Analysis of the Properties of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins Using a Potential-sensitive Fluorescent Probe

A potential-sensitive fluorescent probe, 3,3-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K⁺ ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K⁺ ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.     

Leucine Transport Is Affected by Bacillus thuringiensis Cry1 Toxins in Brush Border Membrane Vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) Midgut

The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K⁺/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K⁺ gradient. Experiments with the voltage-sensitive fluorescent dye 3,3′-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K⁺ gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.     

Differential Effects of Ionic Strength,Divalent Cations and pH on the Pore-forming Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of Cry1Ac and Cry1Ca were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced Cry1Ac activity but decreased considerably that of Cry1Ca. In vesicles, Cry1Ac had a stronger pore-forming ability than Cry1Ca at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of Cry1Ac relative to that of Cry1Ca. The activity of Cry1Ca, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited Cry1Ac activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for Cry1Ac activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of Cry1Ac and Cry1Ca, stress the importance of electrostatic interactions in the mechanism of pore formation by B. thuringiensis toxins.     

Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say)

Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins. In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R₁, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R₁ in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R₁. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R₁ is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R₁ was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.     

Bacillus thuringiensis ssp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure

Bacillus thuringiensis ssp. israelensis (Bti) has been used worldwide for the control of dipteran insect pests. This bacterium produces several Cry and Cyt toxins that individually show activity against mosquitoes but together show synergistic effect. Previous work demonstrated that Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a membrane-bound receptor. In the case of Cry toxins active against lepidopteran insects, receptor interaction triggers the formation of a pre-pore oligomer that is responsible for pore formation and toxicity. In this work we report that binding of Cry11Aa to Cyt1Aa facilitates the formation of a Cry11Aa pre-pore oligomeric structure that is capable of forming pores in membrane vesicles. Cry11Aa and Cyt1A point mutants affected in binding and in synergism had a correlative effect on the formation of Cry11Aa pre-pore oligomer and on pore-formation activity of Cry11Aa. These data further support that Cyt1Aa interacts with Cry11Aa and demonstrate the molecular mechanism by which Cyt1Aa synergizes or suppresses resistance to Cry11Aa, by providing a binding site for Cry11Aa that will result in an efficient formation of Cry11Aa pre-pore that inserts into membranes and forms ionic pores.     

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.     

Influence of the biophysical and biochemical environment on the kinetics of pore formation by Cry toxins

The effect of Bacillus thuringiensis toxins on the permeability of the luminal membrane of Manduca sexta midgut columnar epithelial cells is strongly influenced by several biophysical and biochemical factors, including pH, ionic strength, and divalent cations, suggesting an important role for electrostatic interactions. The influence of these factors can differ greatly, however, depending on the toxin being studied, even for closely related toxins such as Cry1Ac and Cry1Ca. In the present study, the possibility of using temperature changes as a tool for controlling the rate and extent of pore formation in midgut brush border membrane vesicles was evaluated. Lowering temperature gradually decreased the rate of pore formation, but had little effect on the permeability of vesicles previously incubated with toxin at room temperature. The formation of new pores, following incubation of the vesicles with toxin, could thus be almost abolished by rapidly cooling the vesicles to 2 degrees C. Using this approach, changes in the rate of pore formation could be more easily distinguished from alterations in the properties of the pores formed, thus allowing a more detailed analysis of the kinetics and mechanism of pore formation.     

Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K⁺ ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K⁺ ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation

Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices α4 and α5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.     

Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.     

Toxicity, Binding, and Permeability Analyses of Four Bacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with ¹²⁵I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. ¹²⁵I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.     

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pH _i ) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K⁺ and Na⁺ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pH _i depends strongly on the activity of a K⁺/H⁺ exchanger. In the absence of toxin, removal of K⁺ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pH _i equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pH _i . B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H⁺, K⁺ and Na⁺.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm

Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa–QD probes compared to CrylAc–QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa–QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins.