Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Tonic and phasic guanidinium toxin-block of skeletal muscle Na channels expressed in Mammalian cells

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.     

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture.

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.     

An enhanced chemiluminescence enzyme immunoassay for serum progesterone

A competitive enhanced luminescent enzyme immunoassay for serum progesterone is described, which is based on a 11 alpha-hydroxyprogesterone 11-hemisuccinyl-horseradish peroxidase conjugate and a black polystyrene microtitre plate sensitised with anti-progesterone IgG. Bound label was determined using a mixture of 4-iodophenol, luminol and peroxide, and the light emitted from the wells of the plate quantitated using a luminescent plate reader. The assay was sensitive (detection limit 0.5 pg), precise (CV 2.7 - 9.0% in the concentration range 4.3-67.7 nM) and showed good correlation (r = 0.99) with a conventional radioimmunoassay.     

Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors

A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Microtitre plate hybridization system for detection of thermophilic Campylobacter rRNA

A microtitre plate nucleic acid probe hybridization system was developed for the detection of ribosomal RNA from thermophilic Campylobacter (Camp. jejuni, Camp. coli, Camp. lari and Camp. upsaliensis). A specific DNA probe obtained by amplification of 23S rRNA sequences using the polymerase chain reaction technique was immobilized on a microtitre plate, and used for hybridization with target 23S rRNA from cell lysates. The RNA-DNA hybrids thus formed in the wells were detected by an immunoenzymatic assay using a monoclonal antiRNA-DNA hybrid antibody. The sensitivity of this system was 2.7 x 10(4) cells ml(-1). This simple, sensitive and inexpensive hybridization and immunoenzymatic assay system should facilitate the detection of Campylobacter in food and clinical samples.     

A simple and safe method for single HLA-antigen-typing by a solid phase assay

A rapid solid phase assay for detection of single HLA-antigens on platelets was developed. The platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After incubation with commercially available anti-HLA-sera the bound anti-HLA-specific antibodies directed against HLA-antigens present on the platelets were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an HLA-antigen, was indicated by a slight red cell adherence over the reaction surface. In the absence of the HLA-antigen no binding occurred and the indicator red cells formed a small red disc-like pellet.     

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons.

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.     

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding (KD = 1.9 nM) of [3H]saxitonin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4 degrees C. The saturable binding is specifically inhibited by tetrodotoxin with a K1 = 0.30 nM. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttxs), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [3H]saxitonin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.     

Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.     

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel

Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier delta-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I-labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1-5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhalphaIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K(i)s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K(i)=267 nM) and site 4 in rat brain synaptosomes (K(i)=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel.     

Interaction of pyrethroids with the Na channel in mammalian neuronal cells in culture

The interaction of a series of pyrethroids with the Na⁺ channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a ²²Na⁺ influx approach. By themselves, pyrethroids do not stimulate ²²Na⁺ entry through the Na⁺ channel (or the stimulation they give is too small to be analyzed). However, they stimulate ²²Na⁺ entry when used in conjuction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na⁺ channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on ²²Na⁺ influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na⁺ channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor.Some of the pyrethroids used in this study bind to the Na⁺ channel but are unable to stimulate ²²Na⁺ entry. These inactive compounds behave as antagonists of the active pyrethroids.An electrophysiological approach has shown that pyrethroids by themselves are active on the Na⁺ channel of mammalian neurones, and essentially confirm the conclusions made from ²²Na⁺ flux measurements.Pyrethroids are also active on C9 cells in which Na⁺ channels are ‘silent’, that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na⁺ channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na⁺ channel.     

Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C.