Toxin Binding Sites Research Articles

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)

Single-channel current/voltage relationships of two kinds of Na+ channel in vertebrate sensory neurons.

The electrical signals of nerve and muscle are fundamentally dependent on the voltage-gated Na+ channel, which is responsible for the rising phase of the action potential. At least two kinds of Na+ channel are expressed in the membrane of frog dorsal root ganglion (DRG) cells: Na+ channels with fast kinetics that are blocked by tetrodotoxin (TTX) at high affinity, and Na+ channels with slower kinetics that are insensitive to TTX. Recordings of single-channel currents from frog DRG cells, under conditions favoring Na+ as the charge carrier, reveal two distinct amplitudes of single-channel events. With 300 mM external Na+, single-channel events that can be measured in the presence of 1 microM TTX have a slope conductance 7.5 pS. In the absence of TTX, events with a slope conductance of 14.9 pS dominate. Ensemble averages of the smaller single-channel events display the slower kinetics characteristic of the macroscopic TTX-insensitive Na+ currents, and ensemble averages of the larger events display the faster kinetics characteristic of the TTX-sensitive currents. The results are consistent with the idea that the toxin-binding site is sufficiently close to the pore to influence ion permeation.

S-2-5 - Structure of ion channels and the binding sites for drugs and toxins.

S-2-5 - Structure of ion channels and the binding sites for drugs and toxins.

Analyses of the staphylococcal toxin binding sites on MHC class II molecules

Analyses of the staphylococcal toxin binding sites on MHC class II molecules

Effect of lipidic factors on membrane cholesterol topology — mode of binding of θ-toxin to cholesterol in liposomes

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for θ-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglvcerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14–18 carbon-hydrocarbons chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for θ-toxin.

Monoclonal antibody for Joro spider toxin (JSTX) binding site

Monoclonal antibody for Joro spider toxin (JSTX) binding site

Scyllatoxin, a blocker of calcium-activated potassium channels: structure-function relationships and brain localization of the binding sites

Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor.

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.

A Cytolysin, Θ-Toxin, Preferentially Binds to Membrane Cholesterol Surrounded by Phospholipids with 18-Carbon Hydrocarbon Chains in Cholesterol-Rich Region1

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes. II. Extrinsic influence of cations.

Use-dependent declines of Na+ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na+), divalent (Ca2+, Mg2+) and trivalent (La3+) cations on use dependence were found: Increasing the Ca2+ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca2+. Doubling the external Na+ concentration in 0.2 mM Ca2+ solutions did not initiate phasic block. External Mg2+ ions induced a smaller, and La3+ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La3+. The static block of Na+ currents by La3+ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na+ channel whose affinity for TTX or STX depends on 1) the gating conformation of the channel, probably the inactivation and ii) the occupancy of a blocking site by di- or trivalent external cations.

Neoglycolipid analogs of ganglioside GM1 as functional receptors of cholera toxin

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD binding site of diphtheria toxin

8-Azidoadenine and 8-azidoadenosine, two photoactivatable derivatives of adenine and adenosine, are competitive inhibitors of diphtheria toxin of similar potency with respect to their parent compounds. On irradiation, the two tritium-labeled photoactivatable azidoadenines bind covalently and specifically to an enzymic fragment of diphtheria toxin that is known to bind to NAD. This photolabeling is protected by the enzyme substrate NAD. The radiolabeled protein was fragmented, and the radioactive fragments were sequenced. Tyr-65 is labeled specifically by both photoreagents, and its labeling was reduced strongly when NAD was present during irradiation. Labeling is also reduced strongly by adenine, adenosine, and nicotinamide. These results suggest that Tyr-65 is at the NAD binding site of diphtheria toxin and that the competitive inhibitors adenine, adenosine, and nicotinamide bind to the same portion of the catalytic center of the toxin.

