Spontaneous output of acetylcholine from rat diaphragm preparations declines after treatment with botulinum toxin

A gas chromatographic spectrometric assay was used to measure tissue and released acetylcholine and choline in diaphragm preparations of rats previously injected with botulinum toxin type A. Botulinum intoxication was found not to alter the acetylcholine content of rat diaphragms in vivo or in fully paralyzed muscles in vitro. This result provides direct support for the hypothesis that botulinum toxin blocks transmitter release without affecting acetylcholine synthesis. However, in diaphragm preparations in vitro, this toxin was found to inhibit not only the evoked release of acetylcholine but also the spontaneous "leakage" of acetylcholine that is measured at rest. Additional experiments were performed to characterize this action of the toxin. The magnitude of the decline in resting acetylcholine output appears to be too large to be accounted for solely by the known effect of botulinum toxin to reduce the frequency of miniature endplate potentials. The mechanism of this action of botulinum toxin remains an enigma.     

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa.     

Anti-insect scorpion toxins: historical account, activities and prospects

Some toxins from scorpion venoms, much more toxic to insects than to other animal classes, possess high affinity to Na+ channels. These anti-insect scorpion toxins have been divided into: 1) alpha toxins which lack strict selectivity for insects, do not compete with following groups of anti-insect toxins, resemble other alpha scorpion toxins by their structure and their ability, as alpha anemone toxins, to prolong insect axonal action potential durations through a drastic slowing down of the Na+ current inactivation, 2) excitatory insect selective scorpion toxins which induce in blowfly larvae an immediate fast paralysis; in isolated cockroach axons, they depolarize and induce a sustained repetitive activity of short (normal) action potentials through a shift of Na+ activation mechanism towards more negative potentials and some decrease of inactivation at these potential values, 3) depressant insect selective neurotoxins which cause a slow progressive flaccid paralysis of larvae, depolarize insect axons and reduce or even suppress evoked action potentials; resting depolarizations which are antagonized by a post-application of TTX, are due to the opening of sodium channels at very negative potential values and to the suppression of their inactivation mechanism. The decrease of the maximal Na+ conductance following flaccid toxin action may be understood if toxin-modified channels opened at very negative potentials values remain open (or re-open) for much longer times than in control conditions and pass by substate less conductant states. Anti-insect scorpion toxins become of major interest into insect neurophysiology and also into insect pest control, due to their specific target sites and to the recent constructions of insecticidal baculovirus expressions of several of these toxins.     

A novel test for fecal occult blood by the hybridization method with specific human deoxyribonucleic acid in leucocytes: preliminary experiment

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.     

NMR structures and activity of a novel alpha-like toxin from the scorpion Leiurus quinquestriatus hebraeus

NMR structures of a new toxin from the scorpion Leiurus quinquestriatus hebraeus (Lqh III) have been investigated in conjunction with its pharmacological properties. This toxin is proposed to belong to a new group of scorpion toxins, the alpha-like toxins that target voltage-gated sodium channels with specific properties compared with the classical alpha-scorpion toxins. Electrophysiological analysis showed that Lqh III inhibits a sodium current inactivation in the cockroach axon, but induces in addition a resting depolarization due to a slowly decaying tail current atypical to other alpha-toxin action. Binding studies indicated that radiolabeled Lqh III binds with a high degree of affinity (Ki=2.2 nM) on cockroach sodium channels and that the alpha-toxin from L quinquestriatus hebraeus highly active on insects (LqhalphaIT) and alpha-like toxins compete at low concentration for its receptor binding site, suggesting that the alpha-like toxin receptor site is partially overlapping with the receptor site 3. Conversely, in rat brain, Lqh III competes for binding of the most potent anti-mammal alpha-toxin from Androctonus australis Hector venom (AaH II) only at very high concentration. The NMR structures were used for the scrutiny of the similarities and differences with representative scorpion alpha-toxins targeting the voltage-gated sodium channels of either mammals or insects. Three turn regions involved in the functional binding site of the anti-insect LqhalphaIT toxin reveal significant differences in the Lqh III structure. The electrostatic charge distribution in the Lqh III toxin is also surprisingly different when compared with the anti-mammal alpha-toxin AaH II. Similarities in the electrostatic charge distribution are, however, recognized between alpha-toxins highly active on insects and the alpha-like toxin Lqh III. This affords additional important elements to the definition of the new alpha-like group of scorpion toxins and the mammal versus insect scorpion toxin selectivities.     

Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I(K,Ca)) were investigated in ramified murine brain macrophages. In order to induce I(K,Ca) the intracellular concentration of nominal free Ca2+ was adjusted to 1 microM. The Ca2+-activated K+ current of brain macrophages did not show any voltage dependence at test potentials between -120 and +30 mV. A tenfold change in extracellular K+ concentration shifted the reversal potential of I(K,Ca) by 51 mV. The bee venom toxin apamin applied at concentrations of up to 1 microM did not affect I(K,Ca). Ca2+-activated K+ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3 nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I(K,Ca) at concentrations between 1 and 50 nM. Tetraethylammonium (TEA) blocked 8.0% of I(K,Ca) at a concentration of 1 mM, whereas 31.4% of current was blocked by 10 mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2 mM for their ability to block I(K,Ca). La3+ reduced I(K,Ca) by 72.8%, whereas Cd2+ decreased I(K,Ca) by 17.4%; in contrast, Ni2+ did not have any effect on I(K,Ca). Ba2+ applied at a concentration of 1 mM reduced I(K,Ca) voltage-dependently at hyperpolarizing potentials.     

Mechanism of neuromuscular blockade induced by phenthonium, a quaternary derivative of (-)-hyoscyamine, in skeletal muscles

1. The mechanisms underlying the postjunctional blockade induced by phenthonium [N-(4-phenyl) phenacyl 1-hyoscyamine] were investigated in mammalian and amphibian muscles. This muscarinic antagonist was previously shown to enhance specifically the spontaneous acetylcholine (ACh) release at concentrations that blocked neuromuscular transmission. 2. In both rat diaphragm and frog sartorius muscles, phenthonium (Phen, 1-100 microM) depressed the muscle twitches elicited by nerve stimulation (IC50: 23 microM and 5 microM, respectively), and blocked the nerve-evoked muscle action potential. The neuromuscular blockade was not reversed after incubation with neostigmine. 3. Equal concentrations of Phen decreased the rate of rise and prolonged the falling phase of the directly elicited action potential in frog sartorius muscle fibres, indicating that the drug also affects the sodium and potassium conductance. 4. Phen (50 and 100 microM) protected the ACh receptor against alpha-bungarotoxin (BUTX) blockade in the mouse diaphragm allowing recording of endplate potentials and action potentials after 5 h wash with physiological salt solution. 5. Phen (10-100 microM) produced a concentration- and voltage-dependent decrease of the endplate current (e.p.c.), and induced nonlinearity of the current-voltage relationship. At high concentrations Phen also shortened the decay time constant of e.p.c (tau(e.p.c.)) and reduced its voltage sensitivity. 6. At the same range of concentrations, Phen also reduced the initial rate of [125I]-BUTX binding to junctional ACh receptors of the rat diaphragm (apparent dissociation constant = 24 microM), the relationship between the degree of inhibition and antagonist concentration being that expected for a competitive mechanism. 7. It is concluded that Phen affects the electrical excitability of the muscle fibre membrane, and blocks neuromuscular transmission through a mechanism that affects the agonist binding to its recognition site and ionic channel conductance of the nicotinic ACh receptor.     

Depressant insect selective neurotoxins from scorpion venom: chemistry, action, and gene cloning

The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.     

