Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.     

The action of Cu2+ on Bacillus thuringiensis growth investigated by microcalorimetry

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B. thuringiensis growth was studied. Copper has been regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu2+ of different concentration have different effects on B. thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B. thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B. thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml.     

Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage- dependent Ca2+ binding reactions

The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ion. In this scheme, the two Ca2+ binding reactions are voltage dependent, whereas the open-closed transition is voltage independent. The kinetic constant derived for this scheme gives an accurate theoretical fit to the observed equilibrium open-state probability. The results provide evidence for a novel regulatory mechanism for the activity of an ion channel: modulation by voltage of the binding of an agonist molecule, in this case, Ca2+ ion.     

δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, notvery K⁺ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba²⁺ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2–40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis δ-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.     

Effects of phospholipid surface charge on ion conduction in the K+ channel of sarcoplasmic reticulum.

Single-channel K+ currents through sarcoplasmic reticulum K+ channels were compared after reconstitution into planar bilayers formed from neutral or negatively charged phospholipids. In neutral bilayers, the channel conductance saturates with K+ concentration according to a rectangular hyperbola, with half-saturation at 40 mM K+, and maximum conductance of 220 pS. In negatively charged bilayers (70% phosphatidylserine/30% phosphatidylethanolamine), the conductance is, at a given K+ concentration, higher than in neutral bilayers. This effect of negative surface charge is increasingly pronounced at lower ionic strength. The maximum conductance at high K+ approaches 220 pS in negative bilayers, and the channel's ionic selectivity is unaffected by lipid charge. The divalent channel blocker " bisQ11 " causes discrete blocking events in both neutral and negatively charged bilayers; the apparent rate constant of blocking is sensitive to surface charge, while the unblocking rate is largely unaffected. Bilayers containing a positively charged phosphatidylcholine analogue led to K+ conductances lower than those seen in neutral bilayers. The results are consistent with a simple mechanism in which the local K+ concentration sensed by the channel's entryway is determined by both the bulk K+ concentration and the bulk lipid surface potential, as given by the Gouy-Chapman model of the electrified interface. To be described by this approach, the channel's entryway must be assumed to be located 1-2 nm away from the lipid surface, on both sides of the membrane.     

Calcium release from isolated sarcoplasmic reticulum due to 4,4'-dithiodipyridine

The effects of SH reagents on Ca2+ release from sarcoplasmic reticulum (SR) vesicles were examined by the tracer method using 45Ca2+. Among the various SH reagents tested, 4,4'-dithiodipyridine (PDS) was found to induce Ca2+ release most specifically from the heavy fraction of SR vesicles. Further, the following results were obtained. (i) PDS bound covalently to proteins in the SR membrane and induced Ca2+ release. (ii) The Ca2+ release was further enhanced by ATP and caffeine, but inhibited by procaine, ruthenium red and various divalent cations. (iii) PDS enhanced the Ca2+ release in the whole range of Ca2+ concentrations tested. (iv) Choline permeability was also enhanced by PDS. Further, the electrical conductance of the Ca2+-induced Ca2+ release channels was studied by incorporating them into lipid bilayers and it was found that PDS increased the probability of opening of the channels. These results suggest that PDS binds to certain SH groups of the Ca2+-induced Ca2+ release channels in the SR membrane and thus induces Ca2+ release.     

A high conductance cationic channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

A previously undescribed plasma membrane cation channel from Phaseolus vulgaris bean roots was studied after its incorporation into planar lipid bilayers. The channel allows the passage of monovalent cations excluding the flux of both anions (Cl-) and divalent cations (Ca2+). The channel presents a high ( approximately 213 pS) conductance in (300 mM Kcis+)/ (150 mMKtrans+) conditions. The probability of opening (Po) is low at all the tested voltages, but it increases significantly at trans-negative potentials. Permeability ratios (Pcation/PK+) under bi-ionic conditions follow the sequence: K+ (1.0)>NH4+ (0.86)>Na+ (0.78). Under the same conditions, the conductance ratios (gamma cation/gamma K+) follow the sequence: NH4+ (1.1) > or = K+ (1.0)>Na+ (0.80). The low probability of opening exhibited by the channel upon its incorporation into a lipid bilayer makes it a candidate to regulation by (and therefore participation in) cellular signalling networks.     

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.     

Effect of divalent cations on the structure of dipalmitoylphosphatidylcholine and phosphatidylcholine/phosphatidylglycerol bilayers: an 2H-NMR study

The interactions of CaCl2 or MgCl2 with multilamellar phospholipid bilayers were studied by 2H-NMR. Two model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers and (2) bilayers composed of a mixture of phosphatidylcholine and phosphatidylglycerol at a molar ratio of 5:1. Addition of 0.25 M CaCl2 to DPPC bilayers resulted in significant uniform increase of the order parameters of the lipid side chains; the effect of 0.25 M MgCl2 was insignificant. Both phosphatidylcholine and phosphatidylglycerol components of the mixed bilayers were affected by the presence of 0.25 M CaCl2 and, to a much smaller degree, by MgCl2. The addition of Ca2+ induced significantly larger increase of the order parameters of the phosphatidylcholine component. The results are consistent with the long-range effects of Ca2+ binding on the packing of the lipid membranes.     