Effects of receptor concentration, media pH and storage on nicotinic receptor-transmitted signal in a fiber-optic biosensor

The optical signal generated by a nicotinic acetylcholine receptor (nAChR)-based optical biosensor was dependent on the density of toxin binding sites (i.e. receptors) immobilized on the surface of the fiber. The maximum density of nAChR receptors adsorbed on the optic fibers, measured using ( 125I)-α-bungarotoxin (the α-neurotoxin of the Bungarus snake venom) binding, was 6·2 pmol of receptor sites per fiber. Incubation time, that was required for maximal noncovalent immobilization of the receptor protein on the fiber, was <10 min. Immobilization of the nAChR protein on the quartz fiber was affected by pH of the medium, with pH 3·5 as the optimal. Stored in phosphate buffered saline (PBS) or in a dry nitrogen atmosphere, the nAChR optical sensor showed no loss of activity over the first 3 days, then showed a slow but gradual loss in activity (45% over the next 30 days).

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor

The Shaker K + channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K + channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K + channels and may contribute to the formation of the ion conduction pore.

Voltage-sensitive Na+ channels in mammalian peripheral nerves detected using Scorpion toxins

The localization of voltage-sensitive sodium channels was investigated in mouse, rat and rabbit sciatic nerves using iodinated alpha- and beta-Scorpion toxins (ScTx) as specific probes. Saturable specific binding for a beta-ScTx was detected in mouse sciatic nerve homogenates (Kd = 90 pM, binding site capacity = 90 fmol mg-1 protein). LM autoradiographic studies demonstrated that the two types of ScTx stained the Ranvier nodes of the myelinated fibres, and also showed a clear but weaker labelling of the unmyelinated Remak bundles. In the sciatic nerve, which is widely considered as a model 'myelinated nerve', the nodal membrane represented only a small fraction of the total axonal membranes (0.2% and 0.05% for mouse and rabbit sciatic nerves respectively). Therefore, despite their high channel density, nodal membranes contribute only a small proportion of the total labelling by beta-ScTx (15% and 2.3% for mouse and rabbit sciatic nerves respectively), with the major contribution to labelling arising from unmyelinated axons. The distribution of specific binding sites for a beta-Scorpion toxin was then analysed in cross-sections of rabbit sciatic nerve at the EM level. The quantitative analysis of autoradiograms involved three methods, the 50% probability circle method, and two cross-fire analyses using either systematically distributed hypothetical sources or hypothetical sources only located on the plasma membranes of axons and of Schwann cells associated with unmyelinated Remak bundles. No specific beta-Scorpion toxin binding sites were detected at the plasma membrane of Schwann cells from either myelinated fibres or unmyelinated bundles, or at the internodal surface of myelinated axons. Sites were only detected at the surface of unmyelinated axons and at nodal axolemma. Their density in unmyelinated axons was found to be in the range of 1-6 per micron2 of plasma membrane surface area by combining quantitative EM autoradiography and stereological measurements.

Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat

The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20 μM), nicotine (5 μM) or DMPP (20 μM) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10 μM) but reversibly blocked by hexamethonium (20 μM). Although labeling with fluorescent α-bungarotoxin (BGT) demosntrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type.

Expression and characterization of the chick nicotinic acetylcholine receptor α‐subunit in insect cells using a baculovirus vector

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.

Gamma Interferon‐Induced Specific Binding of Tetanus Toxin on the GG2EE Macrophage Cell Line

We have recently demonstrated that tetanus toxin (TT) selectively inhibited gamma interferon-(IFN-gamma)-induced, but not basal, lysozyme activity in the GG2EE macrophage cell line. By an indirect immunofluorescence technique, we show that tetanus toxin, as well as the BIIb tetanus toxin-derived fragment, selectively binds to IFN-gamma-treated cells but fails to bind to control cells. Moreover, BIIb fragment and TT share the same binding sites, as demonstrated by competition/displacement experiments. These data indicate that IFN-gamma pretreatment results in the acquisition of tetanus toxin binding sites on GG2EE cells by a mechanism(s) yet to be identified.