel-forming and vacuolating activity is unable to induce swelling of endosomes. These data provide a direct evidence that the transmembrane VacA channel mediates an influx of anions into endosomes that stimulates the electrogenic v-ATPase proton pump, leading to their osmotic swelling and transformation into vacuoles.

Diversity in the Oligomeric Channel Structure of the Multidrug Efflux Pumps in Pseudomonas aeruginosa

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.

Elimination of channel-forming activity by insertional inactivation of thep66gene inBorrelia burgdorferi

P66 is a chromosomally encoded 66-kDa integral outer membrane protein of the Lyme disease agent Borrelia burgdorferi exhibiting channel-forming activity. Herein, we inactivated and subsequently complemented the p66 gene in the B31-A (WT) strain. The P66 protein was also inactivated in two other channel-forming protein mutant strains, P13-18 (Deltap13) and Deltabba01, and then compared with the channel-forming activities of wild-type and various p66 mutant strains. We further investigated the ion-selectivity of native, purified P66. In conclusion, we show that the porin activity of P66 is eliminated by insertional inactivation and that this activity can be rescued by gene complementation.

The Mannose Transporter Complex: an Open Door for the Macromolecular Invasion of Bacteria

In this issue of the Journal of Bacteriology, Bieler et al. demonstrate (i) that the toxic activity of microcin E492 is independent of the “route of administration,” viz. whether it is added from the outside (as in nature) or expressed endogenously in the cytoplasm, and (ii) that microcin E492 requires the transporter for mannose to deploy its ion channel-forming activity. This is the strongest and most direct evidence to date that the mannose transporter is involved in microcin activity in gram-negative enterobacteria.

Pseudomonas aeruginosa Porin OprF: PROPERTIES OF THE CHANNEL

Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer (Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220-16229). With the fraction depleted for the single domain conformer, we were unable to detect formation of any channels with well defined conductance levels. With the unfractionated OprF, we saw only rare channel formation. However, with the single domain-enriched fraction of OprF, we observed regular insertion of channels with highly reproducible conductances. Single OprF channels demonstrate rich kinetic behavior exhibiting spontaneous transitions between several subconformations that differ in ionic conductance and radius measured in polymer exclusion experiments. Although we showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we also found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only. Therefore, the low permeability of OprF reported earlier may be due to two factors: mainly to the paucity of the single domain conformer in the OprF population and secondly to the predominance of weakly conductive subconformations within the single domain conformer.

Photorhabdus luminescens toxin‐induced permeability change in Manduca sexta and Tenebrio molitor midgut brush border membrane and in unilamellar phospholipid vesicle

Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.

Actin and amphiphilic polymers influence on channel formation by Syringomycin E in lipid bilayers

The bacterial lipodepsipeptide syringomycin E (SRE) added to one (cis-) side of bilayer lipid membrane forms voltage dependent ion channels. It was found that G-actin increased the SRE-induced membrane conductance due to formation of additional SRE-channels only in the case when actin and SRE were applied to opposite sides of a lipid bilayer. The time course of conductance relaxation depended on the sequence of SRE and actin addition, suggesting that actin binds to the lipid bilayer and binding is a limiting step for SRE-channel formation. G-actin adsorption on the membrane was irreversible. The amphiphilic polymers, Konig's polyanion (KP) and poly(Lys, Trp) (PLT) produced the actin-like effect. It was shown that the increase in the SRE membrane activity was due to hydrophobic interactions between the adsorbing molecules and membrane. Nevertheless, hydrophobic interactions were not sufficient for the increase of SRE channel-forming activity. The dependence of the number of SRE-channels on the concentration of adsorbing species gave an S-shaped curve indicating cooperative adsorption of the species. Kinetic analysis of SRE-channel number growth led to the conclusion that the actin, KP, and PLT molecules form aggregates (domains) on the trans-monolayer. It is suggested that an excess of SRE-channel formation occurs within the regions of the cis-monolayer adjacent to the domains of the adsorbed molecules, which increase the effective concentration of SRE-channel precursors.