New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity

Two new toxins were purified from Leiurus quinquestriatus hebraeus (Lqh) scorpion venom, Lqh II and Lqh III. Lqh II sequence reveals only two substitutions, as compared to AaH II, the most active scorpion alpha-toxin on mammals from Androctounus australis Hector. Lqh III shares 80% sequence identity with the alpha-like toxin Bom III from Buthus occitanus mardochei. Using bioassays on mice and cockroach coupled with competitive binding studies with 125I-labeled scorpion alpha-toxins on rat brain and cockroach synaptosomes, the animal selectivity was examined. Lqh II has comparable activity to mammals as AaH II, but reveals significantly higher activity to insects attributed to its C-terminal substitution, and competes at low concentration for binding on both mammalian and cockroach sodium channels. Lqh II thus binds to receptor site 3 on sodium channels. Lqh III is active on both insects and mammals but competes for binding only on cockroach. The latter indicates that Lqh III binds to a distinct receptor site. Thus, Lqh II and Lqh III represent two different scorpion toxin groups, the alpha- and alpha-like toxins, respectively, according to the structural and pharmacological criteria. These new toxins may serve as a lead for clarification of the structural basis for insect vs mammal selectivity of scorpion toxins.     

beta-Fructosidases: a new superfamily of glycosyl-hydrolases

Dihydropyridines (DHPs) block L-type Ca2+ channels more potently at depolarized membrane potentials, consistent with high affinity binding to the inactivated state. Nisoldipine (a DHP antagonist) blocks the smooth muscle channel more potently than the cardiac one, a phenomenon observed not only in native channels but also in expressed channels. We examined whether this tissue specificity was attributable to differences of inactivation in the two channel types. We expressed cardiac or smooth muscle alpha1C subunits in combination with beta2a and alpha2/delta subunits in human embryonic kidney cells, and used 2 mM Ca2+ as the permeant ion. This system thus reproduces the in vivo topology and charge carrier of the channels while facilitating comparison of the two alpha1C splice variants. Both voltage-dependent and isoform-specific sensitivity of 10 nM nisoldipine inhibition of the channel were demonstrated, with the use of -100 mV as the holding potential for fully reprimed channels and -65 mV to populate the inactivated state. Under drug-free conditions, we characterized fast inactivation (1-sec prepulses) and slow inactivation (3 min prepulses) in the two isoforms. Inactivation parameters were not statistically different in the two channel isoforms; if anything, cardiac channels tended to inactivate more than the smooth muscle channels at relevant voltages. Likewise, the voltage-dependent activation was identical in the two isoforms. We thus conclude that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. Intrinsic, gating-independent DHP receptor binding affinity differences must be invoked to explain the isoform-specific sensitivity of the DHP block.     

Makatoxin I, a novel toxin isolated from the venom of the scorpion Buthus martensi Karsch, exhibits nitrergic actions

Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 +/- 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 degrees C, 5% CO2 in O2). MkTx I (2 microg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (50 microM). Thus, MkTx I is the first alpha-toxin that can mediate nitrergic responses in the rat isolated ACM.     

The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch

The cDNA library of venomous glands of the scorpion Buthus martensii Karsch (BmK) was constructed. A cDNA encoding a mammalian neurotoxin corresponding to the known alpha-type toxin, BmK M1, was amplified by polymerase chain reaction (PCR) and cloned, and its full-length sequence was determined. The open reading frame encoded the precursor of BmK M1 with 84 amino acid residues, including a signal peptide of 19 residues, a mature toxin of 64 residues and an additional C-terminal residue Arg which might be cleaved off by proteinase postprocessing immediately after protein synthesis. Based on the determined cDNA sequence and using the total DNA of the scorpion as a template, the gene of BmK M1 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 408 base pairs present within the signal peptide region. Both the intron and exon of BmK M1 share about 75% similarity with those of AaH I' another alpha-type mammalian neurotoxin in the scorpion Androctonus australis Hector.     

