Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin,tolaasin

Tolaasin, a pore‐forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin‐induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 °C, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 °C and the temperature was decreased, complete hemolysis was observed even at 4 °C. These results indicate that tolaasin binding to cell membrane is temperature‐sensitive while tolaasin‐induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore‐forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose‐dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin‐induced hemolysis can be explained by a multi‐hit model. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Pseudomonas tolaasii and tolaasin: comparison of symptom induction on a wide range of Agaricus bisporus strains

Abstract A wide range of Agaricus bisporus , including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii . Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development.     

Detoxification process of tolaasins,lipodepsipeptides, by Microbacterium sp. K3-5

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.     

Purification of a pore-forming peptide toxin, tolaasin, produced by Pseudomonas tolaasii 6264

Tolaasin, a pore-forming peptide toxin, is produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. P. tolaasii 6264 was isolated from the oyster mushroom damaged by the disease in Korean. In order to isolate tolaasin molecules, the supernatant of bacterial culture was harvested at the stationary phase of growth. Tolaasin was prepared by ammonium sulfate precipitation and three steps of chromatograpies, including a gel permeation and two ion exchange chromatographies. Specific hemolytic activity of tolaasin was increased from 1.7 to 162.0 HU mg(-1) protein, a 98-fold increase, and the purification yield was 16.3%. Tolaasin preparation obtained at each purification step was analyzed by HPLC and SDS-PAGE. Two major peptides were detected from all chromatographic preparations. Their molecular masses were analyzed by MALDI-TOF mass spectrometry and they were identified as tolaasin I and tolaasin II. These results demonstrate that the method used in this study is simple, time-saving, and successful for the preparation of tolaasin.     

Ion channel and membrane translocation of diphtheria toxin

Abstract Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.     

Pore formation, polymerization, hemolytic and leukotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum

A new toxin of Enterobacter cloacae was purified and studied by SDS-PAGE electrophoresis with the purpose of investigating its ability to generate polymers and their molecular mass. Monomer of 13.3 kDa and structures of multimeric mass were detected. The toxin of 66 kDa was the most abundant form of toxin. This polymer and the monomer were selected to examine blood cells damage. Membrane pores caused by both toxin forms seemed to be of similar dimension (estimated in 3.6 nm by experiments with osmotic protectors) and were able to lyse erythrocytes and leukocytes. The results obtained indicate that polymerization and pore formation are involved in the molecular events that participate in the cytotoxic effects of E. cloacae toxin. Immunization of rabbits with 13.3kDa toxin generated antibody response capable of inhibiting oxidative stress as well as hemolytic and leukotoxic effects. Immunoblotting indicated that monomer and polymer reacted with antihemolysin serum. The importance of E. cloacae toxin "in vivo" was studied in animals by means of assays performed in peritoneum of rats, inoculated with the hemolytic strain (C1) and a non-hemolytic variant (C4). Both strains stimulated infiltration of leukocytes in peritoneum, but C1 caused cell death and lysis wheras assays with C4 maintained the viability of leukocytes even within 5 h after extraction of samples.     

Secondary structure of ShK toxin, a potassium-channel-blocking peptide

Summary Sea anemones possess small K-channel-blocking peptides about the same size as the scorpion K-channel toxins. We have estimated the secondary structure content (33% helix, 26% -sheet) of one of these toxins, ShK toxin, using CD, Raman, and FTIR spectroscopy. A hypothetical 3D structure of the peptide core has been constructed using secondary structure and disulfide-linkage constraints; a single helical segment running from Ala¹⁴ through Leu²⁵ is predicted.     

Computer modelling of the transmembrane channel formed by a CNBr peptide of diphtheria toxin B fragment

Abstract Diphtheria toxin (DT) forms transmembrane, voltage-dependent channels in a planar lipid bilayer. Channels with similar characteristics were obtained with CB1, a cyanogen bromide peptide of diphtheria toxin B fragment (DTB) (res 340–459). Tryptophan 398 is in interaction with the hydrophobic core of the lipid bilayer. Using the Eisenberg method in association with the Shiffer-Edmunson wheel representation, we have identified two amphipathic α-helices within CB1 (res 346–364 and 389–406) that could be involved in the interaction with lipids. Bearing this information in mind, we are providing a model for the structure of the CB1 channel.     

Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into alpha- and beta-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to beta-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins.     

Statistical properties of ion channel records. Part I: relationship to the macroscopic current

Macroscopic ion channel current can be derived by summation of the stochastic records of individual channel currents. In this paper, we present two probability density functions of single channel records that can uniquely determine the macroscopic current regardless of other statistical properties of records or the stochastic model of channel gating (presented often with stationary Markov models). We show that H(t), probability density function of channel opening events (introduced explicitly in this paper), and D(t), probability density function of the open duration (sometimes has named dwell time distribution as well), determine the normalized macroscopic current, G(t), through G(t) = P(t) - H(t) * Q(t) where P(t) is the cumulative density function of H(t), Q(t) is the cumulative density function of D(t), * is the symbol of convolution integral and G(t) is the macroscopic current divided by the amplitude of single channel current and the number of single channel sweeps. Compared to other equations for the macroscopic current, here the macroscopic current is expressed only in terms of the statistical properties of single channel current and not the stochastic model of ion channel gating or a conditioned form of macroscopic current. Single channel currents of an inactivating BK channel were used to validate this relationship experimentally too. In this paper, we used median filters as they can remove the unwanted noise without smoothing the transitions between open and closed states (compare to low pass filters). This filtering leads to more accurate measurement of transition times and less amount of missed events.     