Effect of block architecture on the ability of polyalkylene oxides to overcome multidrug resistance of tumor cells

The relationship between the block architecture of copolymers consisting of poly(ethylene oxide) and poly(propylene oxide), their ability to induce disturbances in lipid bilayer membranes and cause chemosensitization of tumor cells was investigated. It was found that the ability of copolymers to induce disturbances in model membranes composed of egg lecithin increased with the volume of their hydrophobic blocks. Extremely high channel-forming and flippase activity was observed for the copolymer possessing a brush-like structure. The addition of cholesterol into lipid bilayers reduced the effects induced by copolymers, however, to different extents. The sensitivity of copolymer-induced effects to the addition of cholesterol increased with the growth of the critical packing parameter, which was estimated as a ratio of the volume of the whole copolymer chain to the water-accessible area of its polar headgroup and the distance between the hydrophilic and hydrophobic edges of the molecule. The comparison of the copolymers effect on multi-drug resistant human breast adenocarcinoma MCF7/DOX cells showed that two block copolymers RPE caused maximal chemosensitization, compromising between the shape of the amphiphile and the volume of its hydrophobic block.

Lipid-mediated inactivation of colicin E1 channels by calcium ions

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.

Solute traffic across mammalian peroxisomal membrane--single channel conductance monitoring reveals pore-forming activities in peroxisomes.

Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90-96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.

Antistaphylococcal metabolite from <i>Aureobasidium pullulans: production and characterization</i>

Aureobasidium pullulans (NI.3) isolated from the leaves of Dracaena reflexa variegate produced intracellular antimicrobial metabolite the yield of which was 700-800 U from about 0.7-0.85 g of dry biomass. The antistaphyloccocal metabolite showed strong activity against different Staphylococcus spp. The MICs ranged from 1.25 to 3.6 U/ml. The metabolite was only moderately sensitive to temperature. After keeping at 400C and 700C for one hour it lost only 20% and 60% of its activity respectively. However, it was completely inactivated upon exposure to 1210C for 20 min. The antistaphyloccocal metabolite was insensitive to various protein-denaturing detergents and enzymes like trypsin, proteinase K, lipase and lysozyme. The activity was fairly stable over a wide range of pH (5.7–8). When S. aureus was grown in the medium in presence of antimicrobial metabolite (10 U/ml) the number of CFU started to decrease. However, most of the cells had lost their viability after nine hours exposure. A slower killing of the S. aureus was noted when cells were kept in buffer containing antimicrobial metabolite (5 U/ml). Antimicrobial metabolite induced efflux of potassium ions from cells of Staphylococcus indicating the channel forming activity. Keywords: Aureobasidium, antistaphylococcal activity, potassium efflux African Journal of Clinical and Experimental Microbiology Vol. 6 (3) 2005: 177-187

Low and high molecular weight poly( l-lysine)s/poly( l-lysine)–DNA complexes initiate mitochondrial-mediated apoptosis differently

Poly( l-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L-PLL, and high (27.4 kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine “scrambling” and loss of membrane integrity, but later (24 h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed.

Sphingolipids Influence the Sensitivity of Lipid Bilayers to Fungicide, Syringomycin E

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Deltasyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Deltasyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Deltasyr2 versus wild-type sphingolipids. Deltasyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Deltasyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.

α‐Latrotoxin increases spontaneous and depolarization‐evoked exocytosis from pancreatic islet β‐cells

alpha-Latrotoxin (alpha-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of alpha-LT on single human, mouse and canine beta-cells. In isolated and combined measurements, alpha-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca(2+), into the micromolar range, that are dependent on extracellular Ca(2+); (ii) large conductance non-selective cation channels; and (iii) Ca(2+)-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, alpha-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca(2+). These results strongly support the hypothesis that alpha-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) alpha-LT inserts into the plasma membrane to form Ca(2+) permeable channels and promote Ca(2+) entry thereby triggering Ca(2+)-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, alpha-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca(2+)-sensitive pool of insulin granules activated by global cytosolic Ca(2+) concentrations in the range of approximately 1 microm.

Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei

Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.