Zn~(2+)对PTP开放和细胞色素c释放的浓度依赖性双向调节

研究Zn2+对Ca2+介导线粒体通透过渡孔道(PTP)开放和线粒体细胞色素c释放的影响,及其与线粒体膜电位(ΔΨm)和Ca2+介导的线粒体Ca2+释放(mCICR)之间的关系.提取大鼠肝线粒体,通过紫外分光光度仪检测不同浓度Zn2+作用下Ca2+介导的PTP开放状态;采用荧光分光光度仪测定不同浓度Zn2+作用下线粒体膜电位的变化;采用双波长双光束紫外分光光度仪检测不同浓度Zn2+作用下测试体系内Ca2+浓度的变化,以反映线粒体Ca2+的转运情况(即mCICR);通过免疫印迹法检测不同浓度Zn2+作用下Ca2+介导的线粒体细胞色素c的释放.高浓度Zn2+完全抑制Ca2+介导的PTP开放和细胞色素c释放.一定浓度的Zn2+部分抑制Ca2+介导的PTP开放和细胞色素c释放.适当浓度Zn2+自身介导PTP开放和细胞色素c释放.低浓度Zn2+加速Ca2+介导PTP开放和Ca2+释放;高浓度和一定浓度Zn2+分别完全或部分破坏ΔΨm;高浓度Zn2+完全抑制mCICR.当抑制mCICR时,Ca2+和Zn2+对PTP开放和细胞色素c释放的作用完全抑制.结果表明,Zn2+以浓度依赖方式双向调节PTP开放和细胞色素c释放.Zn2+的作用可能与Zn2+破坏ΔΨm和影响mCICR相关.     

The effect of holotoxin A1 on transport of calcium ions across the lipid models of biological membranes

Planar bilayer lipid membranes formed from trepang phospholipids possess an intrinsic Ca2(+)-permeability. These phospholipids dissolved in a non-polar solvent can extract 45Ca2+ from the aqueous to the organic phase. The triterpenic glycoside holotoxin A isolated from the trepang Stichopus japonicus inhibits the Ca2+ flux of lipid bilayers from trepang phospholipids as well as the Ca2+ flux induced in phosphatidylcholine bilayers by the calcium ionophore X-537A. Toxin inhibits the Ca2+ ionophore A23187 induced Ca2+ efflux from phosphatidylcholine liposomes and 45Ca2+ transition from the aqueous to the organic phase. Holotoxin A does not inhibit the 45Ca2+ transfer to the non-polar phase induced by holoturia phospholipids and does not affect the phosphatidylcholine hydroperoxide-induced Ca2+ flux of lipid bilayers. Using the fluorescent probe pyrene, it was demonstrated that toxin increases the microviscosity of liposomal membranes and trepang oocyte "ghosts".     

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum.

Two types of divalent cation conducting channels from rabbit skeletal muscle sarcoplasmic reticulum (SR) were incorporated into planar lipid bilayers. A high conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy density SR fractions. The 100-pS channel was activated by adenine nucleotides and Ca2+ and inhibited by Mg2+ and ruthenium red. A 10-pS calcium and barium conducting channel could be incorporated into planar lipid bilayers from light, intermediate, and heavy density SR vesicles. 10-pS channel activity in bilayers was not dependent on cis Ca2+ and was only weakly dependent on adenine nucleotides. Ruthenium red at concentrations up to 1 mM had no effect and Mg2+ was only marginally effective in inhibiting macroscopic Ba2+ currents from this channel. Calcium releasing activity in intermediate and heavy density SR fractions was assayed according to a rapid quench protocol and compared with the results obtained in the bilayer. Results from this comparison indicate that the 10-pS channel is probably not involved in rapid Ca2+- and adenine nucleotide-induced Ca2+ release from isolated SR vesicles.     

Capsaicin induces cofilin dephosphorylation in human intestinal cells: the triggering role of cofilin in tight-junction signaling

Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.     

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.     

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. I. Transition anomalies of ultrasonic properties

The effect of Ca2+ on a gel-to-liquid crystal transition as well as the mechanical properties of dipalmitoylphosphatidylcholine bilayers was studied by an ultrasonic technique. Transition temperature increased with increase in Ca2+ concentration, whereas the variation of ultrasonic anomalies indicated that dipalmitoylphosphatidylcholine bilayers exhibited maximum pseudocritical fluctuation at a Ca2+ concentration of about 10 mM. Hardening of dipalmitoylphosphatidylcholine membranes due to the Ca2+ binding was observed above 10 mM CaCl2, suggesting the lateral compression of the lipid bilayer by bound Ca2+. Long-range attraction between bound Ca2+ and the head groups of surrounding lipid molecules was proposed from these calcium effects.     

Calcium Inhibits Dihydropyridine-Stimulated Increases in Opening and Unitary Conductance of a Plant Ca<Superscript>2+</Superscript> Channel

We have previously characterized the “RCA” channel (root Ca²⁺ channel), a voltage-dependent, Ca²⁺-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers. Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202–791). However, the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca²⁺, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca²⁺ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202–791 or nifedipine caused an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive interaction between Ca²⁺ and DHPs at one of the Ca²⁺-binding sites involved in Ca²⁺ permeation and that binding of a DHP to one of the Ca²⁺-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant Ca²⁺-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between plant and animal Ca²⁺-permeable channels.     

Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes

The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed.     

A consensus segment in the M2 domain of the hP2X7 receptor shows ion channel activity in planar lipid bilayers and in biological membranes

The P2X₇ receptor (P2X₇R) is an ATP-gated, cation-selective channel permeable to Na⁺, K⁺ and Ca²⁺. This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X₇R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X₇R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X₇R. The conserved part of the M2 domain of P2X₇R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.     

Amino acid substitution in alpha-helix 7 of Cry1Ac delta-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner.

Insecticidal proteins or delta-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight alpha-helices and is implicated in pore formation in insect midgut epithelial membranes. All the delta-endotoxins share a common hydrophobic motif of eight amino acids in alpha-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac delta-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.