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes

Abstract A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal α-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M . non-electrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.     

Statistical properties of ion channel records. Part II: estimation from the macroscopic current

Macroscopic ion channel current is the summation of the stochastic records of individual channel currents and therefore relates to their statistical properties. As a consequence of this relationship, it may be possible to derive certain statistical properties of single channel records or even generate some estimates of the records themselves from the macroscopic current when the direct measurement of single channel currents is not applicable. We present a procedure for generating the single channel records of an ion channel from its macroscopic current when the stochastic process of channel gating has the following two properties: (I) the open duration is independent of the time of opening event and has a single exponential probability density function (pdf), (II) all the channels have the same probability to open at time t. The application of this procedure is considered for cases where direct measurement of single channel records is difficult or impossible. First, the probability density function (pdf) of opening events, a statistical property of single channel records, is derived from the normalized macroscopic current and mean channel open duration. Second, it is shown that under the conditions (I) and (II), a non-stationary Markov model can represent the stochastic process of channel gating. Third, the non-stationary Markov model is calibrated using the results of the first step. The non-stationary formulation increases the model ability to generate a variety of different single channel records compared to common stationary Markov models. The model is then used to generate single channel records and to obtain other statistical properties of the records. Experimental single channel records of inactivating BK potassium channels are used to evaluate how accurately this procedure reconstructs measured single channel sweeps.     

Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.     

A trimeric building block model for Cry toxins in vitro ion channel formation

The crystal (Cry) insecticidal toxins, or δ-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices α4 and α5 in domain I insert into the lipid bilayer as an α-helical hairpin. Mutagenesis studies have assigned an important role to α5 in maintaining oligomerization, and to α4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for α4, but a right handed trimeric α5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.     

Analysis of peptide uptake in Pseudomonas aeruginosa: A fluorescamine labeling procedure

Intracellular products of peptide transport were analyzed after thin layer chromatography separation by a fluorescamine labeling procedure. Dipeptides penetrated Pseudomonas aeruginosa via 2 transport systems, one of high affinity with K_T values from 1–2 μM, and another one of low affinity (K_T about 30 μM). A single transport system assumed tripeptide uptake with K_T varying from 10–30 μM, depending on the tripeptide used. Peptides entering the cells were rapidly hydrolyzed into their constitutive amino acids.     

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 microM), ASIC1a+3 (IC50=2 microM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.     

Noise analysis of an ion channel in the cardiac myocyte--study based on a Markov model

Mechanical contraction of a cardiac muscle cell is related to the electric activation of the plasma membrane. As in the neuron cell, inflow of the Na(+) ions across the cell membrane causes electric activation with amplitude of about 100 mV. However, differently from the nerve cell, the action potential lasts a few hundred milliseconds before repolarization. Moreover, several types of K(+) channel such as the classical inward rectifier K(+) channel, the voltage dependent channel and others are responsible for the formation of the action potential. The mechanism of opening and closing the K(+) channels is not thoroughly elucidated. In the present paper, a four state Markov model with one open and three closed states is studied to obtain open and close probabilities of the gates constituting a specific ionic channel. The probability density functions of durations of opening and closing of the channel are also discussed.     

Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels

Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na_V1.2) sodium channels in a way dependent on the holding potential, and competes with μ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 μM, the recombinant toxin inhibits Na⁺ currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at −80 mV holding potential) and tetrodotoxin-resistant Na⁺ currents (26.2 ± 4.9% at the same holding potential). At −50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 μM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa_V1.2 > rNa_V1.7 ≈ rNa_V1.4 ≥ rNa_V1.3 > mNa_V1.6 ≥ hNa_V1.8. No effect was seen on hNa_V1.5 and on the arthropods isoforms (DmNa_V1, BGNa_V1.1a and VdNa_V1). The IC₅₀ for Na_V1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na_V1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain.     

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

δ-Atracotoxins (δ-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. We have isolated and determined the amino acid sequence of a novel δ-ACTX, designated δ-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with δ-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other δ-ACTXs. However, δ-ACTX-Hv1b is approximately 15–30-fold less potent than other δ-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between δ-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

Use the force,fluke: Ligand-independent gating of Schistosoma mansoni ion channel TRPMPZQ

The parasitic flatworm ion channel, TRPM_PZQ, is a non-selective cation channel that mediates Ca²⁺ entry and membrane depolarization when activated by the anthelmintic drug, praziquantel (PZQ). TRPM_PZQ is conserved in all platyhelminth genomes scrutinized to date, with the sensitivity of TRPM_PZQ in any particular flatworm correlating with the overall sensitivity of the worm to PZQ. Conservation of this channel suggests it plays a role in flatworm physiology, but the nature of the endogenous cues that activate this channel are currently unknown. Here, we demonstrate that TRPM_PZQ is activated in a ligand-independent manner by membrane stretch, with the electrophysiological signature of channel opening events being identical whether evoked by negative pressure, or by PZQ. TRPM_PZQ is therefore a multimodal ion channel gated by both physical and chemical cues. The mechanosensitivity of TRPM_PZQ is one route for endogenous activation of this ion channel that holds relevance for schistosome physiology given the persistent pressures and mechanical cues experienced throughout the parasite life cycle.