En-bloc irradiation of tumours of the head and neck and their lymphatics. I. Technique and dosimetry

The protein, beta-bungarotoxin, a presynaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, is known to inhibit mitochondrial function. Within 30 min after adding the toxin to a rat diaphragmphrenic nerve preparation, the quantal content increased tenfold and the frequency of miniature endplate potentials increased fourfold. No increase in miniature endplate potential frequency was seen in the absence of extracellular calcium. Since mitochondria may be involved in regulating intracellular calcium levels, the rate at which the transmitter release is turned off was studied by measuring delayed release in the presence and absence of toxin. Delayed release is elevated about eightfold by the toxin. If delayed release is due to residual calcium, as has been hypothesized, these data may be explained if the toxin does not alter the amount of calcium which enters the terminal, but rather the rate at which that calcium is removed. Alternatively, a calcium-dependent modification of the release process itself might be produced. The eventual reduction in transmitter output did not appear to result from depletion of the terminal of releaseable packets of transmitter, but does require extracellular calcium.     

Two similar peptides from the venom of the scorpion Pandinus imperator, one highly effective blocker and the other inactive on K+ channels

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important 'point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (rhoO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; rhoO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Treatment of colo-vesical fistulas in one-stage operation

BACKGROUND: Botulinum toxin A is a potent inhibitor of the release of acetylcholine from nerve endings. Local injection of botulinum toxin has recently been suggested to be helpful in sphincter of Oddi dyskinesia by decreasing sphincter of Oddi pressure. AIMS: To explore the mechanism of action of botulinum toxin A on sphincter of Oddi (SO) muscle. METHODS: Four piglets underwent duodenoscopy and SO manometry was performed. After obtaining a baseline pressure, the SO was injected with normal saline and the experiment repeated after one week. The SO was then injected endoscopically with botulinum toxin (40 U) with follow up manometry one week later. The sphincter of Oddi was removed from 10 pigs, cut into three rings, and placed in an organ bath. The force of contraction was measured and registered on a polygraph. Rings were stimulated by 70 V (10 Hz, 0.5 ms) electrical field stimulation for 20 seconds, exogenous acetylcholine (100 microM), and KCl (125 mM). Botulinum toxin (0.1 U/ml) or atropine (1 microM) was added to the incubation medium and the stimulation was repeated. RESULTS: Mean basal SO pressure in the pigs remained unchanged after saline injection but decreased to about 50% of baseline value following botulinum toxin injection (p = 0.04). The contractions induced by direct stimulation of SO smooth muscle with KCl were not significantly affected by either atropine or botulinum toxin. In all rings exogenous acetylcholine induced contractions, which were totally blocked by atropine, but not by botulinum toxin. Electrical field stimulation induced contractions that were inhibited by both atropine and botulinum toxin. CONCLUSION: Botulinum toxin inhibits pig sphincter of Oddi smooth muscle contractions by a presynaptic cholinergic mechanism, similar to that described in skeletal muscle.     

Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.     

Venom variability among several Tityus serrulatus specimens

Individual differences in venom composition among several Tityus serrulatus specimens collected in the same area were examined by enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies raised against whole venom and against the alpha-type (toxin IV-5 or Ts IV) and the beta-type toxin (toxin gamma or Ts VII) were used to study specific variations in the venom. The ELISA results indicated clear differences among the scorpion venoms examined. The lethality (LD50) determined by subcutaneous injections of pooled venom with the same characteristics showed an interesting correlation between the expression level of each component studied and the lethal effect of the venom. Among the groups analysed, the group with the highest concentration of alpha-type toxin showed the highest toxicity. The groups with the lowest level of toxicity were those with a low concentration of alpha-type toxin. The results show that the lethality of the venom varies from specimen to specimen and suggest that alpha-type toxin must be the major lethal component in the whole venom.     

Solution structure of a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing

We report the detailed solution structure of the 7.2 kDa protein CsE-I, a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the alpha-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical alpha-neurotoxin, CsE-v3, and is characterized by a short 2(1/2)-turn alpha-helix and a 3-strand antiparallel beta-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 A. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the alpha- and beta-neurotoxins.